@phdthesis{Kiss2024, author = {Kiss, Andrea}, title = {Moss-associated bacterial and archaeal communities of northern peatlands: key taxa, environmental drivers and potential functions}, doi = {10.25932/publishup-63064}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-630641}, school = {Universit{\"a}t Potsdam}, pages = {XX, 139, liv}, year = {2024}, abstract = {Moss-microbe associations are often characterised by syntrophic interactions between the microorganisms and their hosts, but the structure of the microbial consortia and their role in peatland development remain unknown. In order to study microbial communities of dominant peatland mosses, Sphagnum and brown mosses, and the respective environmental drivers, four study sites representing different successional stages of natural northern peatlands were chosen on a large geographical scale: two brown moss-dominated, circumneutral peatlands from the Arctic and two Sphagnum-dominated, acidic peat bogs from subarctic and temperate zones. The family Acetobacteraceae represented the dominant bacterial taxon of Sphagnum mosses from various geographical origins and displayed an integral part of the moss core community. This core community was shared among all investigated bryophytes and consisted of few but highly abundant prokaryotes, of which many appear as endophytes of Sphagnum mosses. Moreover, brown mosses and Sphagnum mosses represent habitats for archaea which were not studied in association with peatland mosses so far. Euryarchaeota that are capable of methane production (methanogens) displayed the majority of the moss-associated archaeal communities. Moss-associated methanogenesis was detected for the first time, but it was mostly negligible under laboratory conditions. Contrarily, substantial moss-associated methane oxidation was measured on both, brown mosses and Sphagnum mosses, supporting that methanotrophic bacteria as part of the moss microbiome may contribute to the reduction of methane emissions from pristine and rewetted peatlands of the northern hemisphere. Among the investigated abiotic and biotic environmental parameters, the peatland type and the host moss taxon were identified to have a major impact on the structure of moss-associated bacterial communities, contrarily to archaeal communities whose structures were similar among the investigated bryophytes. For the first time it was shown that different bog development stages harbour distinct bacterial communities, while at the same time a small core community is shared among all investigated bryophytes independent of geography and peatland type. The present thesis displays the first large-scale, systematic assessment of bacterial and archaeal communities associated both with brown mosses and Sphagnum mosses. It suggests that some host-specific moss taxa have the potential to play a key role in host moss establishment and peatland development.}, language = {en} } @phdthesis{Hammel2024, author = {Hammel, Alexander}, title = {Establishing the red microalga Porphyridium purpureum as a novel platform for the production of recombinant proteins}, doi = {10.25932/publishup-63270}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-632709}, school = {Universit{\"a}t Potsdam}, pages = {ix, 159}, year = {2024}, abstract = {Microalgae have been recognized as a promising green production platform for recombinant proteins. The majority of studies on recombinant protein expression have been conducted in the green microalga C. reinhardtii. While promising improvement regarding nuclear transgene expression in this alga has been made, it is still inefficient due to epigenetic silencing, often resulting in low yields that are not competitive with other expressor organisms. Other microalgal species might be better suited for high-level protein expression, but are limited in their availability of molecular tools. The red microalga Porphyridium purpureum recently emerged as candidate for the production of recombinant proteins. It is promising in that transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus at a high copy number, thus leading to high expression values in this red alga. In this work, we expand the genetic tools for P. purpureum and investigate parameters that govern efficient transgene expression. We provide an improved transformation protocol to streamline the generation of transgenic lines in this organism. After being able to efficiently generate transgenic lines, we showed that codon usage is a main determinant of high-level transgene expression, not only at the protein level but also at the level of mRNA accumulation. The optimized expression constructs resulted in YFP accumulation up to an unprecedented 5\% of the total soluble protein. Furthermore, we designed new constructs conferring efficient transgene expression into the culture medium, simplifying purification and harvests of recombinant proteins. To further improve transgene expression, we tested endogenous promoters driving the most highly transcribed genes in P. purpureum and found minor increase of YFP accumulation. We employed the previous findings to express complex viral antigens from the hepatitis B virus and the hepatitis C virus in P. purpureum to demonstrate its feasibility as producer of biopharmaceuticals. The viral glycoproteins were successfully produced to high levels and could reach their native confirmation, indicating a functional glycosylation machinery and an appropriate folding environment in this red alga. We could successfully upscale the biomass production of transgenic lines and with that provide enough material for immunization trials in mice that were performed in collaboration. These trials showed no toxicity of neither the biomass nor the purified antigens, and, additionally, the algal-produced antigens were able to elicit a strong and specific immune response. The results presented in this work pave the way for P. purpureum as a new promising producer organism for biopharmaceuticals in the microalgal field.}, language = {en} } @phdthesis{Cheng2024, author = {Cheng, Feng}, title = {Evolution and ontogeny of electric organ discharge in African weakly electric fish genus Campylomormyrus: a genomic and transcriptomic perspective}, doi = {10.25932/publishup-63017}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-630172}, school = {Universit{\"a}t Potsdam}, pages = {176}, year = {2024}, abstract = {The African weakly electric fishes (Mormyridae) exhibit a remarkable adaptive radiation possibly due to their species-specific electric organ discharges (EODs). It is produced by a muscle-derived electric organ that is located in the caudal peduncle. Divergence in EODs acts as a pre-zygotic isolation mechanism to drive species radiations. However, the mechanism behind the EOD diversification are only partially understood. The aim of this study is to explore the genetic basis of EOD diversification from the gene expression level across Campylomormyrus species/hybrids and ontogeny. I firstly produced a high quality genome of the species C. compressirostris as a valuable resource to understand the electric fish evolution. The next study compared the gene expression pattern between electric organs and skeletal muscles in Campylomormyrus species/hybrids with different types of EOD duration. I identified several candidate genes with an electric organ-specific expression, e.g. KCNA7a, KLF5, KCNJ2, SCN4aa, NDRG3, MEF2. The overall genes expression pattern exhibited a significant association with EOD duration in all analyzed species/hybrids. The expression of several candidate genes, e.g. KCNJ2, KLF5, KCNK6 and KCNQ5, possibly contribute to the regulation of EOD duration in Campylomormyrus due to their increasing or decreasing expression. Several potassium channel genes showed differential expression during ontogeny in species and hybrid with EOD alteration, e.g. KCNJ2. I next explored allele specific expression of intragenus hybrids by crossing the duration EOD species C. compressirostris with the medium duration EOD species C. tshokwe and the elongated duration EOD species C. rhynchophorus. The hybrids exhibited global expression dominance of the C. compressirostris allele in the adult skeletal muscle and electric organ, as well as in the juvenile electric organ. Only the gene KCNJ2 showed dominant expression of the allele from C. rhynchophorus, and this was increasingly dominant during ontogeny. It hence supported our hypothesis that KCNJ2 is a key gene of regulating EOD duration. Our results help us to understand, from a genetic perspective, how gene expression effect the EOD diversification in the African weakly electric fish.}, language = {en} } @phdthesis{Stange2024, author = {Stange, Maike}, title = {A study on Coronin-A and Aip1 function in motility of Dictyostelium discoideum and on Aip1 interchangeability between Dictyostelium discoideum and Arabidopsis thaliana}, doi = {10.25932/publishup-62856}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-628569}, school = {Universit{\"a}t Potsdam}, pages = {xiv, 168}, year = {2024}, abstract = {Actin is one of the most highly conserved proteins in eukaryotes and distinct actin-related proteins with filament-forming properties are even found in prokaryotes. Due to these commonalities, actin-modulating proteins of many species share similar structural properties and proposed functions. The polymerization and depolymerization of actin are critical processes for a cell as they can contribute to shape changes to adapt to its environment and to move and distribute nutrients and cellular components within the cell. However, to what extent functions of actin-binding proteins are conserved between distantly related species, has only been addressed in a few cases. In this work, functions of Coronin-A (CorA) and Actin-interacting protein 1 (Aip1), two proteins involved in actin dynamics, were characterized. In addition, the interchangeability and function of Aip1 were investigated in two phylogenetically distant model organisms. The flowering plant Arabidopsis thaliana (encoding two homologs, AIP1-1 and AIP1-2) and in the amoeba Dictyostelium discoideum (encoding one homolog, DdAip1) were chosen because the functions of their actin cytoskeletons may differ in many aspects. Functional analyses between species were conducted for AIP1 homologs as flowering plants do not harbor a CorA gene. In the first part of the study, the effect of four different mutation methods on the function of Coronin-A protein and the resulting phenotype in D. discoideum was revealed in two genetic knockouts, one RNAi knockdown and a sudden loss-of-function mutant created by chemical-induced dislocation (CID). The advantages and disadvantages of the different mutation methods on the motility, appearance and development of the amoebae were investigated, and the results showed that not all observed properties were affected with the same intensity. Remarkably, a new combination of Selection-Linked Integration and CID could be established. In the second and third parts of the thesis, the exchange of Aip1 between plant and amoeba was carried out. For A. thaliana, the two homologs (AIP1-1 and AIP1-2) were analyzed for functionality as well as in D. discoideum. In the Aip1-deficient amoeba, rescue with AIP1-1 was more effective than with AIP1-2. The main results in the plant showed that in the aip1-2 mutant background, reintroduced AIP1-2 displayed the most efficient rescue and A. thaliana AIP1-1 rescued better than DdAip1. The choice of the tagging site was important for the function of Aip1 as steric hindrance is a problem. The DdAip1 was less effective when tagged at the C-terminus, while the plant AIP1s showed mixed results depending on the tag position. In conclusion, the foreign proteins partially rescued phenotypes of mutant plants and mutant amoebae, despite the organisms only being very distantly related in evolutionary terms.}, language = {en} } @phdthesis{Stiegler2023, author = {Stiegler, Jonas}, title = {Mobile link functions in unpredictable agricultural landscapes}, doi = {10.25932/publishup-62202}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-622023}, school = {Universit{\"a}t Potsdam}, pages = {155}, year = {2023}, abstract = {Animal movement is a crucial aspect of life, influencing ecological and evolutionary processes. It plays an important role in shaping biodiversity patterns, connecting habitats and ecosystems. Anthropogenic landscape changes, such as in agricultural environments, can impede the movement of animals by affecting their ability to locate resources during recurring movements within home ranges and, on a larger scale, disrupt migration or dispersal. Inevitably, these changes in movement behavior have far-reaching consequences on the mobile link functions provided by species inhabiting such extensively altered matrix areas. In this thesis, I investigate the movement characteristics and activity patterns of the European hare (Lepus europaeus), aiming to understand their significance as a pivotal species in fragmented agricultural landscapes. I reveal intriguing results that shed light on the importance of hares for seed dispersal, the influence of personality traits on behavior and space use, the sensitivity of hares to extreme weather conditions, and the impacts of GPS collaring on mammals' activity patterns and movement behavior. In Chapter I, I conducted a controlled feeding experiment to investigate the potential impact of hares on seed dispersal. By additionally utilizing GPS data of hares in two contrasting landscapes, I demonstrated that hares play a vital role, acting as effective mobile linkers for many plant species in small and isolated habitat patches. The analysis of seed intake and germination success revealed that distinct seed traits, such as density, surface area, and shape, profoundly affect hares' ability to disperse seeds through endozoochory. These findings highlight the interplay between hares and plant communities and thus provide valuable insights into seed dispersal mechanisms in fragmented landscapes. By employing standardized behavioral tests in Chapter II, I revealed consistent behavioral responses among captive hares while simultaneously examining the intricate connection between personality traits and spatial patterns within wild hare populations. This analysis provides insights into the ecological interactions and dynamics within hare populations in agricultural habitats. Examining the concept of animal personality, I established a link between personality traits and hare behavior. I showed that boldness, measured through standardized tests, influences individual exploration styles, with shy and bold hares exhibiting distinct space use patterns. In addition to providing valuable insights into the role of animal personality in heterogeneous environments, my research introduced a novel approach demonstrating the feasibility of remotely assessing personality types using animal-borne sensors without additional disturbance of the focal individual. While climate conditions severely impact the activity and, consequently, the fitness of wildlife species across the globe, in Chapter III, I uncovered the sensitivity of hares to temperature, humidity, and wind speed during their peak reproduction period. I found a strong response in activity to high temperatures above 25°C, with a particularly pronounced effect during temperature extremes of over 35°C. The non-linear relationship between temperature and activity was characterized by contrasting responses observed for day and night. These findings emphasize the vulnerability of hares to climate change and the potential consequences for their fitness and population dynamics with the ongoing rise of temperature. Since such insights can only be obtained through capturing and tagging free-ranging animals, I assessed potential impacts and the recovery process post-collar attachment in Chapter IV. For this purpose, I examined the daily distances moved and the temporal-associated activity of 1451 terrestrial mammals out of 42 species during their initial tracking period. The disturbance intensity and the speed of recovery varied across species, with herbivores, females, and individuals captured and collared in relatively secluded study areas experiencing more pronounced disturbances due to limited anthropogenic influences. Mobile linkers are essential for maintaining biodiversity as they influence the dynamics and resilience of ecosystems. Furthermore, their ability to move through fragmented landscapes makes them a key component for restoring disturbed sites. Individual movement decisions determine the scale of mobile links, and understanding variations in space use among individuals is crucial for interpreting their functions. Climate change poses further challenges, with wildlife species expected to adjust their behavior, especially in response to high-temperature extremes, and comprehending the anthropogenic influence on animal movements will remain paramount to effective land use planning and the development of successful conservation strategies. This thesis provides a comprehensive ecological understanding of hares in agricultural landscapes. My research findings underscore the importance of hares as mobile linkers, the influence of personality traits on behavior and spatial patterns, the vulnerability of hares to extreme weather conditions, and the immediate consequences of collar attachment on mammalian movements. Thus, I contribute valuable insights to wildlife conservation and management efforts, aiding in developing strategies to mitigate the impact of environmental changes on hare populations. Moreover, these findings enable the development of methodologies aimed at minimizing the impacts of collaring while also identifying potential biases in the data, thereby benefiting both animal welfare and the scientific integrity of localization studies.}, language = {en} } @phdthesis{Schaefer2024, author = {Sch{\"a}fer, Marj{\"a}nn Helena}, title = {Untersuchungen zur Evolution der 15-Lipoxygenase (ALOX15) bei S{\"a}ugetieren und funktionelle Charakterisierung von Knock-in-M{\"a}usen mit humanisierter Reaktionsspezifit{\"a}t der 15-Lipoxygenase-2 (Alox15b)}, doi = {10.25932/publishup-62034}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-620340}, school = {Universit{\"a}t Potsdam}, pages = {XVII, 280}, year = {2024}, abstract = {Arachidons{\"a}urelipoxygenasen (ALOX-Isoformen) sind Lipid-peroxidierenden Enzyme, die bei der Zelldifferenzierung und bei der Pathogenese verschiedener Erkrankungen bedeutsam sind. Im menschlichen Genom gibt es sechs funktionelle ALOX-Gene, die als Einzelkopiegene vorliegen. F{\"u}r jedes humane ALOX-Gen gibt es ein orthologes Mausgen. Obwohl sich die sechs humanen ALOX-Isoformen strukturell sehr {\"a}hnlich sind, unterscheiden sich ihre funktionellen Eigenschaften deutlich voneinander. In der vorliegenden Arbeit wurden vier unterschiedliche Fragestellungen zum Vorkommen, zur biologischen Rolle und zur Evolutionsabh{\"a}ngigkeit der enzymatischen Eigenschaften von S{\"a}ugetier-ALOX-Isoformen untersucht: 1) Spitzh{\"o}rnchen (Tupaiidae) sind evolution{\"a}r n{\"a}her mit dem Menschen verwandt als Nagetiere und wurden deshalb als Alternativmodelle f{\"u}r die Untersuchung menschlicher Erkrankungen vorgeschlagen. In dieser Arbeit wurde erstmals der Arachidons{\"a}urestoffwechsel von Spitzh{\"o}rnchen untersucht. Dabei wurde festgestellt, dass im Genom von Tupaia belangeri vier unterschiedliche ALOX15-Gene vorkommen und die Enzyme sich hinsichtlich ihrer katalytischen Eigenschaften {\"a}hneln. Diese genomische Vielfalt, die weder beim Menschen noch bei M{\"a}usen vorhanden ist, erschwert die funktionellen Untersuchungen zur biologischen Rolle des ALOX15-Weges. Damit scheint Tupaia belangeri kein geeigneteres Tiermodel f{\"u}r die Untersuchung des ALOX15-Weges des Menschen zu sein. 2) Entsprechend der Evolutionshypothese k{\"o}nnen S{\"a}ugetier-ALOX15-Orthologe in Arachidons{\"a}ure-12-lipoxygenierende- und Arachidons{\"a}ure-15-lipoxygenierende Enzyme eingeteilt werden. Dabei exprimieren S{\"a}ugetierspezies, die einen h{\"o}heren Evolutionsgrad als Gibbons aufweisen, Arachidons{\"a}ure-15-lipoxygenierende ALOX15-Orthologe, w{\"a}hrend evolution{\"a}r weniger weit entwickelte S{\"a}ugetiere Arachidons{\"a}ure-12 lipoxygenierende Enzyme besitzen. In dieser Arbeit wurden elf neue ALOX15-Orthologe als rekombinante Proteine exprimiert und funktionell charakterisiert. Die erhaltenen Ergebnisse f{\"u}gen sich widerspruchsfrei in die Evolutionshypothese ein und verbreitern deren experimentelle Basis. Die experimentellen Daten best{\"a}tigen auch das Triadenkonzept. 3) Da humane und murine ALOX15B-Orthologe unterschiedliche funktionelle Eigenschaften aufweisen, k{\"o}nnen Ergebnisse aus murinen Krankheitsmodellen zur biologischen Rolle der ALOX15B nicht direkt auf den Menschen {\"u}bertragen werden. Um die ALOX15B-Orthologen von Maus und Mensch funktionell einander anzugleichen, wurden im Rahmen der vorliegenden Arbeit Knock-in M{\"a}use durch die In vivo Mutagenese mittels CRISPR/Cas9-Technik hergestellt. Diese exprimieren eine humanisierte Mutante (Doppelmutation von Tyrosin603Asparagins{\"a}ure+Histidin604Valin) der murinen Alox15b. Diese M{\"a}use waren lebens- und fortpflanzungsf{\"a}hig, zeigten aber geschlechtsspezifische Unterschiede zu ausgekreuzten Wildtyp-Kontrolltieren im Rahmen ihre Individualentwicklung. 4) In vorhergehenden Untersuchungen zur Rolle der ALOX15B in Rahmen der Entz{\"u}ndungsreaktion wurde eine antiinflammatorische Wirkung des Enzyms postuliert. In der vorliegenden Arbeit wurde untersucht, ob eine Humanisierung der murinen Alox15b die Entz{\"u}ndungsreaktion in zwei verschiedenen murinen Entz{\"u}ndungsmodellen beeinflusst. Eine Humanisierung der murinen Alox15b f{\"u}hrte zu einer verst{\"a}rkten Ausbildung von Entz{\"u}ndungssymptomen im induzierten Dextran-Natrium-Sulfat-Kolitismodell. Im Gegensatz dazu bewirkte die Humanisierung der Alox15b eine Abschw{\"a}chung der Entz{\"u}ndungssymptome im Freund'schen Adjuvans Pfoten{\"o}demmodell. Diese Daten deuten darauf hin, dass sich die Rolle der ALOX15B in verschiedenen Entz{\"u}ndungsmodellen unterscheidet.}, language = {de} } @phdthesis{Yildiz2023, author = {Yildiz, Tugba}, title = {Dissecting the role of the TusA protein for cell functionality and FtsZ ring assembly in Escherichia coli}, doi = {10.25932/publishup-61713}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-617135}, school = {Universit{\"a}t Potsdam}, pages = {XI, 171}, year = {2023}, abstract = {In this work, the role of the TusA protein was investigated for the cell functionality and FtsZ ring assembly in Escherichia coli. TusA is the tRNA-2-thiouridine synthase that acts as a sulfur transferase in tRNA thiolation for the formation of 2-thiouridine at the position 34 (wobble base) of tRNALys, tRNAGlu and tRNAGln. It binds the persulfide form of sulfur and transfers it to further proteins during mnm5s2U tRNA modification at wobble position and for Moco biosynthesis. With this thiomodification of tRNA, the ribosome binding is more efficient and frameshifting is averted during the protein translation. Previous studies have revealed an essential role of TusA in bacterial cell physiology since deletion of the tusA gene resulted in retarded growth and filamentous cells during the exponential growth phase in a rich medium which suddenly disappeared during the stationary phase. This indicates a problem in the cell division process. Therefore the focus of this work was to investigate the role of TusA for cell functionality and FtsZ ring formation and thus the cell separation. The reason behind the filamentous growth of the tusA mutant strain was investigated by growth and morphological analyses. ΔtusA cells showed a retarded growth during the exponential phase compared to the WT strain. Also, morphological analysis of ΔtusA cells confirmed the filamentous cell shape. The growth and cell division defects in ΔtusA indicated a defect in FtsZ protein as a key player of cell division. The microscopic investigation revealed that filamentous ΔtusA cells possessed multiple DNA parts arranged next to each other. This suggested that although the DNA replication occurred correctly, there was a defect in the step where FtsZ should act; probably FtsZ is unable to assemble to the ring structure or the assembled ring is not able to constrict. All tested mutant strains (ΔtusD, ΔtusE and ΔmnmA) involved in the mnm5s2U34 tRNA modification pathway shared the similar retarded growth and filamentous cell shape like ΔtusA strain. Thus, the cell division defect arises from a defect in mnm5s2U34 tRNA thiolation. Since the FtsZ ring formation was supposed to be defective in filaments, a possible intracellular interaction of TusA and FtsZ was examined by fluorescent (EGFP and mCherry) fusion proteins expression and FRET. FtsZ expressing tusA mutant (DE3) cells showed a red mCherry signal at the cell poles, indicating that FtsZ is still in the assembling phase. Interestingly, the cellular region of EGFP-TusA fusion protein expressed in ΔtusA (DE3) was conspicuous; the EGFP signal was spread throughout the whole cell and, in addition, a slight accumulation of the EGFP-TusA fluorescence was detectable at the cell poles, the same part of the cell as for mCherry-FtsZ. Thus, this strongly suggested an interaction of TusA and FtsZ. Furthermore, the cellular FtsZ and Fis concentrations, and their change during different growth phases were determined via immunoblotting. All tested deletion strains of mnm5s2U34 tRNA modification show high cellular FtsZ and Fis levels in the exponential phase, shifting to the later growth phases. This shift reflects the retarded growth, whereby the deletion strains reach later the exponential phase. Conclusively, the growth and cell division defect, and thus the formation of filaments, is most likely caused by changes in the cellular FtsZ and Fis concentrations. Finally, the translation efficiencies of certain proteins (RpoS, Fur, Fis and mFis) in tusA mutant and in additional gene deletion strains were studied whether they were affected by using unmodified U34 tRNAs of Lys, Glu and Gln. The translation efficiency is decreased in mnm5s2U34 tRNA modification-impaired strains in addition to their existing growth and cell division defect due to the elimination of these three amino acids. Finally, these results confirm and reinforce the importance of Lys, Glu and Gln and the mnm5s2U34 tRNA thiolation for efficient protein translation. Thus, these findings verify that the translation of fur, fis and rpoS is regulated by mnm5s2U34 tRNA modifications, which is growth phase-dependent. In total, this work showed the importance of the role of TusA for bacterial cell functionality and physiology. The deletion of the tusA gene disrupted a complex regulatory network within the cell, that most influenced by the decreased translation of Fis and RpoS, caused by the absence of mnm5s2U34 tRNA modifications. The disruption of RpoS and Fis cellular network influences in turn the cellular FtsZ level in the early exponential phase. Finally, the reduced FtsZ concentration leads to elongated, filamentous E. coli cells, which are unable to divide.}, language = {en} } @phdthesis{Sureshkumar2023, author = {Sureshkumar, Priyavathi}, title = {Erweiterung der zellbasierten Calcium-Imaging-Methode im eukaryotischen zellfreien Proteinsynthese-System f{\"u}r die transient-receptor-potential (TRP) - Ionenkan{\"a}le}, doi = {10.25932/publishup-61987}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-619872}, school = {Universit{\"a}t Potsdam}, pages = {x, 110}, year = {2023}, abstract = {Die Fluoreszenz-Calcium-Imaging-Methode wird auch heute noch als g{\"a}ngige Methode verwendet, vor allem wegen der geringeren Kosten f{\"u}r das Wirkstoffscreening in der pharmazeutischen Forschung, wobei Ionenkan{\"a}le sowie einige der G-Protein gekoppelte Rezeptoren (GPCRs) die Mehrzahl der Wirkstoffziele ansprechen. Die zellfreie Synthese eukaryotischer Proteine hat nicht die Nachteile, die bei der {\"U}berexpression dieser ionenpermeablen Proteine in Zellen auftreten k{\"o}nnen, wie z. B. Zelltoxizit{\"a}t, geringere Proteinexpression und die Beseitigung der exprimierten Proteine aufgrund ver{\"a}nderter Dom{\"a}nen sowie die zeitaufw{\"a}ndige Pflege von Zelllinien. Die Synthese von Ionenkan{\"a}len in zellfreien Proteinsyntheseplattformen f{\"u}r das k{\"u}nftige Wirkstoffscreening ist noch in der Grundlagenforschung. Obwohl die Fluoreszenz-Calcium-Imaging-Methode in zellbasierten Assays weit verbreitet ist, wurde diese Methode bisher noch nicht in zellfreien Proteinexpressionssystemen verwendet. Insgesamt ist die neue Anwendung der Calcium-Imaging-Methode in eukaryontischen zellfreien Systemen eine Voraussetzung f{\"u}r die schnelle pharmakologische Analyse von Wirkstoffen. Das erste Ziel dieser wissenschaftlichen Arbeit bestand darin, die grundlegenden Prinzipien der Calcium-Imaging-Methode zur Untersuchung von Ionenkan{\"a}len in zellbasierten Systemen zu untersuchen. Hierf{\"u}r wurden zwei Tumorzelllinien des Auges verwendet, und zwar benigne Pterygiumzellen und maligne Aderhautmelanom 92.1 Zellen. In diesen Studien wurde die Interaktion zwischen den nativ {\"u}berexprimierten transient-receptor-potential-Ionenkan{\"a}len (TRPs) wie TRP Vanilliod 1 (TRPV1) (Capsaicinrezeptor) und TRP Melastatin 8 (TRPM8) (Mentholrezeptor) in diesen Tumorzellen nach Zugabe von verschiedenen Medikamenten und Hormonen untersucht. Das zweite Ziel dieser Arbeit war es, den Calcium-Mechanismus von GPCRs in den Zellen zu untersuchen. Zu diesem Zweck wurde Mas, ein GPCR und Angiotensin (1-7) -Hormonrezeptor, aus dem renin-angiotensin-aldosteron-system (RAAS) in der Human Embryonic Kidney-293 (HEK293) Zelllinie {\"u}berexprimiert. In dieser Studie wurden insbesondere die Aktivierung klassischer GPCR-Signalwege wie Phospholipase C und Proteinkinase C durch Angiotensin-(1-7) {\"u}ber Mas und die Beteiligung von TRP-Kan{\"a}len nachgewiesen. Die zellbasierte-Calcium-Imaging-Methode f{\"u}r chemische Calcium-Indikatoren ließ sich aufgrund der Anwesenheit einer großen Menge cytosolischer Carboxylesterasen gut anwenden. Carboxylesterase ist das wichtigste Enzym in der Calcium Imaging Methode, das die Verarbeitung chemischen Calcium-Farbstoffe behandelt. Dieses Enzym fehlt jedoch in Mikrosomen, die als Basismembran f{\"u}r die Integration synthetisierter Ionenkan{\"a}le in eukaryontischen zellfreien Systemen verwendet werden. Das dritte Ziel dieser Forschungsarbeit war die Umsetzung der zellbasierten Calcium-Imaging Methode und der Calcium-Signalwege in zellfreie Systeme. Hier wurde die zellfrei synthetisierte Carboxylesterase in Mikrosomen von Spodoptera frugiperda (Sf21) als praktikables Calcium-Imaging-Werkzeug etabliert, um sowohl native ionenpermeable Proteine als auch zellfrei-synthetisierte Ionenkan{\"a}le zu untersuchen. Die Enzymaktivit{\"a}t der zellfrei-synthetisierten Carboxylesterase in Mikrosomen wurde durch Esterase-Assays und den Calcium-Fluoreszenzfarbstoff Fluo-5N Acetoxymethylester (Fluo-5N AM) Belastungstests nachgewiesen. Das Calcium-Imaging der nativ vorhandenen Ca2+-ATPase des sarkoplasmatischen/endoplasmatischen Retikulums (SERCA) und der Ryanodin-Rezeptoren (RyR) in den Mikrosomen sowie der zell-frei exprimierten TRP-Ionenkan{\"a}le wurden mit dem Fura-5N-AM- Fluoreszenzfarbstoff in mit Carboxylesterase vorsynthetisierten Mikrosomen nachgewiesen. Zusammenfassend l{\"a}sst sich sagen, dass das Prinzip der zellbasierten Calcium-Imaging -Methode vielversprechend an das eukaryotische zellfreie Sf21-System angepasst werden konnte, um Ionenkan{\"a}le zu analysieren. Nach entsprechender Forschung k{\"o}nnte die etablierte Methode in Zukunft auch auf andere Membranproteine ausgeweitet werden. Dies umfasst die Untersuchung anderer zell-frei exprimierte GPCRs oder anderer Ionenkan{\"a}le wie Kalium-, Natrium- und Chlorid-Ionenkan{\"a}le.}, language = {de} } @phdthesis{CalderonQuinonez2023, author = {Calder{\´o}n Qui{\~n}{\´o}nez, Ana Patricia}, title = {Ecology and conservation of the jaguar (Panthera onca) in Central America}, doi = {10.25932/publishup-61367}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-613671}, school = {Universit{\"a}t Potsdam}, pages = {140}, year = {2023}, abstract = {Conservation of the jaguar relies on holistic and transdisciplinary conservation strategies that integratively safeguard essential, connected habitats, sustain viable populations and their genetic exchange, and foster peaceful human-jaguar coexistence. These strategies define four research priorities to advance jaguar conservation throughout the species' range. In this thesis I provide several relevant ecological and sociological insights into these research priorities, each addressed in a separate chapter. I focus on the effects of anthropogenic landscapes on jaguar habitat use and population gene flow, spatial patterns of jaguar habitat suitability and functional population connectivity, and on innovative governance approaches which can work synergistically to help achieve human-wildlife conviviality. Furthermore, I translate these insights into recommendations for conservation practice by providing tools and suggestions that conservation managers and stakeholders can use to implement local actions but also make broad scale conservation decisions in Central America. In Chapter 2, I model regional habitat use of jaguars, producing spatially-explicit maps for management of key areas of habitat suitability. Using an occupancy model of 13-year-camera-trap occurrence data, I show that human influence has the strongest impact on jaguar habitat use, and that Jaguar Conservation Units are the most important reservoirs of high quality habitat in this region. I build upon these results by zooming in to an area of high habitat suitability loss in Chapter 3, northern Central America. Here I study the drivers of jaguar gene flow and I produce spatially-explicit maps for management of key areas of functional population connectivity in this region. I use microsatellite data and pseudo-optimized multiscale, multivariate resistance surfaces of gene flow to show that jaguar gene flow is influenced by environmental, and even more strongly, by human influence variables; and that the areas of lowest gene flow resistance largely coincide with the location of the Jaguar Conservation Units. Given that human activities significantly impact jaguar habitat use and gene flow, securing viable jaguar populations in anthropogenic landscapes also requires fostering peaceful human-wildlife coexistence. This is a complex challenge that cannot be met without transdisciplinary academic research and cross-sectoral, collaborative governance structures that effectively respond to the multiple challenges of such coexistence. With this in mind, I focus in Chapter 4 on carnivore conservation initiatives that apply transformative governance approaches to enact transformative change towards human-carnivore coexistence. Using the frameworks of transformative biodiversity governance and convivial conservation, I highlight in this chapter concrete pathways, supported by more inclusive, democratic forms of conservation decision-making and participation that promote truly transformative changes towards human-jaguar conviviality.}, language = {en} } @phdthesis{FloresCastellanos2023, author = {Flores Castellanos, Junio}, title = {Potato tuber (Solanum tuberosum L. cv Desiree) — characterization of starch interacting proteins and maltodextrin metabolism}, doi = {10.25932/publishup-61505}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-615055}, school = {Universit{\"a}t Potsdam}, pages = {XV, 69}, year = {2023}, abstract = {Starch is a biopolymer for which, despite its simple composition, understanding the precise mechanism behind its formation and regulation has been challenging. Several approaches and bioanalytical tools can be used to expand the knowledge on the different parts involved in the starch metabolism. In this sense, a comprehensive analysis targeting two of the main groups of molecules involved in this process: proteins, as effectors/regulators of the starch metabolism, and maltodextrins as starch components and degradation products, was conducted in this research work using potato plants (Solanum tuberosum L. cv. Desiree) as model of study. On one side, proteins physically interacting to potato starch were isolated and analyzed through mass spectrometry and western blot for their identification. Alternatively, starch interacting proteins were explored in potato tubers from transgenic plants having antisense inhibition of starch-related enzymes and on tubers stored under variable environmental conditions. Most of the proteins recovered from the starch granules corresponded to previously described proteins having a specific role in the starch metabolic pathway. Another set of proteins could be grouped as protease inhibitors, which were found weakly interacting to starch. Variations in the protein profile obtained after electrophoresis separation became clear when tubers were stored under different temperatures, indicating a differential expression of proteins in response to changing environmental conditions. On the other side, since maltodextrin metabolism is thought to be involved in both starch initiation and degradation, soluble maltooligosaccharide content in potato tubers was analyzed in this work under diverse experimental variables. For this, tuber disc samples from wild type and transgenic lines strongly repressing either the plastidial or cytosolic form of the -glucan phosphorylase and phosphoglucomutase were incubated with glucose, glucose-6-phosphate, and glucose-1-phosphate solutions to evaluate the influence of such enzymes on the conversion of the carbon sources into soluble maltodextrins, in comparison to wild-type samples. Relative maltodextrin amounts analyzed through capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF) revealed that tuber discs could immediately uptake glucose-1-phosphate and use it to produce maltooligosaccharides with a degree of polymerization of up to 30 (DP30), in contrast to transgenic tubers with strong repression of the plastidial glucan phosphorylase. The results obtained from the maltodextrin analysis support previous indications that a specific transporter for glucose-1-phosphate may exist in both the plant cells and the plastidial membranes, thereby allowing a glucose-6-phosphate independent transport. Furthermore, it confirms that the plastidial glucan phosphorylase is responsible for producing longer maltooligosaccharides in the plastids by catalyzing a glucan polymerization reaction when glucose-1-phosphate is available. All these findings contribute to a better understanding of the role of the plastidial glucan phosphorylase as a key enzyme directly involved in the synthesis and degradation of glucans and their implication on starch metabolism.}, language = {en} } @phdthesis{Stanke2023, author = {Stanke, Sandra}, title = {AC electrokinetic immobilization of influenza viruses and antibodies on nanoelectrode arrays for on-chip immunoassays}, doi = {10.25932/publishup-61716}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-617165}, school = {Universit{\"a}t Potsdam}, pages = {x, 115}, year = {2023}, abstract = {In the present thesis, AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, were demonstrated as a simple and fast method to functionalize the surface of nanoelectrodes with submicrometer sized biological objects. These nanoelectrodes have a cylindrical shape with a diameter of 500 nm arranged in an array of 6256 electrodes. Due to its medical relevance influenza virus as well as anti-influenza antibodies were chosen as a model organism. Common methods to bring antibodies or proteins to biosensor surfaces are complex and time-consuming. In the present work, it was demonstrated that by applying AC electric fields influenza viruses and antibodies can be immobilized onto the nanoelectrodes within seconds without any prior chemical modification of neither the surface nor the immobilized biological object. The distribution of these immobilized objects is not uniform over the entire array, it exhibits a decreasing gradient from the outer row to the inner ones. Different causes for this gradient have been discussed, such as the vortex-shaped fluid motion above the nanoelectrodes generated by, among others, electrothermal fluid flow. It was demonstrated that parts of the accumulated material are permanently immobilized to the electrodes. This is a unique characteristic of the presented system since in the literature the AC electrokinetic immobilization is almost entirely presented as a method just for temporary immobilization. The spatial distribution of the immobilized viral material or the anti-influenza antibodies at the electrodes was observed by either the combination of fluorescence microscopy and deconvolution or by super-resolution microscopy (STED). On-chip immunoassays were performed to examine the suitability of the functionalized electrodes as a potential affinity-based biosensor. Two approaches were pursued: A) the influenza virus as the bio-receptor or B) the influenza virus as the analyte. Different sources of error were eliminated by ELISA and passivation experiments. Hence, the activity of the immobilized object was inspected by incubation with the analyte. This resulted in the successful detection of anti-influenza antibodies by the immobilized viral material. On the other hand, a detection of influenza virus particles by the immobilized anti-influenza antibodies was not possible. The latter might be due to lost activity or wrong orientation of the antibodies. Thus, further examinations on the activity of by AC electric fields immobilized antibodies should follow. When combined with microfluidics and an electrical read-out system, the functionalized chips possess the potential to serve as a rapid, portable, and cost-effective point-of-care (POC) device. This device can be utilized as a basis for diverse applications in diagnosing and treating influenza, as well as various other pathogens.}, language = {en} } @phdthesis{Pruefer2023, author = {Pr{\"u}fer, Mareike}, title = {Charakterisierung und wechselfeldgest{\"u}tzte Herstellung von Enzym-Nanoarrays}, doi = {10.25932/publishup-61232}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612329}, school = {Universit{\"a}t Potsdam}, pages = {104}, year = {2023}, abstract = {Dielektrophorese ist die Manipulation polarisierbarer Partikel durch inhomogene elektrische Wechselfelder. In dieser Arbeit wurden drei verschiedene Enzyme durch Dielektrophorese immobilisiert und anschließend hinsichtlich ihrer katalytischen Aktivit{\"a}t untersucht: Meerrettichperoxidase, Cholinoxidase aus Alcaligenes sp. und Glucoseoxidase aus Aspergillus niger. Die Immobilisierung erfolgte durch Dielektrophorese auf nano-Elektrodenarrays aus Wolfram-Zylindern mit 500 nm Durchmesser oder aus Titannitrid-Ringen mit 20 nm Breite. Die Immobilisierung der Enzyme konnte fluoreszenzmikroskopisch entweder anhand der intrinsischen Fluoreszenz oder aufgrund einer Fluoreszenzmarkierung vor oder nach der Immobilisierung f{\"u}r alle getesteten Enzyme nachgewiesen werden. Die Messung der Enzymaktivit{\"a}t erfolgte quantitativ durch den direkten oder indirekten Nachweis des gebildeten Produktes oder, im Falle der Cholinoxidase, durch Beobachtung der intrinsischen Fluoreszenz des Cofaktors FAD, die vom Oxidationszustand dieses Enzyms abh{\"a}ngt. F{\"u}r die Meerrettichperoxidase konnte so eine hohe erhaltene Enzymaktivit{\"a}t nach der Immobilisierung nachgewiesen werden. Die Aktivit{\"a}t der permanent immobilisierten Fraktion der Meerrettichperoxidase entsprach bis zu 47 \% der h{\"o}chstm{\"o}glichen Aktivit{\"a}t einer Monolage dieses Enzyms auf den Elektroden des Chips. Diese Aktivit{\"a}t kann als aktive, aber zuf{\"a}llig gegen{\"u}ber der Oberfl{\"a}che ausgerichtete Enzymschicht interpretiert werden. F{\"u}r die permanent immobilisierte Glucoseoxidase wurde nur eine Aktivit{\"a}t entsprechend <1,3 \% der Aktivit{\"a}t einer solchen Enzymschicht detektiert, w{\"a}hrend f{\"u}r die immobilisierte Cholinoxidase gar keine Aktivit{\"a}t nachgewiesen werden konnte. Die Aktivit{\"a}t der durch DEP immobilisierten Enzyme konnte somit quantitativ bestimmt werden. Der Anteil an erhaltener Aktivit{\"a}t h{\"a}ngt dabei stark vom verwendeten Enzym ab.}, language = {de} } @phdthesis{Dreymann2023, author = {Dreymann, Nico}, title = {Identification and functional characterization of aptamers targeting human urokinase and NDM-1 for therapeutic and diagnostic applications}, doi = {10.25932/publishup-61291}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612919}, school = {Universit{\"a}t Potsdam}, pages = {IX, 130}, year = {2023}, abstract = {Aptamers are single-stranded DNA (ssDNA) or RNA molecules that can bind specifically and with high affinity to target molecules due to their unique three-dimensional structure. For this reason, they are often compared to antibodies and sometimes even referred to as "chemical antibodies". They are simple and inexpensive to synthesize, easy to modify, and smaller than conventional antibodies. Enzymes, especially hydrolases, are interesting targets in this context. This class of enzymes is capable of hydrolytically cleaving various macromolecules such as proteins, as well as smaller molecules such as antibiotics. Hence, they play an important role in many biological processes including diseases and their treatment. Hydrolase detection as well as the understanding of their function is therefore of great importance for diagnostics and therapy. Due to their various desirable features compared to antibodies, aptamers are being discussed as alternative agents for analytical and diagnostic use in various applications. The use of aptamers in therapy is also frequently investigated, as the binding of aptamers can have effects on the catalytic activity, protein-protein interactions, or proteolytic cascades. Aptamers are generated by an in vitro selection process. Potential aptamer candidates are selected from a pool of enriched nucleic acid sequences with affinity to the target, and their binding affinity and specificity is investigated. This is one of the most important steps in aptamer generation to obtain specific aptamers with high affinity for use in analytical and diagnostic applications. The binding properties or binding domains and their effects on enzyme functions form the basis for therapeutic applications. In this work, the binding properties of DNA aptamers against two different hydrolases were investigated. In view of their potential utility for analytical methods, aptamers against human urokinase (uPA) and New Delhi metallo-β-lactamase-1 (NDM-1) were evaluated for their binding affinity and specificity using different methods. Using the uPA aptamers, a protocol for measuring the binding kinetics of an aptamer-protein-interaction by surface plasmon resonance spectroscopy (SPR) was developed. Based on the increased expression of uPA in different types of cancer, uPA is discussed as a prognostic and diagnostic tumor marker. As uPA aptamers showed different binding sites on the protein, microtiter plate-based aptamer sandwich assay systems for the detection of uPA were developed. Because of the function of urokinase in cancer cell proliferation and metastasis, uPA is also discussed as a therapeutic target. In this regard, the different binding sites of aptamers showed different effects on uPA function. In vitro experiments demonstrated both inhibition of uPA binding to its receptor as well as the inhibition of uPA catalytic activity for different aptamers. Thus, in addition to their specificity and affinity for their targets, the utility of the aptamers for potential diagnostic and therapeutic applications was demonstrated. First, as an alternative inhibitor of human urokinase for therapeutic purposes, and second, as valuable recognition molecules for the detection of urokinase, as a prognostic and diagnostic marker for cancer, and for NDM-1 to detect resistance to carbapenem antibiotics.}, language = {en} } @phdthesis{Stuebler2023, author = {St{\"u}bler, Sabine}, title = {Mathematical model of the mucosal immune response to study inflammatory bowel diseases and their treatments}, doi = {10.25932/publishup-61230}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612301}, school = {Universit{\"a}t Potsdam}, pages = {xiv, 194}, year = {2023}, abstract = {Inflammatory bowel diseases (IBD), characterised by a chronic inflammation of the gut wall, develop as consequence of an overreacting immune response to commensal bacteria, caused by a combination of genetic and environmental conditions. Large inter-individual differences in the outcome of currently available therapies complicate the decision for the best option for an individual patient. Predicting the prospects of therapeutic success for an individual patient is currently only possible to a limited extent; for this, a better understanding of possible differences between responders and non-responders is needed. In this thesis, we have developed a mathematical model describing the most important processes of the gut mucosal immune system on the cellular level. The model is based on literature data, which were on the one hand used (qualitatively) to choose which cell types and processes to incorporate and to derive the model structure, and on the other hand (quantitatively) to derive the parameter values. Using ordinary differential equations, it describes the concentration-time course of neutrophils, macrophages, dendritic cells, T cells and bacteria, each subdivided into different cell types and activation states, in the lamina propria and mesenteric lymph nodes. We evaluate the model by means of simulations of the healthy immune response to salmonella infection and mucosal injury. A virtual population includes IBD patients, which we define through their initially asymptomatic, but after a trigger chronically inflamed gut wall. We demonstrate the model's usefulness in different analyses: (i) The comparison of virtual IBD patients with virtual healthy individuals shows that the disease is elicited by many small or fewer large changes, and allows to make hypotheses about dispositions relevant for development of the disease. (ii) We simulate the effects of different therapeutic targets and make predictions about the therapeutic outcome based on the pre-treatment state. (iii) From the analysis of differences between virtual responders and non-responders, we derive hypotheses about reasons for the inter-individual variability in treatment outcome. (iv) For the example of anti-TNF-alpha therapy, we analyse, which alternative therapies are most promising in case of therapeutic failure, and which therapies are most suited for combination therapies: For drugs also directly targeting the cytokine levels or inhibiting the recruitment of innate immune cells, we predict a low probability of success when used as alternative treatment, but a large gain when used in a combination treatment. For drugs with direct effects on T cells, via modulation of the sphingosine-1-phosphate receptor or inhibition of T cell proliferation, we predict a considerably larger probability of success when used as alternative treatment, but only a small additional gain when used in a combination therapy.}, language = {en} } @phdthesis{Petrich2023, author = {Petrich, Annett}, title = {Quantitative fluorescence microscopy methods to investigate molecular interactions and dynamics in living cells}, doi = {10.25932/publishup-61180}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-611800}, school = {Universit{\"a}t Potsdam}, pages = {244}, year = {2023}, abstract = {Biomolecules such as proteins and lipids have vital roles in numerous cellular functions, including biomolecule transport, protein functions, cellular homeostasis and biomembrane integrity. Traditional biochemistry methods do not provide precise information about cellular biomolecule distribution and behavior under native environmental conditions since they are not transferable to live cell samples. Consequently, this can lead to inaccuracies in quantifying biomolecule interactions due to potential complexities arising from the heterogeneity of native biomembranes. To overcome these limitations, minimal invasive microscopic techniques, such as fluorescence fluctuation spectroscopy (FFS) in combination with fluorescence proteins (FPs) and fluorescence lipid analogs, have been developed. FFS techniques and membrane property sensors enable the quantification of various parameters, including concentration, dynamics, oligomerization, and interaction of biomolecules in live cell samples. In this work, several FFS approaches and membrane property sensors were implemented and employed to examine biological processes of diverse context. Multi-color scanning fluorescence fluctuation spectroscopy (sFCS) was used the examine protein oligomerization, protein-protein interactions (PPIs) and protein dynamics at the cellular plasma membrane (PM). Additionally, two-color number and brightness (N\&B) analysis was extended with the cross-correlation analysis in order to quantify hetero-interactions of proteins in the PM with very slow motion, which would not accessible with sFCS due strong initial photobleaching. Furthermore, two semi-automatic analysis pipelines were designed: spectral F{\"o}rster resonance energy transfer (FRET) analysis to study changes in membrane charge at the inner leaflet of the PM, and spectral generalized polarization (GP) imaging and spectral phasor analysis to monitor changes in membrane fluidity and order. An important parameter for studying PPIs is molecular brightness, which directly determines oligomerization and can be extracted from FFS data. However, FPs often display complex photophysical transitions, including dark states. Therefore, it is crucial to characterize FPs for their dark-states to ensure reliable oligomerization measurements. In this study, N\&B and sFCS analysis were applied to determine photophysical properties of novel green FPs under different conditions (i.e., excitation power and pH) in living cells. The results showed that the new FPs, mGreenLantern (mGL) and Gamillus, exhibited the highest molecular brightness at the cost of lower photostability. The well-established monomeric enhanced green fluorescent protein (mEGFP) remained the best option to investigate PPIs at lower pH, while mGL was best suited for neutral pH, and Gamillus for high pH. These findings provide guidance for selecting an appropriate FP to quantify PPIs via FFS under different environmental conditions. Next, several biophysical fluorescence microscopy approaches (i.e., sFCS, GP imaging, membrane charge FRET) were employed to monitor changes in lipid-lipid-packing in biomembranes in different biological context. Lipid metabolism in cancer cells is known to support rapid proliferation and metastasis. Therefore, targeting lipid synthesis or membrane integrity holds immense promise as an anticancer strategy. However, the mechanism of action of the novel agent erufosine (EPC3) on membrane stability is not fully under stood. The present work revealed that EPC3 reduces lipid packing and composition as well as increased membrane fluidity and dynamic, hence, modifies lipid-lipid-interaction. These effects on membrane integrity were likely triggered by modulations in lipid metabolism and membrane organization. In the case of influenza A virus (IAV) infection, regulation of lipid metabolism is crucial for multiple steps in IAV replication and is related to the pathogenicity of IAV. Here, it is shown for the first time that IAV infection triggers a local enrichment of negatively charged lipids at the inner leaflet of the PM, which decreases membrane fluidity and dynamic, as well as increases lipid packing at the assembly site in living cells. This suggests that IAV alters lipid-lipid interactions and organization at the PM. Overall, this work highlights the potential of biophysical techniques as a screening platform for studying membrane properties in living cells at the single-cell level. Finally, this study addressed remaining questions about the early stage of IAV assembly. The recruitment of matrix protein 1 (M1) and its interaction with other viral surface proteins, hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2), has been a subject of debate due to conflicting results. In this study, different FFS approaches were performed in transfected cells to investigate interactions between IAV proteins themselves and host factors at the PM. FFS measurements revealed that M2 interacts strongly with M1, leading to the translocation of M1 to the PM. This interaction likely took place along the non-canonical pathway, as evidenced by the detection of an interaction between M2 and the host factor LC3-II, leading to the recruitment of LC3-II to the PM. Moreover, weaker interaction was observed between HA and membrane-bound M1, and no interaction between NA and M1. Interestingly, higher oligomeric states of M1 were only detectable in infected cells. These results indicate that M2 initiates virion assembly by recruiting M1 to the PM, which may serve as a platform for further interactions with viral proteins and host factors.}, language = {en} } @phdthesis{Pramanik2023, author = {Pramanik, Shreya}, title = {Protein reconstitution in giant vesicles}, doi = {10.25932/publishup-61278}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-612781}, school = {Universit{\"a}t Potsdam}, pages = {VIII, 132}, year = {2023}, abstract = {Das Leben auf der Erde ist vielf{\"a}ltig und reicht von einzelligen Organismen bis hin zu mehrzelligen Lebewesen wie dem Menschen. Obwohl es Theorien dar{\"u}ber gibt, wie sich diese Organismen entwickelt haben k{\"o}nnten, verstehen wir nur wenig dar{\"u}ber, wie "Leben" aus Molek{\"u}len entstanden ist. Die synthetische Bottom-up-Biologie zielt darauf ab, minimale Zellen zu schaffen, indem sie verschiedene Module wie Kompartimentierung, Wachstum, Teilung und zellul{\"a}re Kommunikation kombiniert. Alle lebenden Zellen haben eine Membran, die sie von dem sie umgebenden w{\"a}ssrigen Medium trennt und sie sch{\"u}tzt. Dar{\"u}ber hinaus haben alle eukaryotischen Zellen Organellen, die von intrazellul{\"a}ren Membranen umschlossen sind. Jede Zellmembran besteht haupts{\"a}chlich aus einer Lipiddoppelschicht mit Membranproteinen. Lipide sind amphiphile Molek{\"u}le, die molekulare Doppelschichten aus zwei Lipid-Monoschichten oder Bl{\"a}ttchen bilden. Die hydrophoben Ketten der Lipide sind einander zugewandt, w{\"a}hrend ihre hydrophilen Kopfgruppen die Grenzfl{\"a}chen zur w{\"a}ssrigen Umgebung bilden. Riesenvesikel sind Modellmembransysteme, die Kompartimente mit einer Gr{\"o}ße von mehreren Mikrometern bilden und von einer einzigen Lipiddoppelschicht umgeben sind. Die Gr{\"o}ße der Riesenvesikel ist mit der Gr{\"o}ße von Zellen vergleichbar und macht sie zu guten Membranmodellen, die mit einem Lichtmikroskop untersucht werden k{\"o}nnen. Allerdings fehlen den Riesenvesikelmembranen nach der ersten Pr{\"a}paration Membranproteine, die in weiteren Pr{\"a}parationsschritten in diese Membranen eingebaut werden m{\"u}ssen. Je nach Protein kann es entweder {\"u}ber Ankerlipide an eines der Membranbl{\"a}ttchen gebunden oder {\"u}ber seine Transmembrandom{\"a}nen in die Lipiddoppelschicht eingebaut werden. Diese Arbeit befasst sich mit der Herstellung von Riesenvesikeln und der Rekonstitution von Proteinen in diesen Vesikeln. Außerdem wird ein mikrofluidischer Chip entworfen, der in verschiedenen Experimenten verwendet werden kann. Die Ergebnisse dieser Arbeit werden anderen Forschern helfen, die Protokolle f{\"u}r die Herstellung von GUVs zu verstehen, Proteine in GUVs zu rekonstituieren und Experimente mit dem mikrofluidischen Chip durchzuf{\"u}hren. Auf diese Weise wird die vorliegende Arbeit f{\"u}r das langfristige Ziel von Nutzen sein, die verschiedenen Module der synthetischen Biologie zu kombinieren, um eine Minimalzelle zu schaffen.}, language = {en} } @phdthesis{Ogunkola2023, author = {Ogunkola, Moses}, title = {Role of the tRNA thiouridine modification protein (TUM1) as a sulfurtransferase in humans}, doi = {10.25932/publishup-61135}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-611357}, school = {Universit{\"a}t Potsdam}, pages = {XII, 82}, year = {2023}, abstract = {Sulfur is essential for the functionality of some important biomolecules in humans. Biomolecules like the Iron-sulfur clusters, tRNAs, Molybdenum cofactor, and some vitamins. The trafficking of sulfur involves proteins collectively called sulfurtransferase. Among these are TUM1, MOCS3, and NFS1. This research investigated the role of TUM1 for molybdenum cofactor biosynthesis and cytosolic tRNA thiolation in humans. The rhodanese-like protein MOCS3 and the L-cysteine desulfurase (NFS1) have been previously demonstrated to interact with TUM1. These interactions suggested a dual function of TUM1 in sulfur transfer for Moco biosynthesis and cytosolic tRNA thiolation. TUM1 deficiency has been implicated to be responsible for a rare inheritable disorder known as mercaptolactate cysteine disulfiduria (MCDU), which is associated with a mental disorder. This mental disorder is similar to the symptoms of sulfite oxidase deficiency which is characterised by neurological disorders. Therefore, the role of TUM1 as a sulfurtransferase in humans was investigated, in CRISPR/Cas9 generated TUM1 knockout HEK 293T cell lines. For the first time, TUM1 was implicated in Moco biosynthesis in humans by quantifying the intermediate product cPMP and Moco using HPLC. Comparing the TUM1 knockout cell lines to the wild-type, accumulation and reduction of cPMP and Moco were observed respectively. The effect of TUM1 knockout on the activity of a Moco-dependent enzyme, Sulfite oxidase, was also investigated. Sulfite oxidase is essential for the detoxification of sulfite to sulfate. Sulfite oxidase activity and protein abundance were reduced due to less availability of Moco. This shows that TUM1 is essential for efficient sulfur transfer for Moco biosynthesis. Reduction in cystathionin -lyase in TUM1 knockout cells was quantified, a possible coping mechanism of the cell against sulfite production through cysteine catabolism. Secondly, the involvement of TUM1 in tRNA thio-modification at the wobble Uridine-34 was reported by quantifying the amount of mcm5s2U and mcm5U via HPLC. The reduction and accumulation of mcm5s2U and mcm5U in TUM1 knockout cells were observed in the nucleoside analysis. Herein, exogenous treatment with NaHS, a hydrogen sulfide donor, rescued the Moco biosynthesis, cytosolic tRNA thiolation, and cell proliferation deficits in TUM1 knockout cells. Further, TUM1 was shown to impact mitochondria bioenergetics through the measurement of the oxygen consumption rate and extracellular acidification rate (ECAR) via the seahorse cell Mito stress analyzer. Reduction in total ATP production was also measured. This reveals how important TUM1 is for H2S biosynthesis in the mitochondria of HEK 293T. Finally, the inhibition of NFS1 in HEK 293T and purified NFS1 protein by 2-methylene 3-quinuclidinone was demonstrated via spectrophotometric and radioactivity quantification. Inhibition of NFS1 by MQ further affected the iron-sulfur cluster-dependent enzyme aconitase activity.}, language = {en} } @phdthesis{MendesFerreira2023, author = {Mendes Ferreira, Clara}, title = {Indirect, tri-trophic effects of fear on biodiversity}, doi = {10.25932/publishup-61102}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-611020}, school = {Universit{\"a}t Potsdam}, pages = {119}, year = {2023}, abstract = {Predator-forager interactions are a major factor in evolutionary adaptation of many species, as predators need to gain energy by consuming prey species, and foragers needs to avoid the worst fate of mortality while still consuming resources for energetic gains. In this evolutionary arms race, the foragers have constantly evolved anti-predator behaviours (e.g. foraging activity changes). To describe all these complex changes, researchers developed the framework of the landscape of fear, that is, the spatio-temporal variation of perceived predation risk. This concept simplifies all the involved ecological processes into one framework, by integrating animal biology and distribution with habitat characteristics. Researchers can then evaluate the perception of predation risk in prey species, what are the behavioural responses of the prey and, therefore, understand the cascading effects of landscapes of fear at the resource levels (tri-trophic effects). Although tri-trophic effects are well studied at the predator-prey interaction level, little is known on how the forager-resource interactions are part of the overall cascading effects of landscapes of fear, despite the changes of forager feeding behaviour - that occur with perceived predation risk - affecting directly the level of the resources. This thesis aimed to evaluate the cascading effects of the landscape of fear on biodiversity of resources, and how the feeding behaviour and movement of foragers shaped the final resource species composition (potential coexistence mechanisms). We studied the changes caused by landscapes of fear on wild and captive rodent communities and evaluated: the cascading effects of different landscapes of fear on a tri-trophic system (I), the effects of fear on a forager's movement patterns and dietary preferences (II) and cascading effects of different types of predation risk (terrestrial versus avian, III). In Chapter I, we applied a novel measure to evaluate the cascading effects of fear at the level of resources, by quantifying the diversity of resources left after the foragers gave-up on foraging (diversity at the giving-up density). We tested the measure at different spatial levels (local and regional) and observed that with decreased perceived predation risk, the density and biodiversity of resources also decreased. Foragers left a very dissimilar community of resources based on perceived risk and resources functional traits, and therefore acted as an equalising mechanism. In Chapter II, we wanted to understand further the decision-making processes of rodents in different landscapes of fear, namely, in which resource species rodents decided to forage on (based on three functional traits: size, nutrients and shape) and how they moved depending on perceived predation risk. In safe landscapes, individuals increased their feeding activity and movements and despite the increased costs, they visited more often patches that were further away from their central-place. Despite a preference for the bigger resources regardless of risk, when perceived predation risk was low, individuals changed their preference to fat-rich resources. In Chapter III, we evaluated the cascading effects of two different types of predation risk in rodents: terrestrial (raccoon) versus avian predation risk. Raccoon presence or absence did not alter the rodents feeding behaviour in different landscapes of fear. Rodent's showed risk avoidance behaviours towards avian predators (spatial risk avoidance), but not towards raccoons (lack of temporal risk avoidance). By analysing the effects of fear in tri-trophic systems, we were able to deepen the knowledge of how non-consumptive effects of predators affect the behaviour of foragers, and quantitatively measure the cascading effects at the level of resources with a novel measure. Foragers are at the core of the ecological processes and responses to the landscape of fear, acting as variable coexistence agents for resource species depending on perceived predation risk. This newly found measures and knowledge can be applied to more trophic chains, and inform researchers on biodiversity patterns originating from landscapes of fear.}, language = {en} } @phdthesis{Riedel2023, author = {Riedel, Soraya Lisanne}, title = {Development of electrochemical antibody-based and enzymatic assays for mycotoxin analysis in food}, doi = {10.25932/publishup-60747}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-607477}, school = {Universit{\"a}t Potsdam}, pages = {XV, 95}, year = {2023}, abstract = {Electrochemical methods are promising to meet the demand for easy-to-use devices monitoring key parameters in the food industry. Many companies run own lab procedures for mycotoxin analysis, but it is a major goal to simplify the analysis. The enzyme-linked immunosorbent assay using horseradish peroxidase as enzymatic label, together with 3,3',5,5' tetramethylbenzidine (TMB)/H2O2 as substrates allows sensitive mycotoxin detection with optical detection methods. For the miniaturization of the detection step, an electrochemical system for mycotoxin analysis was developed. To this end, the electrochemical detection of TMB was studied by cyclic voltammetry on different screen-printed electrodes (carbon and gold) and at different pH values (pH 1 and pH 4). A stable electrode reaction, which is the basis for the further construction of the electrochemical detection system, could be achieved at pH 1 on gold electrodes. An amperometric detection method for oxidized TMB, using a custom-made flow cell for screen-printed electrodes, was established and applied for a competitive magnetic bead-based immunoassay for the mycotoxin ochratoxin A. A limit of detection of 150 pM (60 ng/L) could be obtained and the results were verified with optical detection. The applicability of the magnetic bead-based immunoassay was tested in spiked beer using a handheld potentiostat connected via Bluetooth to a smartphone for amperometric detection allowing to quantify ochratoxin A down to 1.2 nM (0.5 µg/L). Based on the developed electrochemical detection system for TMB, the applicability of the approach was demonstrated with a magnetic bead-based immunoassay for the ergot alkaloid, ergometrine. Under optimized assay conditions a limit of detection of 3 nM (1 µg/L) was achieved and in spiked rye flour samples ergometrine levels in a range from 25 to 250 µg/kg could be quantified. All results were verified with optical detection. The developed electrochemical detection method for TMB gives great promise for the detection of TMB in many other HRP-based assays. A new sensing approach, based on an enzymatic electrochemical detection system for the mycotoxin fumonisin B1 was established using an Aspergillus niger fumonisin amine oxidase (AnFAO). AnFAO was produced recombinantly in E. coli as maltose-binding protein fusion protein and catalyzes the oxidative deamination of fumonisins, producing hydrogen peroxide. It was found that AnFAO has a high storage and temperature stability. The enzyme was coupled covalently to magnetic particles, and the enzymatically produced H2O2 in the reaction with fumonisin B1 was detected amperometrically in a flow injection system using Prussian blue/carbon electrodes and the custom-made wall-jet flow cell. Fumonisin B1 could be quantified down to 1.5 µM (≈ 1 mg/L). The developed system represents a new approach to detect mycotoxins using enzymes and electrochemical methods.}, language = {en} } @phdthesis{Soltani2023, author = {Soltani, Ouad}, title = {BLF1-Mode of Action in Barley Leaf Size Control}, doi = {10.25932/publishup-60705}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-607054}, school = {Universit{\"a}t Potsdam}, pages = {110}, year = {2023}, abstract = {Establishment of final leaf size in plants represents a complex mechanism that relies on the precise regulation of two interconnected cellular processes, cell division and cell expansion. In previous work, the barley protein BROAD LEAF1 (BLF1) was identified as a novel negative regulator of cell proliferation, that mainly limits leaf growth in the width direction. Here I identified a novel RING/U-box protein that interacts with BLF1 through a yeast two hybrid screen. Using BiFC, Co-IP and FRET I confirmed the interaction of the two proteins in planta. Enrichment of the BLF1-mEGFP fusion protein and the increase of the FRET signal upon MG132 treatment of tobacco plants, together with an in vivo ubiquitylation assay in bacteria, confirmed that the RING/U-box E3 interacts with BLF1 to mediate its ubiquitylation and degradation by the 26S proteasome system. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome by bortezomib treatment on BLF1-vYFP transgenic barley plants also resulted in an enrichment of the BLF1 protein. I thus demonstrated that RING/U-box E3 is colocalized with BLF1 in nuclei and negatively regulates BLF1 protein levels. Analysis of ring-e3_1 knock-out mutants suggested the involvement of the RING/U-box E3 gene in leaf growth control, although the effect was mainly on leaf length. Together, my results suggest that proteasomal degradation, possibly mediated by RING/U-box E3, contributes to fine-tuning BLF1 protein-level in barley.}, language = {en} } @phdthesis{Carrasco2023, author = {Carrasco, Tomas}, title = {Genome structure analysis and patterns of transposable elements evolution in the slow-evolving Testudines clade}, doi = {10.25932/publishup-60657}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-606577}, school = {Universit{\"a}t Potsdam}, pages = {144}, year = {2023}, abstract = {Transposable elements (TEs) are loci that can replicate and multiply within the genome of their host. Within the host, TEs through transposition are responsible for variation on genomic architecture and gene regulation across all vertebrates. Genome assemblies have increased in numbers in recent years. However, to explore in deep the variations within different genomes, such as SNPs (single nucleotide polymorphism), INDELs (Insertion-deletion), satellites and transposable elements, we need high-quality genomes. Studies of molecular markers in the past 10 years have limitations to correlate with biological differences because molecular markers rely on the accuracy of the genomic resources. This has generated that a substantial part of the studies of TE in recent years have been on high quality genomic resources such as Drosophila, zebrafinch and maize. As testudine have a slow mutation rate lower only to crocodilians, with more than 300 species, adapted to different environments all across the globe, the testudine clade can help us to study variation. Here we propose Testudines as a clade to study variation and the abundance of TE on different species that diverged a long time ago. We investigated the genomic diversity of sea turtles, identifying key genomic regions associated to gene family duplication, specific expansion of particular TE families for Dermochelyidae and that are important for phenotypic differentiation, the impact of environmental changes on their populations, and the dynamics of TEs within different lineages. In chapter 1, we identify that despite high levels of genome synteny within sea turtles, we identified that regions of reduced collinearity and microchromosomes showed higher concentrations of multicopy gene families, as well as genetic distances between species, indicating their potential importance as sources of variation underlying phenotypic differentiation. We found that differences in the ecological niches occupied by leatherback and green turtles have led to contrasting evolutionary paths for their olfactory receptor genes. We identified in leatherback turtles a long-term low population size. Nonetheless, we identify no correlation between the regions of reduced collinearity with abundance of TEs or an accumulation of a particular TE group. In chapter 2, we identified that sea turtle genomes contain a significant proportion of TEs, with differences in TE abundance between species, and the discovery of a recent expansion of Penelope-like elements (PLEs) in the highly conserved sea turtle genome provides new insights into the dynamics of TEs within Testudines. In chapter 3, we compared the proportion of TE across the Testudine clade, and we identified that the proportion of transposable elements within the clade is stable, regardless of the quality of the assemblies. However, we identified that the proportion of TEs orders has correlation with genome quality depending of their expanded abundancy. For retrotransposon, a highly abundant element for this clade, we identify no correlation. However, for DNA elements a rarer element on this clade, correlate with the quality of the assemblies. Here we confirm that high-quality genomes are fundamental for the study of transposable element evolution and the conservation within the clade. The detection and abundance of specific orders of TEs are influenced by the quality of the genomes. We identified that a reduction in the population size on D. coriacea had left signals of long-term low population sizes on their genomes. On the same note we identified an expansion of TE on D. coriacea, not present in any other member of the available genomes of Testudines, strongly suggesting that it is a response of deregulation of TE on their genomes as consequences of the low population sizes. Here we have identified important genomic regions and gene families for phenotypic differentiation and highlighted the impact of environmental changes on the populations of sea turtles. We stated that accurate classification and analysis of TE families are important and require high-quality genome assemblies. Using TE analysis we manage to identify differences in highly syntenic species. These findings have significant implications for conservation and provide a foundation for further research into genome evolution and gene function in turtles and other vertebrates. Overall, this study contributes to our understanding of evolutionary change and adaptation mechanisms.}, language = {en} } @phdthesis{Malchow2023, author = {Malchow, Anne-Kathleen}, title = {Developing an integrated platform for predicting niche and range dynamics}, doi = {10.25932/publishup-60273}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-602737}, school = {Universit{\"a}t Potsdam}, pages = {xiv, 169}, year = {2023}, abstract = {Species are adapted to the environment they live in. Today, most environments are subjected to rapid global changes induced by human activity, most prominently land cover and climate changes. Such transformations can cause adjustments or disruptions in various eco-evolutionary processes. The repercussions of this can appear at the population level as shifted ranges and altered abundance patterns. This is where global change effects on species are usually detected first. To understand how eco-evolutionary processes act and interact to generate patterns of range and abundance and how these processes themselves are influenced by environmental conditions, spatially-explicit models provide effective tools. They estimate a species' niche as the set of environmental conditions in which it can persist. However, the currently most commonly used models rely on static correlative associations that are established between a set of spatial predictors and observed species distributions. For this, they assume stationary conditions and are therefore unsuitable in contexts of global change. Better equipped are process-based models that explicitly implement algorithmic representations of eco-evolutionary mechanisms and evaluate their joint dynamics. These models have long been regarded as difficult to parameterise, but an increased data availability and improved methods for data integration lessen this challenge. Hence, the goal of this thesis is to further develop process-based models, integrate them into a complete modelling workflow, and provide the tools and guidance for their successful application. With my thesis, I presented an integrated platform for spatially-explicit eco-evolutionary modelling and provided a workflow for their inverse calibration to observational data. In the first chapter, I introduced RangeShiftR, a software tool that implements an individual-based modelling platform for the statistical programming language R. Its open-source licensing, extensive help pages and available tutorials make it accessible to a wide audience. In the second chapter, I demonstrated a comprehensive workflow for the specification, calibration and validation of RangeShiftR by the example of the red kite in Switzerland. The integration of heterogeneous data sources, such as literature and monitoring data, allowed to successfully calibrate the model. It was then used to make validated, spatio-temporal predictions of future red kite abundance. The presented workflow can be adopted to any study species if data is available. In the third chapter, I extended RangeShiftR to directly link demographic processes to climatic predictors. This allowed me to explore the climate-change responses of eight Swiss breeding birds in more detail. Specifically, the model could identify the most influential climatic predictors, delineate areas of projected demographic suitability, and attribute current population trends to contemporary climate change. My work shows that the application of complex, process-based models in conservation-relevant contexts is feasible, utilising available tools and data. Such models can be successfully calibrated and outperform other currently used modelling approaches in terms of predictive accuracy. Their projections can be used to predict future abundances or to assess alternative conservation scenarios. They further improve our mechanistic understanding of niche and range dynamics under climate change. However, only fully mechanistic models, that include all relevant processes, allow to precisely disentangle the effects of single processes on observed abundances. In this respect, the RangeShiftR model still has potential for further extensions that implement missing influential processes, such as species interactions. Dynamic, process-based models are needed to adequately model a dynamic reality. My work contributes towards the advancement, integration and dissemination of such models. This will facilitate numeric, model-based approaches for species assessments, generate ecological insights and strengthen the reliability of predictions on large spatial scales under changing conditions.}, language = {en} } @phdthesis{Parry2023, author = {Parry, Victor}, title = {From individual to community level: Assessing swimming movement, dispersal and fitness of zooplankton}, doi = {10.25932/publishup-59769}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-597697}, school = {Universit{\"a}t Potsdam}, pages = {ix, 118}, year = {2023}, abstract = {Movement is a mechanism that shapes biodiversity patterns across spatialtemporal scales. Thereby, the movement process affects species interactions, population dynamics and community composition. In this thesis, I disentangled the effects of movement on the biodiversity of zooplankton ranging from the individual to the community level. On the individual movement level, I used video-based analysis to explore the implication of movement behavior on preypredator interactions. My results showed that swimming behavior was of great importance as it determined their survival in the face of predation. The findings also additionally highlighted the relevance of the defense status/morphology of prey, as it not only affected the prey-predator relationship by the defense itself but also by plastic movement behavior. On the community movement level, I used a field mesocosm experiment to explore the role of dispersal (time i.e., from the egg bank into the water body and space i.e., between water bodies) in shaping zooplankton metacommunities. My results revealed that priority effects and taxon-specific dispersal limitation influenced community composition. Additionally, different modes of dispersal also generated distinct community structures. The egg bank and biotic vectors (i.e. mobile links) played significant roles in the colonization of newly available habitat patches. One crucial aspect that influences zooplankton species after arrival in new habitats is the local environmental conditions. By using common garden experiments, I assessed the performance of zooplankton communities in their home vs away environments in a group of ponds embedded within an agricultural landscape. I identified environmental filtering as a driving factor as zooplankton communities from individual ponds developed differently in their home and away environments. On the individual species level, there was no consistent indication of local adaptation. For some species, I found a higher abundance/fitness in their home environment, but for others, the opposite was the case, and some cases were indifferent. Overall, the thesis highlights the links between movement and biodiversity patterns, ranging from the individual active movement to the community level.}, language = {en} } @phdthesis{Courtin2023, author = {Courtin, J{\´e}r{\´e}my}, title = {Biodiversity changes in Siberia between quaternary glacial and interglacial stages}, doi = {10.25932/publishup-59584}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-595847}, school = {Universit{\"a}t Potsdam}, pages = {vi, 199}, year = {2023}, abstract = {Der vom Menschen verursachte Klimawandel wirkt sich auf die biologische Vielfalt der Erde und damit auf die {\"O}kosysteme und ihre Leistungen aus. Die {\"O}kosysteme in den hohen Breitengraden sind aufgrund der verst{\"a}rkten Erw{\"a}rmung an den Polen noch st{\"a}rker betroffen als der Rest der n{\"o}rdlichen Hemisph{\"a}re. Dennoch ist es schwierig, die Dynamik von {\"O}kosystemen in den hohen Breitengraden vorherzusagen, da die Wechselwirkungen zwischen abiotischen und biotischen Komponenten sehr komplex sind. Da die Vergangenheit der Schl{\"u}ssel zur Zukunft ist, ist die Interpretation vergangener {\"o}kologischer Ver{\"a}nderungen m{\"o}glich, um laufende Prozesse besser zu verstehen. Im Quart{\"a}r durchlief das Pleistoz{\"a}n mehrere glaziale und interglaziale Phasen, welche die {\"O}kosysteme der Vergangenheit beeinflussten. W{\"a}hrend des letzten Glazials bedeckte die pleistoz{\"a}ne Steppentundra den gr{\"o}ßten Teil der unvergletscherten n{\"o}rdlichen Hemisph{\"a}re und verschwand parallel zum Aussterben der Megafauna am {\"U}bergang zum Holoz{\"a}n (vor etwa 11 700 Jahren). Der Ursprung des R{\"u}ckgangs der Steppentundra ist nicht gut erforscht, und die Kenntnis {\"u}ber die Mechanismen, die zu den Ver{\"a}nderungen in den vergangenen Lebensgemeinschaften und {\"O}kosystemen gef{\"u}hrt haben, ist von hoher Priorit{\"a}t, da sie wahrscheinlich mit denen vergleichbar sind, die sich auf moderne {\"O}kosysteme auswirken. Durch die Entnahme von See- oder Permafrostkernsedimenten kann die vergangene Artenvielfalt an den {\"U}berg{\"a}ngen zwischen Eis- und Zwischeneiszeiten untersucht werden. Sibirien und Beringia waren der Ursprung der Ausbreitung der Steppentundra, weshalb die Untersuchung dieses Gebiets hohe Priorit{\"a}t hat. Bis vor kurzem waren Makrofossilien und Pollen die g{\"a}ngigsten Methoden. Sie dienen der Rekonstruktion vergangener Ver{\"a}nderungen in der Zusammensetzung der Bev{\"o}lkerung, haben aber ihre Grenzen und Schw{\"a}chen. Seit Ende des 20. Jahrhunderts kann auch sediment{\"a}re alte DNA (sedaDNA) untersucht werden. Mein Hauptziel war es, durch den Einsatz von sedaDNA-Ans{\"a}tzen wissenschaftliche Beweise f{\"u}r Ver{\"a}nderungen in der Zusammensetzung und Vielfalt der {\"O}kosysteme der n{\"o}rdlichen Hemisph{\"a}re am {\"U}bergang zwischen den quart{\"a}ren Eiszeiten und Zwischeneiszeiten zu liefern. In dieser Arbeit liefere ich Momentaufnahmen ganzer alter {\"O}kosysteme und beschreibe die Ver{\"a}nderungen in der Zusammensetzung zwischen Quart{\"a}rglazialen und Interglazialen und best{\"a}tige die Vegetationszusammensetzung sowie die r{\"a}umlichen und zeitlichen Grenzen der pleistoz{\"a}nen Steppentundra. Ich stelle einen allgemeinen Verlust der Pflanzenvielfalt fest, wobei das Aussterben der Pflanzen parallel zum Aussterben der Megafauna verlief. Ich zeige auf, wie der Verlust der biotischen Widerstandsf{\"a}higkeit zum Zusammenbruch eines zuvor gut etablierten Systems f{\"u}hrte, und diskutiere meine Ergebnisse im Hinblick auf den laufenden Klimawandel. Mit weiteren Arbeiten zur Eingrenzung von Verzerrungen und Grenzen kann sedaDNA parallel zu den etablierteren Makrofossilien- und Pollenans{\"a}tzen verwendet werden oder diese sogar ersetzen, da meine Ergebnisse die Robustheit und das Potenzial von sedaDNA zur Beantwortung neuer pal{\"a}o{\"o}kologischer Fragen wie Ver{\"a}nderungen der Pflanzenvielfalt und -verluste belegen und Momentaufnahmen ganzer alter Biota liefern.}, language = {en} } @phdthesis{Derežanin2023, author = {Derežanin, Lorena}, title = {Contribution of structural variation to adaptive evolution of mammalian genomes}, doi = {10.25932/publishup-59144}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-591443}, school = {Universit{\"a}t Potsdam}, pages = {188}, year = {2023}, abstract = {Following the extinction of dinosaurs, the great adaptive radiation of mammals occurred, giving rise to an astonishing ecological and phenotypic diversity of mammalian species. Even closely related species often inhabit vastly different habitats, where they encounter diverse environmental challenges and are exposed to different evolutionary pressures. As a response, mammals evolved various adaptive phenotypes over time, such as morphological, physiological and behavioural ones. Mammalian genomes vary in their content and structure and this variation represents the molecular mechanism for the long-term evolution of phenotypic variation. However, understanding this molecular basis of adaptive phenotypic variation is usually not straightforward. The recent development of sequencing technologies and bioinformatics tools has enabled a better insight into mammalian genomes. Through these advances, it was acknowledged that mammalian genomes differ more, both within and between species, as a consequence of structural variation compared to single-nucleotide differences. Structural variant types investigated in this thesis - such as deletion, duplication, inversion and insertion, represent a change in the structure of the genome, impacting the size, copy number, orientation and content of DNA sequences. Unlike short variants, structural variants can span multiple genes. They can alter gene dosage, and cause notable gene expression differences and subsequently phenotypic differences. Thus, they can lead to a more dramatic effect on the fitness (reproductive success) of individuals, local adaptation of populations and speciation. In this thesis, I investigated and evaluated the potential functional effect of structural variations on the genomes of mustelid species. To detect the genomic regions associated with phenotypic variation I assembled the first reference genome of the tayra (Eira barbara) relying on linked-read sequencing technology to achieve a high level of genome completeness important for reliable structural variant discovery. I then set up a bioinformatics pipeline to conduct a comparative genomic analysis and explore variation between mustelid species living in different environments. I found numerous genes associated with species-specific phenotypes related to diet, body condition and reproduction among others, to be impacted by structural variants. Furthermore, I investigated the effects of artificial selection on structural variants in mice selected for high fertility, increased body mass and high endurance. Through selective breeding of each mouse line, the desired phenotypes have spread within these populations, while maintaining structural variants specific to each line. In comparison to the control line, the litter size has doubled in the fertility lines, individuals in the high body mass lines have become considerably larger, and mice selected for treadmill performance covered substantially more distance. Structural variants were found in higher numbers in these trait-selected lines than in the control line when compared to the mouse reference genome. Moreover, we have found twice as many structural variants spanning protein-coding genes (specific to each line) in trait-selected lines. Several of these variants affect genes associated with selected phenotypic traits. These results imply that structural variation does indeed contribute to the evolution of the selected phenotypes and is heritable. Finally, I suggest a set of critical metrics of genomic data that should be considered for a stringent structural variation analysis as comparative genomic studies strongly rely on the contiguity and completeness of genome assemblies. Because most of the available data used to represent reference genomes of mammalian species is generated using short-read sequencing technologies, we may have incomplete knowledge of genomic features. Therefore, a cautious structural variation analysis is required to minimize the effect of technical constraints. The impact of structural variants on the adaptive evolution of mammalian genomes is slowly gaining more focus but it is still incorporated in only a small number of population studies. In my thesis, I advocate the inclusion of structural variants in studies of genomic diversity for a more comprehensive insight into genomic variation within and between species, and its effect on adaptive evolution.}, language = {en} } @phdthesis{Goethel2023, author = {G{\"o}thel, Markus}, title = {Entwicklung eines Verfahrens zur Generierung von spezifischen monoklonalen Antik{\"o}rpern gegen Mikroorganismen basierend auf in silico Epitopanalysen}, doi = {10.25932/publishup-58801}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-588017}, school = {Universit{\"a}t Potsdam}, pages = {XVI, 113}, year = {2023}, abstract = {Monoklonale Antik{\"o}rper (mAK) sind eines der wichtigsten Biomolek{\"u}le f{\"u}r die Umweltanalytik und die medizinische Diagnostik. F{\"u}r die Detektion von Mikroorganismen bilden sie die Grundlage f{\"u}r ein schnelles und pr{\"a}zises Testverfahren. Bis heute gibt es, aufgrund des hohen zeitlichen und materiellen Aufwandes und der unspezifischen Immunisierungsstrategien, nur wenige mAK, die spezifisch Mikroorganismen erkennen. Zu diesem Zweck sollte ein anwendbares Verfahren f{\"u}r die Generierung von mAK gegen Mikroorganismen entwickelt werden, welches anhand von Escherichia coli O157:H7 und Legionella pneumophila validiert wurde. In dieser Dissertation konnten neue Oberfl{\"a}chenstrukturen auf den Mikroorganismen mittels vergleichender Genomanalysen und in silico Epitopanalysen identifiziert werden. Diese wurden in das Virush{\"u}llprotein VP1 integriert und f{\"u}r eine gezielte Immunisierungsstrategie verwendet. F{\"u}r die Bestimmung antigenspezifischer antik{\"o}rperproduzierender Hybridome wurde ein Immunf{\"a}rbeprotokoll entwickelt und etabliert, um die Hybridome im Durchflusszytometer zu sortieren. In der vorliegenden Studie konnten f{\"u}r E. coli O157:H7 insgesamt 53 potenzielle Proteinkandidaten und f{\"u}r L. pneumophila 38 Proteine mithilfe der bioinformatischen Analyse identifiziert werden. F{\"u}nf verschiedene potenzielle Epitope wurden f{\"u}r E. coli O157:H7 und drei verschiedenen f{\"u}r L. pneumophila ausgew{\"a}hlt und f{\"u}r die Immunisierung mit chim{\"a}ren VP1 verwendet. Alle Immunseren zeigten eine antigenspezifische Immunantwort. Aus den nachfolgend generierten Hybridomzellen konnten mehrere Antik{\"o}rperkandidaten gewonnen werden, welche in Charakterisierungsstudien eine starke Bindung zu E. coli O157:H7 bzw. L. pneumophila vorwiesen. Kreuzreaktivit{\"a}ten zu anderen relevanten Mikroorganismen konnten keine bzw. nur in geringem Maße festgestellt werden. Folglich konnte der hier beschriebene interdisziplin{\"a}re Ansatz zur Generierung spezifischer mAK gegen Mikroorganismen nachweislich spezifische mAK hervorbringen und ist als hocheffizienter Arbeitsablauf f{\"u}r die Herstellung von Antik{\"o}rpern gegen Mikroorganismen einsetzbar.}, language = {de} } @phdthesis{Gerling2022, author = {Gerling, Marten Tobias}, title = {A microfluidic system for high-precision image-based live cell sorting using dielectrophoretic forces}, doi = {10.25932/publishup-58742}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-587421}, school = {Universit{\"a}t Potsdam}, pages = {vii, 87, VI}, year = {2022}, abstract = {An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods. Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality. In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly. With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95\% and about 85\% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system. The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.}, language = {en} } @phdthesis{Leins2023, author = {Leins, Johannes A.}, title = {Combining model detail with large scales}, doi = {10.25932/publishup-58283}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-582837}, school = {Universit{\"a}t Potsdam}, pages = {xv, 168}, year = {2023}, abstract = {The global climate crisis is significantly contributing to changing ecosystems, loss of biodiversity and is putting numerous species on the verge of extinction. In principle, many species are able to adapt to changing conditions or shift their habitats to more suitable regions. However, change is progressing faster than some species can adjust, or potential adaptation is blocked and disrupted by direct and indirect human action. Unsustainable anthropogenic land use in particular is one of the driving factors, besides global heating, for these ecologically critical developments. Precisely because land use is anthropogenic, it is also a factor that could be quickly and immediately corrected by human action. In this thesis, I therefore assess the impact of three climate change scenarios of increasing intensity in combination with differently scheduled mowing regimes on the long-term development and dispersal success of insects in Northwest German grasslands. The large marsh grasshopper (LMG, Stethophyma grossum, Linn{\´e} 1758) is used as a species of reference for the analyses. It inhabits wet meadows and marshes and has a limited, yet fairly good ability to disperse. Mowing and climate conditions affect the development and mortality of the LMG differently depending on its life stage. The specifically developed simulation model HiLEG (High-resolution Large Environmental Gradient) serves as a tool for investigating and projecting viability and dispersal success under different climate conditions and land use scenarios. It is a spatially explicit, stage- and cohort-based model that can be individually configured to represent the life cycle and characteristics of terrestrial insect species, as well as high-resolution environmental data and the occurrence of external disturbances. HiLEG is a freely available and adjustable software that can be used to support conservation planning in cultivated grasslands. In the three case studies of this thesis, I explore various aspects related to the structure of simulation models per se, their importance in conservation planning in general, and insights regarding the LMG in particular. It became apparent that the detailed resolution of model processes and components is crucial to project the long-term effect of spatially and temporally confined events. Taking into account conservation measures at the regional level has further proven relevant, especially in light of the climate crisis. I found that the LMG is benefiting from global warming in principle, but continues to be constrained by harmful mowing regimes. Land use measures could, however, be adapted in such a way that they allow the expansion and establishment of the LMG without overly affecting agricultural yields. Overall, simulation models like HiLEG can make an important contribution and add value to conservation planning and policy-making. Properly used, simulation results shed light on aspects that might be overlooked by subjective judgment and the experience of individual stakeholders. Even though it is in the nature of models that they are subject to limitations and only represent fragments of reality, this should not keep stakeholders from using them, as long as these limitations are clearly communicated. Similar to HiLEG, models could further be designed in such a way that not only the parameterization can be adjusted as required, but also the implementation itself can be improved and changed as desired. This openness and flexibility should become more widespread in the development of simulation models.}, language = {en} } @phdthesis{KoikkarahAji2023, author = {Koikkarah Aji, Amit}, title = {Quantitative sub cellular characterization of Hantavirus structural proteins}, doi = {10.25932/publishup-58661}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-586612}, school = {Universit{\"a}t Potsdam}, pages = {101}, year = {2023}, abstract = {Hantaviruses (HVs) are a group of zoonotic viruses that infect human beings primarily through aerosol transmission of rodent excreta and urine samplings. HVs are classified geographically into: Old World HVs (OWHVs) that are found in Europe and Asia, and New World HVs (NWHVs) that are observed in the Americas. These different strains can cause severe hantavirus diseases with pronounced renal syndrome or severe cardiopulmonary system distress. HVs can be extremely lethal, with NWHV infections reaching up to 40 \% mortality rate. HVs are known to generate epidemic outbreaks in many parts of the world including Germany, which has seen periodic HV infections over the past decade. HV has a trisegmented genome. The small segment (S) encodes the nucleocapsid protein (NP), the middle segment (M) encodes the glycoproteins (GPs) Gn and Gc which forms up to tetramers and primarily monomers \\& dimers upon independent expression respectively and large segment (L) encodes RNA dependent RNA polymerase (RdRp). Interactions between these viral proteins are crucial in providing mechanistic insights into HV virion development. Despite best efforts, there continues to be lack of quantification of these associations in living cells. This is required in developing the mechanistic models for HV viral assembly. This dissertation focuses on three key questions pertaining to the initial steps of virion formation that primarily involves the GPs and NP. The research investigations in this work were completed using Fluorescence Correlation Spectroscopy (FCS) approaches. FCS is frequently used in assessing the biophysical features of bio-molecules including protein concentration and diffusion dynamics and circumvents the requirement of protein overexpression. FCS was primarily applied in this thesis to evaluate protein multimerization, at single cell resolution. The first question addressed which GP spike formation model proposed by Hepojoki et al.(2010) appropriately describes the evidence in living cells. A novel in cellulo assay was developed to evaluate the amount of fluorescently labelled and unlabeled GPs upon co-expression. The results clearly showed that Gn and Gc initially formed a heterodimeric Gn:Gc subunit. This sub-unit then multimerizes with congruent Gn:Gc subunits to generate the final GP spike. Based on these interactions, models describing the formation of GP complex (with multiple GP spike subunits) were additionally developed. HV GP assembly primarily takes place in the Golgi apparatus (GA) of infected cells. Interestingly, NWHV GPs are hypothesized to assemble at the plasma membrane (PM). This led to the second research question in this thesis, in which a systematic comparison between OWHV and NWHV GPs was conducted to validate this hypothesis. Surprisingly, GP localization at the PM was congruently observed with OWHV and NWHV GPs. Similar results were also discerned with OWHV and NWHV GP localization in the absence of cytoskeletal factors that regulate HV trafficking in cells. The final question focused on quantifying the NP-GP interactions and understanding their influence of NP and GP multimerization. Gc mutlimers were detected in the presence of NP and complimented by the presence of localized regions of high NP-Gc interactions in the perinuclear region of living cells. Gc-CT domain was shown to influence NP-Gc associations. Gn, on the other hand, formed up to tetrameric complexes, independent from the presence of NP. The results in this dissertation sheds light on the initial steps of HV virion formation by quantifying homo and heterotypic interactions involving NP and GPs, which otherwise are very difficult to perform. Finally, the in cellulo methodologies implemented in this work can be potentially extended to understand other key interactions involved in HV virus assembly.}, language = {en} } @phdthesis{Pellegrino2022, author = {Pellegrino, Antonio}, title = {miRNA profiling for diagnosis of chronic pain in polyneuropathy}, doi = {10.25932/publishup-58385}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-583858}, school = {Universit{\"a}t Potsdam}, pages = {viii, 97, xi}, year = {2022}, abstract = {This dissertation aimed to determine differential expressed miRNAs in the context of chronic pain in polyneuropathy. For this purpose, patients with chronic painful polyneuropathy were compared with age matched healthy patients. Taken together, all miRNA pre library preparation quality controls were successful and none of the samples was identified as an outlier or excluded for library preparation. Pre sequencing quality control showed that library preparation worked for all samples as well as that all samples were free of adapter dimers after BluePippin size selection and reached the minimum molarity for further processing. Thus, all samples were subjected to sequencing. The sequencing control parameters were in their optimal range and resulted in valid sequencing results with strong sample to sample correlation for all samples. The resulting FASTQ file of each miRNA library was analyzed and used to perform a differential expression analysis. The differentially expressed and filtered miRNAs were subjected to miRDB to perform a target prediction. Three of those four miRNAs were downregulated: hsa-miR-3135b, hsa-miR-584-5p and hsa-miR-12136, while one was upregulated: hsa-miR-550a-3p. miRNA target prediction showed that chronic pain in polyneuropathy might be the result of a combination of miRNA mediated high blood flow/pressure and neural activity dysregulations/disbalances. Thus, leading to the promising conclusion that these four miRNAs could serve as potential biomarkers for the diagnosis of chronic pain in polyneuropathy. Since TRPV1 seems to be one of the major contributors of nociception and is associated with neuropathic pain, the influence of PKA phosphorylated ARMS on the sensitivity of TRPV1 as well as the part of AKAP79 during PKA phosphorylation of ARMS was characterized. Therefore, possible PKA-sites in the sequence of ARMS were identified. This revealed five canonical PKA-sites: S882, T903, S1251/52, S1439/40 and S1526/27. The single PKA-site mutants of ARMS revealed that PKA-mediated ARMS phosphorylation seems not to influence the interaction rate of TRPV1/ARMS. While phosphorylation of ARMST903 does not increase the interaction rate with TRPV1, ARMSS1526/27 is probably not phosphorylated and leads to an increased interaction rate. The calcium flux measurements indicated that the higher the interaction rate of TRPV1/ARMS, the lower the EC50 for capsaicin of TRPV1, independent of the PKA phosphorylation status of ARMS. In addition, the western blot analysis confirmed the previously observed TRPV1/ARMS interaction. More importantly, AKAP79 seems to be involved in the TRPV1/ARMS/PKA signaling complex. To overcome the problem of ARMS-mediated TRPV1 sensitization by interaction, ARMS was silenced by shRNA. ARMS silencing resulted in a restored TRPV1 desensitization without affecting the TRPV1 expression and therefore could be used as new topical therapeutic analgesic alternative to stop ARMS mediated TRPV1 sensitization.}, language = {en} } @phdthesis{Schlossarek2023, author = {Schlossarek, Dennis}, title = {Identification of dynamic protein-metabolite complexes in saccharomyces cerevisiae using co-fractionation mass spectrometry}, doi = {10.25932/publishup-58282}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-582826}, school = {Universit{\"a}t Potsdam}, pages = {123}, year = {2023}, abstract = {Cells are built from a variety of macromolecules and metabolites. Both, the proteome and the metabolome are highly dynamic and responsive to environmental cues and developmental processes. But it is not their bare numbers, but their interactions that enable life. The protein-protein (PPI) and protein-metabolite interactions (PMI) facilitate and regulate all aspects of cell biology, from metabolism to mitosis. Therefore, the study of PPIs and PMIs and their dynamics in a cell-wide context is of great scientific interest. In this dissertation, I aim to chart a map of the dynamic PPIs and PMIs across metabolic and cellular transitions. As a model system, I study the shift from the fermentative to the respiratory growth, known as the diauxic shift, in the budding yeast Saccharomyces cerevisiae. To do so, I am applying a co-fractionation mass spectrometry (CF-MS) based method, dubbed protein metabolite interactions using size separation (PROMIS). PROMIS, as well as comparable methods, will be discussed in detail in chapter 1. Since PROMIS was developed originally for Arabidopsis thaliana, in chapter 2, I will describe the adaptation of PROMIS to S. cerevisiae. Here, the obtained results demonstrated a wealth of protein-metabolite interactions, and experimentally validated 225 previously predicted PMIs. Applying orthogonal, targeted approaches to validate the interactions of a proteogenic dipeptide, Ser-Leu, five novel protein-interactors were found. One of those proteins, phosphoglycerate kinase, is inhibited by Ser-Leu, placing the dipeptide at the regulation of glycolysis. In chapter 3, I am presenting PROMISed, a novel web-tool designed for the analysis of PROMIS- and other CF-MS-datasets. Starting with raw fractionation profiles, PROMISed enables data pre-processing, profile deconvolution, scores differences in fractionation profiles between experimental conditions, and ultimately charts interaction networks. PROMISed comes with a user-friendly graphic interface, and thus enables the routine analysis of CF-MS data by non-computational biologists. Finally, in chapter 4, I applied PROMIS in combination with the isothermal shift assay to the diauxic shift in S. cerevisiae to study changes in the PPI and PMI landscape across this metabolic transition. I found a major rewiring of protein-protein-metabolite complexes, exemplified by the disassembly of the proteasome in the respiratory phase, the loss of interaction of an enzyme involved in amino acid biosynthesis and its cofactor, as well as phase and structure specific interactions between dipeptides and enzymes of central carbon metabolism. In chapter 5, I am summarizing the presented results, and discuss a strategy to unravel the potential patterns of dipeptide accumulation and binding specificities. Lastly, I recapitulate recently postulated guidelines for CF-MS experiments, and give an outlook of protein interaction studies in the near future.}, language = {en} } @phdthesis{BysaniKondagari2023, author = {Bysani Kondagari, Viswanada Reddy}, title = {Engineering and evolution of saccharomyces cerevisiae for synthetic formatotrophic growth via the reductive glycine pathway}, doi = {10.25932/publishup-58222}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-582222}, school = {Universit{\"a}t Potsdam}, pages = {124}, year = {2023}, abstract = {Increasing demand for food, healthcare, and transportation arising from the growing world population is accompanied by and driving global warming challenges due to the rise of the atmospheric CO2 concentration. Industrialization for human needs has been increasingly releasing CO2 into the atmosphere for the last century or more. In recent years, the possibility of recycling CO2 to stabilize the atmospheric CO2 concentration and combat rising temperatures has gained attention. Thus, using CO2 as the feedstock to address future world demands is the ultimate solution while controlling the rapid climate change. Valorizing CO2 to produce activated and stable one-carbon feedstocks like formate and methanol and further upgrading them to industrial microbial processes to replace unsustainable feedstocks would be crucial for a future biobased circular economy. However, not all microbes can grow on formate as a feedstock, and those microbes that can grow are not well established for industrial processes. S. cerevisiae is one of the industrially well-established microbes, and it is a significant contributor to bioprocess industries. However, it cannot grow on formate as a sole carbon and energy source. Thus, engineering S. cerevisiae to grow on formate could potentially pave the way to sustainable biomass and value-added chemicals production. The Reductive Glycine Pathway (RGP), designed as the aerobic twin of the anaerobic Reductive Acetyl-CoA pathway, is an efficient formate and CO2 assimilation pathway. The RGP comprises of the glycine synthesis module (Mis1p, Gcv1p, Gcv2p, Gcv3p, and Lpd1p), the glycine to serine conversion module (Shmtp), the pyruvate synthesis module (Cha1p), and the energy supply module (Fdh1p). The RGP requires formate and elevated CO2 levels to operate the glycine synthesis module. In this study, I established the RGP in the yeast system using growth-coupled selection strategies to achieve formate and CO2-dependent biomass formation in aerobic conditions. Firstly, I constructed serine biosensor strains by disrupting the native serine and glycine biosynthesis routes in the prototrophic S288c and FL100 yeast strains and insulated serine, glycine, and one-carbon metabolism from the central metabolic network. These strains cannot grow on glucose as the sole carbon source but require the supply of serine or glycine to complement the engineered auxotrophies. Using growth as a readout, I employed these strains as selection hosts to establish the RGP. Initially, to achieve this, I engineered different serine-hydroxymethyltransferases in the genome of serine biosensor strains for efficient glycine to serine conversion. Then, I implemented the glycine synthesis module of the RGP in these strains for the glycine and serine synthesis from formate and CO2. I successfully conducted Adaptive Laboratory Evolution (ALE) using these strains, which yielded a strain capable of glycine and serine biosynthesis from formate and CO2. Significant growth improvements from 0.0041 h-1 to 0.03695 h-1 were observed during ALE. To validate glycine and serine synthesis, I conducted carbon tracing experiments with 13C formate and 13CO2, confirming that more than 90\% of glycine and serine biosynthesis in the evolved strains occurs via the RGP. Interestingly, labeling data also revealed that 10-15\% of alanine was labelled, indicating pyruvate synthesis from the formate-derived serine using native serine deaminase (Cha1p) activity. Thus, RGP contributes to a small pyruvate pool which is converted to alanine without any selection pressure for pyruvate synthesis from formate. Hence, this data confirms the activity of all three modules of RGP even in the presence of glucose. Further, ALE in glucose limiting conditions did not improve pyruvate flux via the RGP. Growth characterization of these strains showed that the best growth rates were achieved in formate concentrations between 25 mM to 300 mM. Optimum growth required 5\% CO2, and dropped when the CO2 concentration was reduced from 5\% to 2.5\%. Whole-genome sequencing of these evolved strains revealed mutations in genes that encode Gdh1p, Pet9p, and Idh1p. These enzymes might influence intracellular NADPH, ATP, and NADH levels, indicating adjustment to meet the energy demand of the RGP. I reverse-engineered the GDH1 truncation mutation on unevolved serine biosensor strains and reproduced formate dependent growth. To elucidate the effect of the GDH1 mutation on formate assimilation, I reintroduced this mutation in the S288c strain and conducted carbon-tracing experiments to compared formate assimilation between WT and ∆gdh1 mutant strains. Comparatively, enhanced formate assimilation was recorded in the ∆gdh1 mutant strain. Although the 13C carbon tracing experiments confirmed the activity of all three modules of the RGP, the overall pyruvate flux via the RGP might be limited by the supply of reducing power. Hence, in a different approach, I overexpressed the formate dehydrogenase (Fdh1p) for energy supply and serine deaminase (Cha1p) for active pyruvate synthesis in the S288c parental strain and established growth on formate and serine without glucose in the medium. Further reengineering and evolution of this strain with a consistent energy, and formate-derived serine supply for pyruvate synthesis, is essential to achieve complete formatotrophic growth in the yeast system.}, language = {en} } @phdthesis{MartinezSeidel2023, author = {Martinez-Seidel, Federico}, title = {Ribosome Heterogeneity and Specialization during Temperature Acclimation in Plants}, doi = {10.25932/publishup-58072}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-580724}, school = {Universit{\"a}t Potsdam}, pages = {374}, year = {2023}, abstract = {Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation.}, language = {en} } @phdthesis{Steppert2022, author = {Steppert, Isabel}, title = {Entwicklung einer nichtinvasiven Diagnostikmethode zum Nachweis von Infektionserregern}, doi = {10.25932/publishup-57544}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-575441}, school = {Universit{\"a}t Potsdam}, pages = {XIV, 101, LVIII}, year = {2022}, abstract = {Die aktuelle COVID-19-Pandemie zeigt deutlich, wie sich Infektionskrankheiten weltweit verbreiten k{\"o}nnen. Neben Viruserkrankungen breiten sich auch multiresistente bakterielle Erreger weltweit aus. Dementsprechend besteht ein hoher Bedarf, durch fr{\"u}hzeitige Erkennung Erkrankte zu finden und Infektionswege zu unterbrechen. Herk{\"o}mmliche kulturelle Verfahren ben{\"o}tigen minimalinvasive bzw. invasive Proben und dauern f{\"u}r Screeningmaßnahmen zu lange. Deshalb werden schnelle, nichtinvasive Verfahren ben{\"o}tigt. Im klassischen Griechenland verließen sich die {\"A}rzte unter anderem auf ihren Geruchssinn, um Infektionen und andere Krankheiten zu differenzieren. Diese charakteristischen Ger{\"u}che sind fl{\"u}chtige organische Substanzen (VOC), die im Rahmen des Metabolismus eines Organismus entstehen. Tiere, die einen besseren Geruchssinn haben, werden trainiert, bestimmte Krankheitserreger am Geruch zu unterscheiden. Allerdings ist der Einsatz von Tieren im klinischen Alltag nicht praktikabel. Es bietet sich an, auf technischem Weg diese VOCs zu analysieren. Ein technisches Verfahren, diese VOCs zu unterscheiden, ist die Ionenmobilit{\"a}tsspektrometrie gekoppelt mit einer multikapillaren Gaschromatographies{\"a}ule (MCC-IMS). Hier zeigte sich, dass es sich bei dem Verfahren um eine schnelle, sensitive und verl{\"a}ssliche Methode handelt. Es ist bekannt, dass verschiedene Bakterien aufgrund des Metabolismus unterschiedliche VOCs und damit eigene spezifische Ger{\"u}che produzieren. Im ersten Schritt dieser Arbeit konnte gezeigt werden, dass die verschiedenen Bakterien in-vitro nach einer kurzen Inkubationszeitzeit von 90 Minuten anhand der VOCs differenziert werden k{\"o}nnen. Hier konnte analog zur Diagnose in biochemischen Testreihen eine hierarchische Klassifikation der Bakterien erfolgen. Im Gegensatz zu Bakterien haben Viren keinen eigenen Stoffwechsel. Ob virusinfizierte Zellen andere VOCs als nicht-infizierte Zellen freisetzen, wurde an Zellkulturen {\"u}berpr{\"u}ft. Hier konnte gezeigt werden, dass sich die Fingerprints der VOCs in Zellkulturen infizierter Zellen mit Respiratorischen Synzytial-Viren (RSV) von nicht-infizierten Zellen unterscheiden. Virusinfektionen im intakten Organismus unterscheiden sich von den Zellkulturen dadurch, dass hier neben Ver{\"a}nderungen im Zellstoffwechsel auch durch Abwehrmechanismen VOCs freigesetzt werden k{\"o}nnen. Zur {\"U}berpr{\"u}fung, inwiefern sich Infektionen im intakten Organismus ebenfalls anhand VOCs unterscheiden lassen, wurde bei Patienten mit und ohne Nachweis einer Influenza A Infektion als auch bei Patienten mit Verdacht auf SARS-CoV-2 (Schweres-akutes-Atemwegssyndrom-Coronavirus Typ 2) Infektion die Atemluft untersucht. Sowohl Influenza-infizierte als auch SARS-CoV-2 infizierte Patienten konnten untereinander und von nicht-infizierten Patienten mittels MCC-IMS Analyse der Atemluft unterschieden werden. Zusammenfassend erbringt die MCC-IMS ermutigende Resultate in der schnellen nichtinvasiven Erkennung von Infektionen sowohl in vitro als auch in vivo.}, language = {de} } @phdthesis{Drago2022, author = {Drago, Claudia}, title = {Microplastics in the environment: Assessing the ingestion and effect of microplastics on freshwater rotifers in an environmental scenario}, doi = {10.25932/publishup-57335}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-573356}, school = {Universit{\"a}t Potsdam}, pages = {xv, 116}, year = {2022}, abstract = {Microplastics in the environments are estimated to increase in the near future due to increasing consumption of plastic product and also due to further fragmentation in small pieces. The fate and effects of MP once released into the freshwater environment are still scarcely studied, compared to the marine environment. In order to understand possible effect and interaction of MPs in freshwater environment, planktonic zooplankton organisms are very useful for their crucial trophic role. In particular freshwater rotifers are one of the most abundant organisms and they are the interface between primary producers and secondary consumers. The aim of my thesis was to investigate the ingestion and the effect of MPs in rotifers from a more natural scenario and to individuate processes such as the aggregation of MPs, the food dilution effect and the increasing concentrations of MPs that could influence the final outcome of MPs in the environment. In fact, in a near natural scenario MPs interaction with bacteria and algae, aggregations together with the size and concentration are considered drivers of ingestion and effect. The aggregation of MPs makes smaller MPs more available for rotifers and larger MPs less ingested. The negative effect caused by the ingestion of MPs was modulated by their size but also by the quantity and the quality of food that cause variable responses. In fact, rotifers in the environment are subjected to food limitation and the presence of MPs could exacerbate this condition and decrease the population and the reproduction input. Finally, in a scenario incorporating an entire zooplanktonic community, MPs were ingested by most individuals taking into account their feeding mode but also the concentration of MPs, which was found to be essential for the availability of MPs. This study highlights the importance to investigate MPs from a more environmental perspective, this in fact could provide an alternative and realistic view of effect of MPs in the ecosystem.}, language = {en} } @phdthesis{Tegtmeier2022, author = {Tegtmeier, Laura}, title = {Functional analysis of ENTH domain proteins}, doi = {10.25932/publishup-57004}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-570049}, school = {Universit{\"a}t Potsdam}, pages = {106}, year = {2022}, abstract = {In plant cells, subcellular transport of cargo proteins relies to a large extent on post-Golgi transport pathways, many of which are mediated by clathrin-coated vesicles (CCVs). Vesicle formation is facilitated by different factors like accessory proteins and adaptor protein complexes (APs), the latter serving as a bridge between cargo proteins and the coat protein clathrin. One type of accessory proteins is defined by a conserved EPSIN N-TERMINAL HOMOLOGY (ENTH) domain and interacts with APs and clathrin via motifs in the C-terminal part. In Arabidopsis thaliana, there are three closely related ENTH domain proteins (EPSIN1, 2 and 3) and one highly conserved but phylogenetically distant outlier, termed MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1). In case of the trans-Golgi network (TGN) located MTV1, clathrin association and a role in vacuolar transport have been shown previously (Sauer et al. 2013). In contrast, for EPSIN1 and EPSIN2 limited functional and localization data were available; and EPSIN3 remained completely uncharacterized prior to this study (Song et al. 2006; Lee et al. 2007). The molecular details of ENTH domain proteins in plants are still unknown. In order to systematically characterize all four ENTH proteins in planta, we first investigated expression and subcellular localization by analysis of stable reporter lines under their endogenous promotors. Although all four genes are ubiquitously expressed, their subcellular distribution differs markedly. EPSIN1 and MTV1 are located at the TGN, whereas EPSIN2 and EPSIN3 are associated with the plasma membrane (PM) and the cell plate. To examine potential functional redundancy, we isolated knockout T-DNA mutant lines and created all higher order mutant combinations. The clearest evidence for functional redundancy was observed in the epsin1 mtv1 double mutant, which is a dwarf displaying overall growth reduction. These findings are in line with the TGN localization of both MTV1 and EPS1. In contrast, loss of EPSIN2 and EPSIN3 does not result in a growth phenotype compared to wild type, however, a triple knockout of EPSIN1, EPSIN2 and EPSIN3 shows partially sterile plants. We focused mainly on the epsin1 mtv1 double mutant and addressed the functional role of these two genes in clathrin-mediated vesicle transport by comprehensive molecular, biochemical, and genetic analyses. Our results demonstrate that EPSIN1 and MTV1 promote vacuolar transport and secretion of a subset of cargo. However, they do not seem to be involved in endocytosis and recycling. Importantly, employing high-resolution imaging, genetic and biochemical experiments probing the relationship of the AP complexes, we found that EPSIN1/AP1 and MTV1/AP4 define two spatially and molecularly distinct subdomains of the TGN. The AP4 complex is essential for MTV1 recruitment to the TGN, whereas EPSIN1 is independent of AP4 but presumably acts in an AP1-dependent framework. Our findings suggest that this ENTH/AP pairing preference is conserved between animals and plants.}, language = {en} } @phdthesis{Oberkofler2022, author = {Oberkofler, Vicky}, title = {Molecular basis of HS memory in Arabidopsis thaliana}, doi = {10.25932/publishup-56954}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569544}, school = {Universit{\"a}t Potsdam}, pages = {181}, year = {2022}, abstract = {Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years. On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first. This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains. In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.}, language = {en} } @phdthesis{Folikumah2022, author = {Folikumah, Makafui Yao}, title = {Stimuli-promoted in situ formation of hydrogels with thiol/thioester containing peptide precursors}, doi = {10.25932/publishup-56971}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569713}, school = {Universit{\"a}t Potsdam}, pages = {159}, year = {2022}, abstract = {Hydrogels are potential synthetic ECM-like substitutes since they provide functional and structural similarities compared to soft tissues. They can be prepared by crosslinking of macromolecules or by polymerizing suitable precursors. The crosslinks are not necessarily covalent bonds, but could also be formed by physical interactions such as π-π interactions, hydrophobic interactions, or H-bonding. On demand in situ forming hydrogels have garnered increased interest especially for biomedical applications over preformed gels due to the relative ease of in vivo delivery and filling of cavities. The thiol-Michael addition reaction provides a straightforward and robust strategy for in situ gel formation with its fast reaction kinetics and ability to proceed under physiological conditions. The incorporation of a trigger function into a crosslinking system becomes even more interesting since gelling can be controlled with stimulus of choice. The use of small molar mass crosslinker precursors with active groups orthogonal to thiol-Michael reaction type electrophile provides the opportunity to implement an on-demand in situ crosslinking without compromising the fast reaction kinetics. It was postulated that short peptide sequences due to the broad range structural-function relations available with the different constituent amino acids, can be exploited for the realisation of stimuli-promoted in situ covalent crosslinking and gelation applications. The advantages of this system over conventional polymer-polymer hydrogel systems are the ability tune and predict material property at the molecular level. The main aim of this work was to develop a simplified and biologically-friendly stimuli-promoted in situ crosslinking and hydrogelation system using peptide mimetics as latent crosslinkers. The approach aims at using a single thiodepsipeptide sequence to achieve separate pH- and enzyme-promoted gelation systems with little modification to the thiodepsipeptide sequence. The realization of this aim required the completion of three milestones. In the first place, after deciding on the thiol-Michael reaction as an effective in situ crosslinking strategy, a thiodepsipeptide, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH (TDP) with expected propensity towards pH-dependent thiol-thioester exchange (TTE) activation, was proposed as a suitable crosslinker precursor for pH-promoted gelation system. Prior to the synthesis of the proposed peptide-mimetic, knowledge of the thiol-Michael reactivity of the would-be activated thiol moiety SH-Leu, which is internally embedded in the thiodepsipeptide was required. In line with pKa requirements for a successful TTE, the reactivity of a more acidic thiol, SH-Phe was also investigated to aid the selection of the best thiol to be incorporated in the thioester bearing peptide based crosslinker precursor. Using 'pseudo' 2D-NMR investigations, it was found that only reactions involving SH-Leu yielded the expected thiol-Michael product, an observation that was attributed to the steric hindrance of the bulkier nature of SH-Phe. The fast reaction rates and complete acrylate/maleimide conversion obtained with SH-Leu at pH 7.2 and higher aided the direct elimination of SH-Phe as a potential thiol for the synthesis of the peptide mimetic. Based on the initial studies, for the pH-promoted gelation system, the proposed Ac-Pro-Leu-Gly-SLeu-Leu-Gly-NEtSH was kept unmodified. The subtle difference in pKa values between SH-Leu (thioester thiol) and the terminal cysteamine thiol from theoretical conditions should be enough to effect a 'pseudo' intramolecular TTE. In polar protic solvents and under basic aqueous conditions, TDP successfully undergoes a 'pseudo' intramolecular TTE reaction to yield an α,ω-dithiol tripeptide, HSLeu-Leu-Gly-NEtSH. The pH dependence of thiolate ion generation by the cysteamine thiol aided the incorporation of the needed stimulus (pH) for the overall success of TTE (activation step) - thiol-Michael addition (crosslinking) strategy. Secondly, with potential biomedical applications in focus, the susceptibility of TDP, like other thioesters, to intermolecular TTE reaction was probed with a group of thiols of varying thiol pKa values, since biological milieu characteristically contain peptide/protein thiols. L-cysteine, which is a biologically relevant thiol, and a small molecular weight thiol, methylthioglycolate both with relatively similar thiol pKa, values, led to an increase concentration of the dithiol crosslinker when reacted with TDP. In the presence of acidic thiols (p-NTP and 4MBA), a decrease in the dithiol concentration was observed, an observation that can be attributed to the inability of the TTE tetrahedral intermediate to dissociate into exchange products and is in line with pKa requirements for successful TTE reaction. These results additionally makes TDP more attractive and the potentially the first crosslinker precursor for applications in biologically relevant media. Finally, the ability of TDP to promote pH-sensitive in situ gel formation was probed with maleimide functionalized 4-arm polyethylene glycol polymers in tris-buffered media of varying pHs. When a 1:1 thiol: maleimide molar ratio was used, TDP-PEG4MAL hydrogels formed within 3, 12 and 24 hours at pH values of 8.5, 8.0 and 7.5 respectively. However, gelation times of 3, 5 and 30 mins were observed for the same pH trend when the thiol: maleimide molar was increased to 2:1. A direct correlation of thiol content with G' of the gels at each pH could also be drawn by comparing gels with thiol: maleimide ratios of 1:1 to those with 2:1 thiol: maleimide mole ratios. This is supported by the fact that the storage modulus (G') is linearly dependent on the crosslinking density of the polymer. The values of initial G′ for all gels ranged between (200 - 5000 Pa), which falls in the range of elasticities of certain tissue microenvironments for example brain tissue 200 - 1000 Pa and adipose tissue (2500 - 3500 Pa). Knowledge so far gained from the study on the ability to design and tune the exchange reaction of thioester containing peptide mimetic will give those working in the field further insight into the development of new sequences tailored towards specific applications. TTE substrate design using peptide mimetic as presented in this work has revealed interesting new insights considering the state-of-the-art. Using the results obtained as reference, the strategy provides a possibility to extend the concept to the controlled delivery of active molecules needed for other robust and high yielding crosslinking reactions for biomedical applications. Application for this sequentially coupled functional system could be seen e.g. in the treatment of inflamed tissues associated with urinary tract like bladder infections for which pH levels above 7 were reported. By the inclusion of cell adhesion peptide motifs, the hydrogel network formed at this pH could act as a new support layer for the healing of damage epithelium as shown in interfacial gel formation experiments using TDP and PEG4MAL droplets. The versatility of the thiodepsipeptide sequence, Ac-Pro-Leu-Gly-SLeu-Leu-Gly-(TDPo) was extended for the design and synthesis of a MMP-sensitive 4-arm PEG-TDPo conjugate. The purported cleavage of TDPo at the Gly-SLeu bond yields active thiol units for subsequent reaction of orthogonal Michael acceptor moieties. One of the advantages of stimuli-promoted in situ crosslinking systems using short peptides should be the ease of design of required peptide molecules due to the predictability of peptide functions their sequence structure. Consequently the functionalisation of a 4-arm PEG core with the collagenase active TDPo sequence yielded an MMP-sensitive 4-arm thiodepsipeptide-PEG conjugate (PEG4TDPo) substrate. Cleavage studies using thiol flourometric assay in the presence of MMPs -2 and -9 confirmed the susceptibility of PEG4TDPo towards these enzymes. The resulting time-dependent increase in fluorescence intensity in the presence of thiol assay signifies the successful cleavage of TDPo at the Gly-SLeu bond as expected. It was observed that the cleavage studies with thiol flourometric assay introduces a sigmoid non-Michaelis-Menten type kinetic profile, hence making it difficult to accurately determine the enzyme cycling parameters, kcat and KM . Gelation studies with PEG4MAL at 10 \% wt. concentrations revealed faster gelation with MMP-2 than MMP-9 with 28 and 40 min gelation times respectively. Possible contributions by hydrolytic cleavage of PEG4TDPo has resulted in the gelation of PEG4MAL blank samples but only after 60 minutes of reaction. From theoretical considerations, the simultaneous gelation reaction would be expected to more negatively impact the enzymatic than hydrolytic cleavage. The exact contributions from hydrolytic cleavage of PEG4TDPo would however require additional studies. In summary this new and simplified in situ crosslinking system using peptide-based crosslinker precursors with tuneable properties exhibited in situ crosslinking gelation kinetics on similar levels with already active dithiols reported. The advantageous on-demand functionality associated with its pH-sensitivity and physiological compatibility makes it a strong candidate worth further research as biomedical applications in general and on-demand material synthesis is concerned. Results from MMP-promoted gelation system unveils a simple but unexplored approach for in situ synthesis of covalently crosslinked soft materials, that could lead to the development of an alternative pathway in addressing cancer metastasis by making use of MMP overexpression as a trigger. This goal has so far not being reach with MMP inhibitors despite the extensive work this regard.}, language = {en} } @phdthesis{Eckert2022, author = {Eckert, Silvia}, title = {Trait variation in changing environments: Assessing the role of DNA methylation in non-native plant species}, doi = {10.25932/publishup-56884}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-568844}, school = {Universit{\"a}t Potsdam}, pages = {VIII, 134, CXXX}, year = {2022}, abstract = {The increasing introduction of non-native plant species may pose a threat to local biodiversity. However, the basis of successful plant invasion is not conclusively understood, especially since these plant species can adapt to the new range within a short period of time despite impoverished genetic diversity of the starting populations. In this context, DNA methylation is considered promising to explain successful adaptation mechanisms in the new habitat. DNA methylation is a heritable variation in gene expression without changing the underlying genetic information. Thus, DNA methylation is considered a so-called epigenetic mechanism, but has been studied in mainly clonally reproducing plant species or genetic model plants. An understanding of this epigenetic mechanism in the context of non-native, predominantly sexually reproducing plant species might help to expand knowledge in biodiversity research on the interaction between plants and their habitats and, based on this, may enable more precise measures in conservation biology. For my studies, I combined chemical DNA demethylation of field-collected seed material from predominantly sexually reproducing species and rearing offsping under common climatic conditions to examine DNA methylation in an ecological-evolutionary context. The contrast of chemically treated (demethylated) plants, whose variation in DNA methylation was artificially reduced, and untreated control plants of the same species allowed me to study the impact of this mechanism on adaptive trait differentiation and local adaptation. With this experimental background, I conducted three studies examining the effect of DNA methylation in non-native species along a climatic gradient and also between climatically divergent regions. The first study focused on adaptive trait differentiation in two invasive perennial goldenrod species, Solidago canadensis sensu latu and S. gigantea AITON, along a climate gradient of more than 1000 km in length in Central Europe. I found population differences in flowering timing, plant height, and biomass in the temporally longer-established S. canadensis, but only in the number of regrowing shoots for S. gigantea. While S. canadensis did not show any population structure, I was able to identify three genetic groups along this climatic gradient in S. gigantea. Surprisingly, demethylated plants of both species showed no change in the majority of traits studied. In the subsequent second study, I focused on the longer-established goldenrod species S. canadensis and used molecular analyses to infer spatial epigenetic and genetic population differences in the same specimens from the previous study. I found weak genetic but no epigenetic spatial variation between populations. Additionally, I was able to identify one genetic marker and one epigenetic marker putatively susceptible to selection. However, the results of this study reconfirmed that the epigenetic mechanism of DNA methylation appears to be hardly involved in adaptive processes within the new range in S. canadensis. Finally, I conducted a third study in which I reciprocally transplanted short-lived plant species between two climatically divergent regions in Germany to investigate local adaptation at the plant family level. For this purpose, I used four plant families (Amaranthaceae, Asteraceae, Plantaginaceae, Solanaceae) and here I additionally compared between non-native and native plant species. Seeds were transplanted to regions with a distance of more than 600 kilometers and had either a temperate-oceanic or a temperate-continental climate. In this study, some species were found to be maladapted to their own local conditions, both in non-native and native plant species alike. In demethylated individuals of the plant species studied, DNA methylation had inconsistent but species-specific effects on survival and biomass production. The results of this study highlight that DNA methylation did not make a substantial contribution to local adaptation in the non-native as well as native species studied. In summary, my work showed that DNA methylation plays a negligible role in both adaptive trait variation along climatic gradients and local adaptation in non-native plant species that either exhibit a high degree of genetic variation or rely mainly on sexual reproduction with low clonal propagation. I was able to show that the adaptive success of these non-native plant species can hardly be explained by DNA methylation, but could be a possible consequence of multiple introductions, dispersal corridors and meta-population dynamics. Similarly, my results illustrate that the use of plant species that do not predominantly reproduce clonally and are not model plants is essential to characterize the effect size of epigenetic mechanisms in an ecological-evolutionary context.}, language = {en} } @phdthesis{Milles2022, author = {Milles, Alexander}, title = {Sources and consequences of intraspecific trait variation in movement behaviour}, doi = {10.25932/publishup-56501}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-565011}, school = {Universit{\"a}t Potsdam}, pages = {xvi, 225}, year = {2022}, abstract = {Variation in traits permeates and affects all levels of biological organisation, from within individuals to between species. Yet, intraspecific trait variation (ITV) is not sufficiently represented in many ecological theories. Instead, species averages are often assumed. Especially ITV in behaviour has only recently attracted more attention as its pervasiveness and magnitude became evident. The surge in interest in ITV in behaviour was accompanied by a methodological and technological leap in the field of movement ecology. Many aspects of behaviour become visible via movement, allowing us to observe inter-individual differences in fundamental processes such as foraging, mate searching, predation or migration. ITV in movement behaviour may result from within-individual variability and consistent, repeatable among-individual differences. Yet, questions on why such among-individual differences occur in the first place and how they are integrated with life-history have remained open. Furthermore, consequences of ITV, especially of among-individual differences in movement behaviour, on populations and species communities are not sufficiently understood. In my thesis, I approach timely questions on the sources and consequences of ITV, particularly, in movement behaviour. After outlining fundamental concepts and the current state of knowledge, I approach these questions by using agent-based models to integrate concepts from behavioural and movement ecology and to develop novel perspectives. Modern coexistence theory is a central pillar of community ecology, yet, insufficiently considers ITV in behaviour. In chapter 2, I model a competitive two-species system of ground-dwelling, central-place foragers to investigate the consequences of among-individual differences in movement behaviour on species coexistence. I show that the simulated among-individual differences, which matched with empirical data, reduce fitness differences betweem species, i.e. provide an equalising coexistence mechanism. Furthermore, I explain this result mechanistically and, thus, resolve an apparent ambiguity of the consequences of ITV on species coexistence described in previous studies. In chapter 3, I turn the focus to sources of among-individual differences in movement behaviour and their potential integration with life-history. The pace-of-life syndrome (POLS) theory predicts that the covariation between among-individual differences in behaviour and life-history is mediated by a trade-off between early and late reproduction. This theory has generated attention but is also currently scrutinised. In chapter 3, I present a model which supports a recent conceptual development that suggests fluctuating density-dependent selection as a cause of the POLS. Yet, I also identified processes that may alter the association between movement behaviour and life-history across levels of biological organization. ITV can buffer populations, i.e. reduce their extinction risk. For instance, among-individual differences can mediate portfolio effects or increase evolvability and, thereby, facilitate rapid evolution which can alleviate extinction risk. In chapter 4, I review ITV, environmental heterogeneity, and density-dependent processes which constitute local buffer mechanisms. In the light of habitat isolation, which reduces connectivity between populations, local buffer mechanisms may become more relevant compared to dispersal-related regional buffer mechanisms. In this chapter, I argue that capacities, latencies, and interactions of local buffer mechanisms should motivate more process-based and holistic integration of local buffer mechanisms in theoretical and empirical studies. Recent perspectives propose to apply principles from movement and community ecology to study filamentous fungi. It is an open question whether and how the arrangement and geometry of microstructures select for certain movement traits, and, thus, facilitate coexistence-stabilising niche partitioning. As a coauthor of chapter 5, I developed an agent-based model of hyphal tips navigating in soil-like microstructures along a gradient of soil porosity. By measuring network properties, we identified changes in the optimal movement behaviours along the gradient. Our findings suggest that the soil architecture facilitates niche partitioning. The core chapters are framed by a general introduction and discussion. In the general introduction, I outline fundamental concepts of movement ecology and describe theory and open questions on sources and consequences of ITV in movement behaviour. In the general discussion, I consolidate the findings of the core chapters and critically discuss their respective value and, if applicable, their impact. Furthermore, I emphasise promising avenues for further research.}, language = {en} } @phdthesis{Heinsohn2022, author = {Heinsohn, Natascha}, title = {Development of a fiber-based sensor for the molecular detection of pathogens using Legionella as an example}, doi = {10.25932/publishup-56683}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-566833}, school = {Universit{\"a}t Potsdam}, pages = {X, 175}, year = {2022}, abstract = {Fiber-based microfluidics has undergone many innovative developments in recent years, with exciting examples of portable, cost-effective and easy-to-use detection systems already being used in diagnostic and analytical applications. In water samples, Legionella are a serious risk as human pathogens. Infection occurs through inhalation of aerosols containing Legionella cells and can cause severe pneumonia and may even be fatal. In case of Legionella contamination of water-bearing systems or Legionella infection, it is essential to find the source of the contamination as quickly as possible to prevent further infections. In drinking, industrial and wastewater monitoring, the culture-based method is still the most commonly used technique to detect Legionella contamination. In order to improve the laboratory-dependent determination, the long analysis times of 10-14 days as well as the inaccuracy of the measured values in colony forming units (CFU), new innovative ideas are needed. In all areas of application, for example in public, commercial or private facilities, rapid and precise analysis is required, ideally on site. In this PhD thesis, all necessary single steps for a rapid DNA-based detection of Legionella were developed and characterized on a fiber-based miniaturized platform. In the first step, a fast, simple and device-independent chemical lysis of the bacteria and extraction of genomic DNA was established. Subsequently, different materials were investigated with respect to their non-specific DNA retention. Glass fiber filters proved to be particularly suitable, as they allow recovery of the DNA sample from the fiber material in combination with dedicated buffers and exhibit low autofluorescence, which was important for fluorescence-based readout. A fiber-based electrophoresis unit was developed to migrate different oligonucleotides within a fiber matrix by application of an electric field. A particular advantage over lateral flow assays is the targeted movement, even after the fiber is saturated with liquid. For this purpose, the entire process of fiber selection, fiber chip patterning, combination with printed electrodes, and testing of retention and migration of different DNA samples (single-stranded, double-stranded and genomic DNA) was performed. DNA could be pulled across the fiber chip in an electric field of 24 V/cm within 5 minutes, remained intact and could be used for subsequent detection assays e.g., polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). Fiber electrophoresis could also be used to separate DNA from other components e.g., proteins or cell lysates or to pull DNA through multiple layers of the glass microfiber. In this way, different fragments experienced a moderate, size-dependent separation. Furthermore, this arrangement offers the possibility that different detection reactions could take place in different layers at a later time. Electric current and potential measurements were collected to investigate the local distribution of the sample during migration. While an increase in current signal at high concentrations indicated the presence of DNA samples, initial experiments with methylene blue stained DNA showed a temporal sequence of signals, indicating sample migration along the chip. For the specific detection of a Legionella DNA, a FISH-based detection with a molecular beacon probe was tested on the glass microfiber. A specific region within the 16S rRNA gene of Legionella spp. served as a target. For this detection, suitable reaction conditions and a readout unit had to be set up first. Subsequently, the sensitivity of the probe was tested with the reverse complementary target sequence and the specificity with several DNA fragments that differed from the target sequence. Compared to other DNA sequences of similar length also found in Legionella pneumophila, only the target DNA was specifically detected on the glass microfiber. If a single base exchange is present or if two bases are changed, the probe can no longer distinguish between the DNA targets and non-targets. An analysis with this specificity can be achieved with other methods such as melting point determination, as was also briefly indicated here. The molecular beacon probe could be dried on the glass microfiber and stored at room temperature for more than three months, after which it was still capable of detecting the target sequence. Finally, the feasibility of fiber-based FISH detection for genomic Legionella DNA was tested. Without further processing, the probe was unable to detect its target sequence in the complex genomic DNA. However, after selecting and application of appropriate restriction enzymes, specific detection of Legionella DNA against other aquatic pathogens with similar fragment patterns as Acinetobacter haemolyticus was possible.}, language = {en} } @phdthesis{Ziege2022, author = {Ziege, Ricardo}, title = {Growth dynamics and mechanical properties of E. coli biofilms}, doi = {10.25932/publishup-55986}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-559869}, school = {Universit{\"a}t Potsdam}, pages = {xi, 123}, year = {2022}, abstract = {Biofilms are complex living materials that form as bacteria get embedded in a matrix of self-produced protein and polysaccharide fibres. The formation of a network of extracellular biopolymer fibres contributes to the cohesion of the biofilm by promoting cell-cell attachment and by mediating biofilm-substrate interactions. This sessile mode of bacteria growth has been well studied by microbiologists to prevent the detrimental effects of biofilms in medical and industrial settings. Indeed, biofilms are associated with increased antibiotic resistance in bacterial infections, and they can also cause clogging of pipelines or promote bio-corrosion. However, biofilms also gained interest from biophysics due to their ability to form complex morphological patterns during growth. Recently, the emerging field of engineered living materials investigates biofilm mechanical properties at multiple length scales and leverages the tools of synthetic biology to tune the functions of their constitutive biopolymers. This doctoral thesis aims at clarifying how the morphogenesis of Escherichia coli (E. coli) biofilms is influenced by their growth dynamics and mechanical properties. To address this question, I used methods from cell mechanics and materials science. I first studied how biological activity in biofilms gives rise to non-uniform growth patterns. In a second study, I investigated how E. coli biofilm morphogenesis and its mechanical properties adapt to an environmental stimulus, namely the water content of their substrate. Finally, I estimated how the mechanical properties of E. coli biofilms are altered when the bacteria express different extracellular biopolymers. On nutritive hydrogels, micron-sized E. coli cells can build centimetre-large biofilms. During this process, bacterial proliferation and matrix production introduce mechanical stresses in the biofilm, which release through the formation of macroscopic wrinkles and delaminated buckles. To relate these biological and mechanical phenomena, I used time-lapse fluorescence imaging to track cell and matrix surface densities through the early and late stages of E. coli biofilm growth. Colocalization of high cell and matrix densities at the periphery precede the onset of mechanical instabilities at this annular region. Early growth is detected at this outer annulus, which was analysed by adding fluorescent microspheres to the bacterial inoculum. But only when high rates of matrix production are present in the biofilm centre, does overall biofilm spreading initiate along the solid-air interface. By tracking larger fluorescent particles for a long time, I could distinguish several kinematic stages of E. coli biofilm expansion and observed a transition from non-linear to linear velocity profiles, which precedes the emergence of wrinkles at the biofilm periphery. Decomposing particle velocities to their radial and circumferential components revealed a last kinematic stage, where biofilm movement is mostly directed towards the radial delaminated buckles, which verticalize. The resulting compressive strains computed in these regions were observed to substantially deform the underlying agar substrates. The co-localization of higher cell and matrix densities towards an annular region and the succession of several kinematic stages are thus expected to promote the emergence of mechanical instabilities at the biofilm periphery. These experimental findings are predicted to advance future modelling approaches of biofilm morphogenesis. E. coli biofilm morphogenesis is further anticipated to depend on external stimuli from the environment. To clarify how the water could be used to tune biofilm material properties, we quantified E. coli biofilm growth, wrinkling dynamics and rigidity as a function of the water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that substrates with high water content promote biofilm spreading kinetics, while substrates with low water content promote biofilm wrinkling. The wrinkles observed on biofilm cross-sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, the micro-indentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to the water content of their substrate, which might be used for tuning their material properties in view of further applications. Biofilm material properties further depend on the composition and structure of the matrix of extracellular proteins and polysaccharides. In particular, E. coli biofilms were suggested to present tissue-like elasticity due to a dense fibre network consisting of amyloid curli and phosphoethanolamine-modified cellulose. To understand the contribution of these components to the emergent mechanical properties of E. coli biofilms, we performed micro-indentation on biofilms grown from bacteria of several strains. Besides showing higher dry masses, larger spreading diameters and slightly reduced water contents, biofilms expressing both main matrix components also presented high rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, a lack of amyloid curli fibres provides much higher adhesive energies and more viscoelastic fluid-like material behaviour. Therefore, the combination of amyloid curli and phosphoethanolamine-modified cellulose fibres implies the formation of a composite material whereby the amyloid curli fibres provide rigidity to E. coli biofilms, whereas the phosphoethanolamine-modified cellulose rather acts as a glue. These findings motivate further studies involving purified versions of these protein and polysaccharide components to better understand how their interactions benefit biofilm functions. All three studies depict different aspects of biofilm morphogenesis, which are interrelated. The first work reveals the correlation between non-uniform biological activities and the emergence of mechanical instabilities in the biofilm. The second work acknowledges the adaptive nature of E. coli biofilm morphogenesis and its mechanical properties to an environmental stimulus, namely water. Finally, the last study reveals the complementary role of the individual matrix components in the formation of a stable biofilm material, which not only forms complex morphologies but also functions as a protective shield for the bacteria it contains. Our experimental findings on E. coli biofilm morphogenesis and their mechanical properties can have further implications for fundamental and applied biofilm research fields.}, language = {en} } @phdthesis{Kuerschner2022, author = {K{\"u}rschner, Tobias}, title = {Disease transmission and persistence in dynamic landscapes}, doi = {10.25932/publishup-56468}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-564689}, school = {Universit{\"a}t Potsdam}, pages = {120, LXXIII}, year = {2022}, abstract = {Infectious diseases are an increasing threat to biodiversity and human health. Therefore, developing a general understanding of the drivers shaping host-pathogen dynamics is of key importance in both ecological and epidemiological research. Disease dynamics are driven by a variety of interacting processes such as individual host behaviour, spatiotemporal resource availability or pathogen traits like virulence and transmission. External drivers such as global change may modify the system conditions and, thus, the disease dynamics. Despite their importance, many of these drivers are often simplified and aggregated in epidemiological models and the interactions among multiple drivers are neglected. In my thesis, I investigate disease dynamics using a mechanistic approach that includes both bottom-up effects - from landscape dynamics to individual movement behaviour - as well as top-down effects - from pathogen virulence on host density and contact rates. To this end, I extended an established spatially explicit individual-based model that simulates epidemiological and ecological processes stochastically, to incorporate a dynamic resource landscape that can be shifted away from the timing of host population-dynamics (chapter 2). I also added the evolution of pathogen virulence along a theoretical virulence-transmission trade-off (chapter 3). In chapter 2, I focus on bottom-up effects, specifically how a temporal shift of resource availability away from the timing of biological events of host-species - as expected under global change - scales up to host-pathogen interactions and disease dynamics. My results show that the formation of temporary disease hotspots in combination with directed individual movement acted as key drivers for pathogen persistence even under highly unfavourable conditions for the host. Even with drivers like global change further increasing the likelihood of unfavourable interactions between host species and their environment, pathogens can continue to persist with heir hosts. In chapter 3, I demonstrate that the top-down effect caused by pathogen-associated mortality on its host population can be mitigated by selection for lower virulent pathogen strains when host densities are reduced through mismatches between seasonal resource availability and host life-history events. I chapter 4, I combined parts of both theoretical models into a new model that includes individual host movement decisions and the evolution of pathogenic virulence to simulate pathogen outbreaks in realistic landscapes. I was able to match simulated patterns of pathogen spread to observed patterns from long-term outbreak data of classical swine fever in wild boar in Northern Germany. The observed disease course was best explained by a simulated high virulent strain, whereas sampling schemes and vaccination campaigns could explain differences in the age-distribution of infected hosts. My model helps to understand and disentangle how the combination of individual decision making and evolution of virulence can act as important drivers of pathogen spread and persistence. As I show across the chapters of this thesis, the interplay of both bottom-up and top-down processes is a key driver of disease dynamics in spatially structured host populations, as they ultimately shape host densities and contact rates among moving individuals. My findings are an important step towards a paradigm shift in disease ecology away from simplified assumptions towards the inclusion of mechanisms, such as complex multi-trophic interactions, and their feedbacks on pathogen spread and disease persistence. The mechanisms presented here should be at the core of realistic predictive and preventive epidemiological models.}, language = {en} } @phdthesis{Nwosu2022, author = {Nwosu, Ebuka Canisius}, title = {Sedimentary DNA-based reconstruction of cyanobacterial communities from Lake Tiefer See, NE Germany, for the last 11,000 years}, doi = {10.25932/publishup-56359}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-563590}, school = {Universit{\"a}t Potsdam}, pages = {xxvi, 214}, year = {2022}, abstract = {Climate change and human-driven eutrophication promote the spread of harmful cyanobacteria blooms in lakes worldwide, which affects water quality and impairs the aquatic food chain. In recent times, sedimentary ancient DNA-based (sedaDNA) studies were used to probe how centuries of climate and environmental changes have affected cyanobacterial assemblages in temperate lakes. However, there is a lack of information on the consistency between sediment-deposited cyanobacteria communities versus those of the water column, and on the individual role of natural climatic changes versus human pressure on cyanobacteria community dynamics over multi-millennia time scales. Therefore, this thesis uses sedimentary ancient DNA of Lake Tiefer See in northeastern Germany to trace the deposition of cyanobacteria along the water column into the sediment, and to reconstruct cyanobacteria communities spanning the last 11,000 years using a set of molecular techniques including quantitative PCR, biomarkers, metabarcoding, and metagenome sequence analyses. The results of this thesis proved that cyanobacterial composition and species richness did not significantly differ among different water depths, sediment traps, and surface sediments. This means that the cyanobacterial community composition from the sediments reflects the water column communities. However, there is a skewed sediment deposition of different cyanobacteria groups because of DNA alteration and/or deterioration during transport along the water column to the sediment. Specifically, single filament taxa, such as Planktothrix, are poorly represented in sediments despite being abundant in the water column as shown by an additional study of the thesis on cyanobacteria seasonality. In contrast, aggregate-forming taxa, like Aphanizomenon, are relatively overrepresented in sediment although they are not abundant in the water column. These different deposition patterns of cyanobacteria taxa should be considered in future DNA-based paleolimnological investigations. The thesis also reveals a substantial increase in total cyanobacteria abundance during the Bronze Age which is not apparent in prior phases of the early to middle Holocene and is suggested to be caused by human farming, deforestation, and excessive nutrient addition to the lake. Not only cyanobacterial abundance was influenced by human activity but also cyanobacteria community composition differed significantly between phases of no, moderate, and intense human impact. The data presented in this thesis are the first on sedimentary cyanobacteria DNA since the early Holocene in a temperate lake. The results bring together archaeological, historical climatic, and limnological data with deep DNA-sequencing and paleoecology to reveal a legacy impact of human pressure on lake cyanobacteria populations dating back to approximately 4000 years.}, language = {en} } @phdthesis{Ramm2022, author = {Ramm, Timo}, title = {Entwicklung von Multiplex-Methoden zur Antik{\"o}rper-Charakterisierung und Validierung}, doi = {10.25932/publishup-56153}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-561531}, school = {Universit{\"a}t Potsdam}, pages = {XIV, 138}, year = {2022}, abstract = {Antik{\"o}rper werden in verschiedensten Bereichen, sowohl zu therapeutischen als auch zu diagnostischen und forschungsorientierten Zwecken verwendet. Vor der Verwendung des Antik{\"o}rpers bedarf es der Charakterisierung seiner Eigenschaften, in Bezug auf sein Epitop und sein Bindeverhalten gegen{\"u}ber dem Paratop. Gleichzeitig muss, in Abh{\"a}ngigkeit des Einsatzes, der Antik{\"o}rper, f{\"u}r den gew{\"u}nschten Gebrauch, validiert werden. Zu diesem Zweck wurden in der vorliegenden Arbeit Bead-basierte, multiplexe Testsysteme entworfen, ausgetestet und etabliert mit dem Ziel, eine einfache Screeningmethode zu entwickeln, um eine hohe Anzahl an Proben beziehungsweise Analyten gleichzeitig bestimmen zu k{\"o}nnen. Daf{\"u}r wurden drei verschiedene Herangehensweisen etabliert. So wurden ein phospho-PKA-Substrat Antik{\"o}rper, welcher phosphorylierte Bindemotive der PKA der Form RRxpS erkennt, gleichzeitig mit einer Reihe an Peptide getestet, welche Punktmutationen im Vergleich zur Konsensussequenz enthielten, um den Einfluss einzelner Aminos{\"a}uren auf die Bindung des Antik{\"o}rpers zu untersuchen. Es konnte im Multiplex gezeigt werden, dass die Unterschiede im Antik{\"o}rperbindungsverhalten in Abh{\"a}ngigkeit der Aminos{\"a}ure an verschiedenen P-Positionen detektierbar waren. Mit dem Bead-basierten Multiplexansatz konnten, durch Messungen von Konzentrationsreihen des Antik{\"o}rpers, Bindungskinetiken aufgenommen und diese mit bereits etablierten Methoden verglichen werden. Des Weiteren wurden verschiedene Antik{\"o}rper, welche essenzielle Bestandteile von Bead-basierten Testsystemen darstellten, validiert. Es wurden dabei verschiedene Antik{\"o}rper, welche spezifisch THC und CBD erkennen ausgetestet und anschließend ein kompetitiver Assay zur Detektion von THC und CBD in humanem Serum etabliert, und die Nachweisgrenzen bestimmt. Ferner sollten Pferdeseren von Tieren, welche am Sommerekzem leiden, auf ihren IgE-Gehalt hin bestimmt werden. Daf{\"u}r wurden relevante Proteine rekombinant hergestellt und durch Immobilisierung an Beads im Multiplex mit Serum inkubiert. Die spezifische Bindung des IgE an die Allergen sollte damit messbar gemacht werden k{\"o}nnen. F{\"u}r die Gesamtvalidierung des Testsystems wurden zuvor s{\"a}mtliche Einzelschritte einzeln validiert, um im Anschluss im multiplexen Screening zu vermessen. Die Nutzung von Bead-basierten Multiplexmessungen als eine Plattformtechnologie erleichtert die Charakterisierung von Antik{\"o}rpern sowie ihre Validierung f{\"u}r verschiedene Testsysteme.}, language = {de} } @phdthesis{Demin2022, author = {Demin, Paul}, title = {Blaulicht-aktivierbares Proteinexpressionssystem in Saccharomyces cerevisiae}, doi = {10.25932/publishup-55969}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-559696}, school = {Universit{\"a}t Potsdam}, pages = {127}, year = {2022}, abstract = {Synthetische Transkriptionsfaktoren bestehen wie nat{\"u}rliche Transkriptionsfaktoren aus einer DNA-Bindedom{\"a}ne, die sich spezifisch an die Bindestellensequenz vor dem Ziel-Gen anlagert, und einer Aktivierungsdom{\"a}ne, die die Transkriptionsmaschinerie rekrutiert, sodass das Zielgen exprimiert wird. Der Unterschied zu den nat{\"u}rlichen Transkriptionsfaktoren ist, sowohl dass die DNA-Bindedom{\"a}ne als auch die Aktivierungsdom{\"a}ne wirtsfremd sein k{\"o}nnen und dadurch k{\"u}nstliche Stoffwechselwege im Wirt, gr{\"o}ßtenteils chemisch, induziert werden k{\"o}nnen. Optogenetische synthetische Transkriptionsfaktoren, die hier entwickelt wurden, gehen einen Schritt weiter. Dabei ist die DNA-Bindedom{\"a}ne nicht mehr an die Aktivierungsdom{\"a}ne, sondern mit dem Blaulicht-Photorezeptor CRY2 gekoppelt. Die Aktivierungsdom{\"a}ne wurde mit dem Interaktionspartner CIB1 fusioniert. Unter Blaulichtbestrahlung dimerisieren CRY2 und CIB1 und damit einhergehend die beiden Dom{\"a}nen, sodass ein funktionsf{\"a}higer Transkriptionsfaktor entsteht. Dieses System wurde in die Saccharomyces cerevisiae genomisch integriert. Verifiziert wurde das konstruierte System mit Hilfe des Reporters yEGFP, welcher durchflusszytometrisch detektiert werden konnte. Es konnte gezeigt werden, dass die yEGFP Expression variabel gestaltet werden kann, indem unterschiedlich lange Blaulichtimpulse ausgesendet wurden, die DNA-Bindedom{\"a}ne, die Aktivierungsdom{\"a}ne oder die Anzahl der Bindestellen, an dem sich die DNA-Bindedom{\"a}ne anlagert, ver{\"a}ndert wurden. Um das System f{\"u}r industrielle Anwendungen attraktiv zu gestalten, wurde das System vom Deepwell-Maßstab auf Photobioreaktor-Maßstab hochskaliert. Außerdem erwies sich das Blaulichtsystem sowohl im Laborstamm YPH500 als auch im industriell oft verwendeten Hefestamm CEN.PK als funktional. Des Weiteren konnte ein industrierelevante Protein ebenso mit Hilfe des verifizierten Systems exprimiert werden. Schlussendlich konnte in dieser Arbeit das etablierte Blaulicht-System erfolgreich mit einem Rotlichtsystem kombiniert werden, was zuvor noch nicht beschrieben wurde.}, language = {de} } @phdthesis{Schulte2022, author = {Schulte, Luise}, title = {Dynamics of Larix (Mill.) species in Siberia during the last 50,000 years inferred from sedimentary ancient DNA}, doi = {10.25932/publishup-55878}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-558782}, school = {Universit{\"a}t Potsdam}, pages = {xi, 121}, year = {2022}, abstract = {The deciduous needle tree larch (Larix Mill.) covers more than 80\% of the Asian boreal forests. Only a few Larix species constitute the vast forests and these species differ markedly in their ecological traits, most importantly in their ability to grow on and stabilize underlying permafrost. The pronounced dominance of the summergreen larches makes the Asian boreal forests unique, as the rest of the northern hemisphere boreal forests is almost exclusively dominated by evergreen needle-leaf forests. Global warming is impacting the whole world but is especially pronounced in the arctic and boreal regions. Although adapted to extreme climatic conditions, larch forests are sensitive to varying climatic conditions. By their sheer size, changes in Asian larch forests as range shifts or changes in species composition and the resulting vegetation-climate feedbacks are of global relevance. It is however still uncertain if larch forests will persist under the ongoing warming climate or if they will be replaced by evergreen forests. It is therefore of great importance to understand how these ecosystems will react to future climate warmings and if they will maintain their dominance. One step in the better understanding of larch dynamics is to study how the vast dominant forests developed and why they only established in northern Asia. A second step is to study how the species reacted to past changes in the climate. The first objective of this thesis was to review and identify factors promoting Asian larch dominance. I achieved this by synthesizing and comparing reported larch occurrences and influencing components on the northern hemisphere continents in the present and in the past. The second objective was to find a possibility to directly study past Larix populations in Siberia and specifically their genetic variation, enabling the study of geographic movements. For this, I established chloroplast enrichment by hybridization capture from sedimentary ancient DNA (sedaDNA) isolated from lake sediment records. The third objective was to use the established method to track past larch populations, their glacial refugia during the Last Glacial Maximum (LGM) around 21,000 years before present (ka BP), and their post-glacial migration patterns. To study larch promoting factors, I compared the present state of larch species ranges, areas of dominance, their bioclimatic niches, and the distribution on different extents and thaw depths of permafrost. The species comparison showed that the bioclimatic niches greatly overlap between the American and Asian species and that it is only in the extremely continental climates in which only the Asian larch species can persist. I revealed that the area of dominance is strongly connected to permafrost extent but less linked to permafrost seasonal thaw depths. Comparisons of the paleorecord of larch between the continents suggest differences in the recolonization history. Outside of northern Asia and Alaska, glacial refugial populations of larch were confined to the southern regions and thus recolonization could only occur as migration from south to north. Alaskan larch populations could not establish wide-range dominant forest which could be related to their own genetically depletion as separated refugial population. In Asia, it is still unclear whether or not the northern refugial populations contributed and enhanced the postglacial colonization or whether they were replaced by populations invading from the south in the course of climate warming. Asian larch dominance is thus promoted partly by adaptions to extremely continental climates and by adaptations to grow on continuous permafrost but could be also connected to differences in glacial survival and recolonization history of Larix species. Except for extremely rare macrofossil findings of fossilized cones, traditional methods to study past vegetation are not able to distinguish between larch species or populations. Within the scope of this thesis, I therefore established a method to retrieve genetic information of past larch populations to distinguish between species. Using the Larix chloroplast genome as target, I successfully applied the method of DNA target enrichment by hybridization capture on sedaDNA samples from lake records and showed that it is able to distinguish between larch species. I then used the method on samples from lake records from across Siberia dating back up to 50 ka BP. The results allowed me to address the question of glacial survival and post-glacial recolonization mode in Siberian larch species. The analyzed pattern showed that LGM refugia were almost exclusively constituted by L. gmelinii, even in sites of current L. sibirica distribution. For included study sites, L. sibirica migrated into its extant northern distribution area only in the Holocene. Consequently, the post-glacial recolonization of L. sibirica was not enhanced by northern glacial refugia. In case of sites in extant distribution area of L. gmelinii, the absence of a genetic turn-over point to a continuous population rather than an invasion of southern refugia. The results suggest that climate has a strong influence on the distribution of Larix species and that species may also respond differently to future climate warming. Because species differ in their ecological characteristics, species distribution is also relevant with respect to further feedbacks between vegetation and climate. With this thesis, I give an overview of present and past larch occurrences and evaluate which factors promote their dominance. Furthermore, I provide the tools to study past Larix species and give first important insights into the glacial history of Larix populations.}, language = {en} } @phdthesis{Kahl2022, author = {Kahl, Sandra}, title = {Evolutionary adaptive responses to rapid climate change in plants}, doi = {10.25932/publishup-55648}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-556483}, school = {Universit{\"a}t Potsdam}, pages = {127}, year = {2022}, abstract = {The ongoing climate change is altering the living conditions for many organisms on this planet at an unprecedented pace. Hence, it is crucial for the survival of species to adapt to these changing conditions. In this dissertation Silene vulgaris is used as a model organism to understand the adaption strategies of widely distributed plant species to the current climate change. Especially plant species that possess a wide geographic range are expected to have a high phenotypic plasticity or to show genetic differentiation in response to the different climate conditions they grow in. However, they are often underrepresented in research. In the greenhouse experiment presented in this thesis, I examined the phenotypic responses and plasticity in S. vulgaris to estimate its' adaptation potential. Seeds from 25 wild European populations were collected along a latitudinal gradient and grown in a greenhouse under three different precipitation (65 mm, 75 mm, 90 mm) and two different temperature regimes (18°C, 21°C) that resembled a possible climate change scenario for central Europe. Afterwards different biomass and fecundity-related plant traits were measured. The treatments significantly influenced the plants but did not reveal a latitudinal difference in response to climate treatments for most plant traits. The number of flowers per individual however, showed a stronger plasticity in northern European populations (e.g., Swedish populations) where numbers decreased more drastically with increased temperature and decreased precipitation. To gain an even deeper understanding of the adaptation of S. vulgaris to climate change it is also important to reveal the underlying phylogeny of the sampled populations. Therefore, I analysed their population genetic structure through whole genome sequencing via ddRAD. The sequencing revealed three major genetic clusters in the S. vulgaris populations sampled in Europe: one cluster comprised Southern European populations, one cluster Western European populations and another cluster contained central European populations. A following analysis of experimental trait responses among the clusters to the climate-change scenario showed that the genetic clusters significantly differed in biomass-related traits and in the days to flowering. However, half of the traits showed parallel response patterns to the experimental climate-change scenario. In addition to the potential geographic and genetic adaptation differences to climate change this dissertation also deals with the response differences between the sexes in S. vulgaris. As a gynodioecious species populations of S. vulgaris consist of female and hermaphrodite individuals and the sexes can differ in their morphological traits which is known as sexual dimorphism. As climate change is becoming an important factor influencing plant morphology it remains unclear if and how different sexes may respond in sexually dimorphic species. To examine this question the sex of each individual plant was determined during the greenhouse experiment and the measured plant traits were analysed accordingly. In general, hermaphrodites had a higher number of flowers but a lower number of leaves than females. With regards to the climate change treatment, I found that hermaphrodites showed a milder negative response to higher temperatures in the number of flowers produced and in specific leaf area (SLA) compared to females. Synthesis - The significant treatment response in Silene vulgaris, independent of population origin in most traits suggests a high degree of universal phenotypic plasticity. Also, the three European intraspecific genetic lineages detected showed comparable parallel response patterns in half of the traits suggesting considerable phenotypic plasticity. Hence, plasticity might represent a possible adaptation strategy of this widely distributed species during ongoing and future climatic changes. The results on sexual dimorphism show that females and hermaphrodites are differing mainly in their number of flowers and females are affected more strongly by the experimental climate-change scenario. These results provide a solid knowledge basis on the sexual dimorphism in S. vulgaris under climate change, but further research is needed to determine the long-term impact on the breeding system for the species. In summary this dissertation provides a comprehensive insight into the adaptation mechanisms and consequences of a widely distributed and gynodioecious plant species and leverages our understanding of the impact of anthropogenic climate change on plants.}, language = {en} } @phdthesis{Korovila2022, author = {Korovila, Ioanna}, title = {Role of proteolytic systems in lipotoxicity-induced hepatocellular damage}, doi = {10.25932/publishup-55238}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-552385}, school = {Universit{\"a}t Potsdam}, pages = {117}, year = {2022}, abstract = {Scope: Several studies show that excessive lipid intake can cause hepatic steatosis. To investigate lipotoxicity on cellular level, palmitate (PA) is often used to highly increase lipid droplets (LDs). One way to remove LDs is autophagy, while it is controversially discussed if autophagy is also affected by PA. It is aimed to investigate whether PA-induced LD accumulation can impair autophagy and punicalagin, a natural autophagy inducer from pomegranate, can improve it. Methods and results: To verify the role of autophagy in LD degradation, HepG2 cells are treated with PA and analyzed for LD and perilipin 2 content in presence of autophagy inducer Torin 1 and inhibitor 3-Methyladenine. PA alone seems to initially induce autophagy-related proteins but impairs autophagic-flux in a time-dependent manner, considering 6 and 24 h PA. To examine whether punicalagin can prevent autophagy impairment, cells are cotreated for 24 h with PA and punicalagin. Results show that punicalagin preserves expression of autophagy-related proteins and autophagic flux, while simultaneously decreasing LDs and perilipin 2. Conclusion: Data provide new insights into the role of PA-induced excessive LD content on autophagy and suggest autophagy-inducing properties of punicalagin, indicating that punicalagin can be a health-beneficial compound for future research on lipotoxicity in liver.}, language = {en} } @phdthesis{Kath2022, author = {Kath, Nadja Jeanette}, title = {Functional traits determine biomass dynamics, coexistence and energetics in plankton food webs}, doi = {10.25932/publishup-55123}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-551239}, school = {Universit{\"a}t Potsdam}, pages = {197}, year = {2022}, abstract = {Plankton food webs are the basis of marine and limnetic ecosystems. Especially aquatic ecosystems of high biodiversity provide important ecosystem services for humankind as providers of food, coastal protection, climate regulation, and tourism. Understanding the dynamics of biomass and coexistence in these food webs is a first step to understanding the ecosystems. It also lays the foundation for the development of management strategies for the maintenance of the marine and freshwater biodiversity despite anthropogenic influences. Natural food webs are highly complex, and thus often equally complex methods are needed to analyse and understand them well. Models can help to do so as they depict simplified parts of reality. In the attempt to get a broader understanding of the complex food webs, diverse methods are used to investigate different questions. In my first project, we compared the energetics of a food chain in two versions of an allometric trophic network model. In particular, we solved the problem of unrealistically high trophic transfer efficiencies (up to 70\%) by accounting for both basal respiration and activity respiration, which decreased the trophic transfer efficiency to realistic values of ≤30\%. Next in my second project I turned to plankton food webs and especially phytoplankton traits. Investigating a long-term data set from Lake Constance we found evidence for a trade-off between defence and growth rate in this natural phytoplankton community. I continued working with this data set in my third project focusing on ciliates, the main grazer of phytoplankton in spring. Boosted regression trees revealed that temperature and predators have the highest influence on net growth rates of ciliates. We finally investigated in my fourth project a food web model inspired by ciliates to explore the coexistence of plastic competitors and to study the new concept of maladaptive switching, which revealed some drawbacks of plasticity: faster adaptation led to higher maladaptive switching towards undefended phenotypes which reduced autotroph biomass and coexistence and increased consumer biomass. It became obvious that even well-established models should be critically questioned as it is important not to forget reality on the way to a simplistic model. The results showed furthermore that long-term data sets are necessary as they can help to disentangle complex natural processes. Last, one should keep in mind that the interplay between models and experiments/ field data can deliver fruitful insights about our complex world.}, language = {en} } @phdthesis{Fischer2022, author = {Fischer, Axel}, title = {Investigating the impact of genomic compartments contributing to non-Mendelian inheritance based on high throughput sequencing data}, doi = {10.25932/publishup-54900}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-549001}, school = {Universit{\"a}t Potsdam}, pages = {vii, 122}, year = {2022}, abstract = {More than a century ago the phenomenon of non-Mendelian inheritance (NMI), defined as any type of inheritance pattern in which traits do not segregate in accordance with Mendel's laws, was first reported. In the plant kingdom three genomic compartments, the nucleus, chloroplast, and mitochondrion, can participate in such a phenomenon. High-throughput sequencing (HTS) proved to be a key technology to investigate NMI phenomena by assembling and/or resequencing entire genomes. However, generation, analysis and interpretation of such datasets remain challenging by the multi-layered biological complexity. To advance our knowledge in the field of NMI, I conducted three studies involving different HTS technologies and implemented two new algorithms to analyze them. In the first study I implemented a novel post-assembly pipeline, called Semi-Automated Graph-Based Assembly Curator (SAGBAC), which visualizes non-graph-based assemblies as graphs, identifies recombinogenic repeat pairs (RRPs), and reconstructs plant mitochondrial genomes (PMG) in a semiautomated workflow. We applied this pipeline to assemblies of three Oenothera species resulting in a spatially folded and circularized model. This model was confirmed by PCR and Southern blot analyses and was used to predict a defined set of 70 PMG isoforms. With Illumina Mate Pair and PacBio RSII data, the stoichiometry of the RRPs was determined quantitatively differing up to three-fold. In the second study I developed a post-multiple sequence alignment algorithm, called correlation mapping (CM), which correlates segment-wise numbers of nucleotide changes to a numeric ascertainable phenotype. We applied this algorithm to 14 wild type and 18 mutagenized plastome assemblies within the Oenothera genus and identified two genes, accD and ycf2 that may cause the competitive behavior of plastid genotypes as plastids can be biparental inherited in Oenothera. Moreover, lipid composition of the plastid envelope membrane is affected by polymorphisms within these two genes. For the third study, I programmed a pipeline to investigate a NMI phenomenon, known as paramutation, in tomato by analyzing DNA and bisulfite sequencing data as well as microarray data. We identified the responsible gene (Solyc02g0005200) and were able to fully repress its caused phenotype by heterologous complementation with a paramutation insensitive transgene of the Arabidopsis thaliana orthologue. Additionally, a suppressor mutant shows a globally altered DNA methylation pattern and carries a large deletion leading to a gene fusion involving a histone deacetylase. In conclusion, my developed and implemented algorithms and data analysis pipelines are suitable to investigate NMI and led to novel insights about such phenomena by reconstructing PMGs (SAGBAC) as a requirement to study mitochondria-associated phenotypes, by identifying genes (CM) causing interplastidial competition as well by applying a DNA/Bisulfite-seq analysis pipeline to shed light in a transgenerational epigenetic inheritance phenomenon.}, language = {en} } @phdthesis{Shevtsova2022, author = {Shevtsova, Iuliia}, title = {Recent and future vegetation change in the treeline region of Chukotka (NE Russia) inferred from field data, satellite data and modelling}, doi = {10.25932/publishup-54845}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548452}, school = {Universit{\"a}t Potsdam}, pages = {149}, year = {2022}, abstract = {Vegetation change at high latitudes is one of the central issues nowadays with respect to ongoing climate changes and triggered potential feedback. At high latitude ecosystems, the expected changes include boreal treeline advance, compositional, phenological, physiological (plants), biomass (phytomass) and productivity changes. However, the rate and the extent of the changes under climate change are yet poorly understood and projections are necessary for effective adaptive strategies and forehanded minimisation of the possible negative feedbacks. The vegetation itself and environmental conditions, which are playing a great role in its development and distribution are diverse throughout the Subarctic to the Arctic. Among the least investigated areas is central Chukotka in North-Eastern Siberia, Russia. Chukotka has mountainous terrain and a wide variety of vegetation types on the gradient from treeless tundra to northern taiga forests. The treeline there in contrast to subarctic North America and north-western and central Siberia is represented by a deciduous conifer, Larix cajanderi Mayr. The vegetation varies from prostrate lichen Dryas octopetala L. tundra to open graminoid (hummock and non-hummock) tundra to tall Pinus pumila (Pall.) Regel shrublands to sparse and dense larch forests. Hence, this thesis presents investigations on recent compositional and above-ground biomass (AGB) changes, as well as potential future changes in AGB in central Chukotka. The aim is to assess how tundra-taiga vegetation develops under changing climate conditions particularly in Fareast Russia, central Chukotka. Therefore, three main research questions were considered: 1) What changes in vegetation composition have recently occurred in central Chukotka? 2) How have the above-ground biomass AGB rates and distribution changed in central Chukotka? 3) What are the spatial dynamics and rates of tree AGB change in the upcoming millennia in the northern tundra-taiga of central Chukotka? Remote sensing provides information on the spatial and temporal variability of vegetation. I used Landsat satellite data together with field data (foliage projective cover and AGB) from two expeditions in 2016 and 2018 to Chukotka to upscale vegetation types and AGB for the study area. More specifically, I used Landsat spectral indices (Normalised Difference Vegetation Index (NDVI), Normalised Difference Water Index (NDWI) and Normalised Difference Snow Index (NDSI)) and constrained ordination (Redundancy analysis, RDA) for further k-means-based land-cover classification and general additive model (GAM)-based AGB maps for 2000/2001/2002 and 2016/2017. I also used Tandem-X DEM data for a topographical correction of the Landsat satellite data and to derive slope, aspect, and Topographical Wetness Index (TWI) data for forecasting AGB. Firstly, in 2016, taxa-specific projective cover data were collected during a Russian-German expedition. I processed the field data and coupled them with Landsat spectral Indices in the RDA model that was used for k-means classification. I could establish four meaningful land-cover classes: (1) larch closed-canopy forest, (2) forest tundra and shrub tundra, (3) graminoid tundra and (4) prostrate herb tundra and barren areas, and accordingly, I produced the land cover maps for 2000/2001/2002 and 2016/20017. Changes in land-cover classes between the beginning of the century (2000/2001/2002) and the present time (2016/2017) were estimated and interpreted as recent compositional changes in central Chukotka. The transition from graminoid tundra to forest tundra and shrub tundra was interpreted as shrubification and amounts to a 20\% area increase in the tundra-taiga zone and 40\% area increase in the northern taiga. Major contributors of shrubification are alder, dwarf birch and some species of the heather family. Land-cover change from the forest tundra and shrub tundra class to the larch closed-canopy forest class is interpreted as tree infilling and is notable in the northern taiga. We find almost no land-cover changes in the present treeless tundra. Secondly, total AGB state and change were investigated for the same areas. In addition to the total vegetation AGB, I provided estimations for the different taxa present at the field sites. As an outcome, AGB in the study region of central Chukotka ranged from 0 kg m-2 at barren areas to 16 kg m-2 in closed-canopy forests with the larch trees contributing the highest. A comparison of changes in AGB within the investigated period from 2000 to 2016 shows that the greatest changes (up to 1.25 kg m 2 yr 1) occurred in the northern taiga and in areas where land cover changed to larch closed-canopy forest. Our estimations indicate a general increase in total AGB throughout the investigated tundra-taiga and northern taiga, whereas the tundra showed no evidence of change in AGB within the 15 years from 2002 to 2017. In the third manuscript, potential future AGB changes were estimated based on the results of simulations of the individual-based spatially explicit vegetation model LAVESI using different climate scenarios, depending on Representative Concentration Pathways (RCPs) RCP 2.6, RCP 4.5 and RCP 8.5 with or without cooling after 2300 CE. LAVESI-based AGB was simulated for the current state until 3000 CE for the northern tundra-taiga study area for larch species because we expect the most notable changes to occur will be associated with forest expansion in the treeline ecotone. The spatial distribution and current state of tree AGB was validated against AGB field data, AGB extracted from Landsat satellite data and a high spatial resolution image with distinctive trees visible. The simulation results are indicating differences in tree AGB dynamics plot wise, depending on the distance to the current treeline. The simulated tree AGB dynamics are in concordance with fundamental ecological (emigrational and successional) processes: tree stand formation in simulated results starts with seed dispersion, tree stand establishment, tree stand densification and episodic thinning. Our results suggest mostly densification of existing tree stands in the study region within the current century in the study region and a lagged forest expansion (up to 39\% of total area in the RCP 8.5) under all considered climate scenarios without cooling in different local areas depending on the closeness to the current treeline. In scenarios with cooling air temperature after 2300 CE, forests stopped expanding at 2300 CE (up to 10\%, RCP 8.5) and then gradually retreated to their pre-21st century position. The average tree AGB rates of increase are the strongest in the first 300 years of the 21st century. The rates depend on the RCP scenario, where the highest are as expected under RCP 8.5. Overall, this interdisciplinary thesis shows a successful integration of field data, satellite data and modelling for tracking recent and predicting future vegetation changes in mountainous subarctic regions. The obtained results are unique for the focus area in central Chukotka and overall, for mountainous high latitude ecosystems.}, language = {en} } @phdthesis{Pitzen2022, author = {Pitzen, Valentin}, title = {Weitergef{\"u}hrte funktionelle Charakterisierung des centrosomalen Proteins Cep192 und Untersuchung der Topologie des Centrosoms in Dictyostelium Am{\"o}ben}, doi = {10.25932/publishup-54889}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548891}, school = {Universit{\"a}t Potsdam}, pages = {XI, 104}, year = {2022}, abstract = {Das Centrosom von Dictyostelium ist acentriol{\"a}r aufgebaut, misst ca. 500 nm und besteht aus einer dreischichten Core-Struktur mit umgebender Corona, an der Mikrotubuli nukleieren. In dieser Arbeit wurden das centrosomale Protein Cep192 und m{\"o}gliche Interaktionspartner am Centrosom eingehend untersucht. Die einleitende Lokalisationsuntersuchung von Cep192 ergab, dass es w{\"a}hrend der gesamten Mitose an den Spindelpolen lokalisiert und im Vergleich zu den anderen Strukturproteinen der Core-Struktur am st{\"a}rksten exprimiert ist. Die dauerhafte Lokalisation an den Spindelpolen w{\"a}hrend der Mitose wird f{\"u}r Proteine angenommen, die in den beiden identisch aufgebauten {\"a}ußeren Core-Schichten lokalisieren, die das mitotische Centrosom formen. Ein Knockdown von Cep192 f{\"u}hrte zur Ausbildung von {\"u}berz{\"a}hligen Mikrotubuli-organisierenden Zentren (MTOC) sowie zu einer leicht erh{\"o}hten Ploidie. Deshalb wird eine Destabilisierung des Centrosoms durch die verminderte Cep192-Expression angenommen. An Cep192 wurden zwei kleine Tags, der SpotH6- und BioH6-Tag, etabliert, die mit kleinen fluoreszierenden Nachweiskonjugaten markiert werden konnten. Mit den so getagten Proteinen konnte die hochaufl{\"o}sende Expansion Microscopy f{\"u}r das Centrosom optimiert werden und die Core-Struktur erstmals proteinspezifisch in der Fluoreszenzmikroskopie dargestellt werden. Cep192 lokalisiert dabei in den {\"a}ußeren Core-Schichten. Die kombinierte Markierung von Cep192 und den centrosomalen Proteinen CP39 und CP91 in der Expansion Microscopy erlaubte die Darstellung des dreischichtigen Aufbaus der centrosomalen Core-Struktur, wobei CP39 und CP91 zwischen Cep192 in der inneren Core-Schicht lokalisieren. Auch die Corona wurde in der Expansion Microscopy untersucht: Das Corona-Protein CDK5RAP2 lokalisiert in r{\"a}umlicher N{\"a}he zu Cep192 in der inneren Corona. Ein Vergleich der Corona-Proteine CDK5RAP2, CP148 und CP224 in der Expansion Microscopy ergab unterscheidbare Sublokalisationen der Proteine innerhalb der Corona und relativ zur Core-Struktur. In Biotinylierungsassays mit den centrosomalen Core-Proteinen CP39 und CP91 sowie des Corona-Proteins CDK5RAP2 konnte Cep192 als m{\"o}glicher Interaktionspartner identifiziert werden. Die Ergebnisse dieser Arbeit zeigen die wichtige Funktion des Proteins Cep192 im Dictyostelium-Centrosom und erm{\"o}glichen durch die Kombination aus Biotinylierungsassays und Expansion Microscopy der untersuchten Proteine ein verbessertes Verst{\"a}ndnis der Topologie des Centrosoms.}, language = {de} } @phdthesis{Welsch2022, author = {Welsch, Maryna}, title = {Investigation of the stress tolerance regulatory network integration of the NAC transcription factor JUNGBRUNNEN1 (JUB1)}, doi = {10.25932/publishup-54731}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-547310}, school = {Universit{\"a}t Potsdam}, pages = {XIII, 116}, year = {2022}, abstract = {The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) is an important negative regulator of plant senescence, as well as of gibberellic acid (GA) and brassinosteroid (BR) biosynthesis in Arabidopsis thaliana. Overexpression of JUB1 promotes longevity and enhances tolerance to drought and other abiotic stresses. A similar role of JUB1 has been observed in other plant species, including tomato and banana. Our data show that JUB1 overexpressors (JUB1-OXs) accumulate higher levels of proline than WT plants under control conditions, during the onset of drought stress, and thereafter. We identified that overexpression of JUB1 induces key proline biosynthesis and suppresses key proline degradation genes. Furthermore, bZIP63, the transcription factor involved in proline metabolism, was identified as a novel downstream target of JUB1 by Yeast One-Hybrid (Y1H) analysis and Chromatin immunoprecipitation (ChIP). However, based on Electrophoretic Mobility Shift Assay (EMSA), direct binding of JUB1 to bZIP63 could not be confirmed. Our data indicate that JUB1-OX plants exhibit reduced stomatal conductance under control conditions. However, selective overexpression of JUB1 in guard cells did not improve drought stress tolerance in Arabidopsis. Moreover, the drought-tolerant phenotype of JUB1 overexpressors does not solely depend on the transcriptional control of the DREB2A gene. Thus, our data suggest that JUB1 confers tolerance to drought stress by regulating multiple components. Until today, none of the previous studies on JUB1´s regulatory network focused on identifying protein-protein interactions. We, therefore, performed a yeast two-hybrid screen (Y2H) which identified several protein interactors of JUB1, two of which are the calcium-binding proteins CaM1 and CaM4. Both proteins interact with JUB1 in the nucleus of Arabidopsis protoplasts. Moreover, JUB1 is expressed with CaM1 and CaM4 under the same conditions. Since CaM1.1 and CaM4.1 encode proteins with identical amino acid sequences, all further experiments were performed with constructs involving the CaM4 coding sequence. Our data show that JUB1 harbors multiple CaM-binding sites, which are localized in both the N-terminal and C-terminal regions of the protein. One of the CaM-binding sites, localized in the DNA-binding domain of JUB1, was identified as a functional CaM-binding site since its mutation strongly reduced the binding of CaM4 to JUB1. Furthermore, JUB1 transactivates expression of the stress-related gene DREB2A in mesophyll cells; this effect is significantly reduced when the calcium-binding protein CaM4 is expressed as well. Overexpression of both genes in Arabidopsis results in early senescence observed through lower chlorophyll content and an enhanced expression of senescence-associated genes (SAGs) when compared with single JUB1 overexpressors. Our data also show that JUB1 and CaM4 proteins interact in senescent leaves, which have increased Ca2+ levels when compared to young leaves. Collectively, our data indicate that JUB1 activity towards its downstream targets is fine-tuned by calcium-binding proteins during leaf senescence.}, language = {en} } @phdthesis{WijesinghaAhchige2022, author = {Wijesingha Ahchige, Micha}, title = {Canalization of plant metabolism and yield}, doi = {10.25932/publishup-54884}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548844}, school = {Universit{\"a}t Potsdam}, pages = {VIII, 160}, year = {2022}, abstract = {Plant metabolism is the main process of converting assimilated carbon to different crucial compounds for plant growth and therefore crop yield, which makes it an important research topic. Although major advances in understanding genetic principles contributing to metabolism and yield have been made, little is known about the genetics responsible for trait variation or canalization although the concepts have been known for a long time. In light of a growing global population and progressing climate change, understanding canalization of metabolism and yield seems ever-more important to ensure food security. Our group has recently found canalization metabolite quantitative trait loci (cmQTL) for tomato fruit metabolism, showing that the concept of canalization applies on metabolism. In this work two approaches to investigate plant metabolic canalization and one approach to investigate yield canalization are presented. In the first project, primary and secondary metabolic data from Arabidopsis thaliana and Phaseolus vulgaris leaf material, obtained from plants grown under different conditions was used to calculate cross-environment coefficient of variations or fold-changes of metabolite levels per genotype and used as input for genome wide association studies. While primary metabolites have lower CV across conditions and show few and mostly weak associations to genomic regions, secondary metabolites have higher CV and show more, strong metabolite to genome associations. As candidate genes, both potential regulatory genes as well as metabolic genes, can be found, albeit most metabolic genes are rarely directly related to the target metabolites, suggesting a role for both potential regulatory mechanisms as well as metabolic network structure for canalization of metabolism. In the second project, candidate genes of the Solanum lycopersicum cmQTL mapping are selected and CRISPR/Cas9-mediated gene-edited tomato lines are created, to validate the genes role in canalization of metabolism. Obtained mutants appeared to either have strong aberrant developmental phenotypes or appear wild type-like. One phenotypically inconspicuous mutant of a pantothenate kinase, selected as candidate for malic acid canalization shows a significant increase of CV across different watering conditions. Another such mutant of a protein putatively involved in amino acid transport, selected as candidate for phenylalanine canalization shows a similar tendency to increased CV without statistical significance. This potential role of two genes involved in metabolism supports the hypothesis of structural relevance of metabolism for its own stability. In the third project, a mutant for a putative disulfide isomerase, important for thylakoid biogenesis, is characterized by a multi-omics approach. The mutant was characterized previously in a yield stability screening and showed a variegated leaf phenotype, ranging from green leaves with wild type levels of chlorophyll over differently patterned variegated to completely white leaves almost completely devoid of photosynthetic pigments. White mutant leaves show wild type transcript levels of photosystem assembly factors, with the exception of ELIP and DEG orthologs indicating a stagnation at an etioplast to chloroplast transition state. Green mutant leaves show an upregulation of these assembly factors, possibly acting as overcompensation for partially defective disulfide isomerase, which seems sufficient for proper chloroplast development as confirmed by a wild type-like proteome. Likely as a result of this phenotype, a general stress response, a shift to a sink-like tissue and abnormal thylakoid membranes, strongly alter the metabolic profile of white mutant leaves. As the severity and pattern of variegation varies from plant to plant and may be effected by external factors, the effect on yield instability, may be a cause of a decanalized ability to fully exploit the whole leaf surface area for photosynthetic activity.}, language = {en} } @phdthesis{Moradian2022, author = {Moradian, Hanieh}, title = {Modulation of human macrophage activity by mRNA-mediated genetic engineering}, doi = {10.25932/publishup-54857}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548579}, school = {Universit{\"a}t Potsdam}, pages = {IX, 148}, year = {2022}, abstract = {Macrophages play an integral role for the innate immune system. It is critically important for basic research and therapeutic applications to find approaches to potentially modulate their function as the first line of defense. Transient genetic engineering via delivery of synthetic mRNA can serve for such purposes as a robust, reliable and safe technology to modulate macrophage functions. However, a major drawback particularly in the transfection of sensitive immune cells such as macrophages is the immunogenicity of exogenous IVT-mRNAs. Consequently, the direct modulation of human macrophage activity by mRNA-mediated genetic engineering was the aim of this work. The synthetic mRNA can instruct macrophages to synthesize specific target proteins, which can steer macrophage activity in a tailored fashion. Thus, the focus of this dissertation was to identify parameters triggering unwanted immune activation of macrophages, and to find approaches to minimize such effects. When comparing different carrier types as well as mRNA chemistries, the latter had unequivocally a more pronounced impact on activation of human macrophages and monocytes. Exploratory investigations revealed that the choice of nucleoside chemistry, particularly of modified uridine, plays a crucial role for IVT-mRNA-induced immune activation, in a dose-dependent fashion. Additionally, the contribution of the various 5' cap structures tested was only minor. Moreover, to address the technical aspects of the delivery of multiple genes as often mandatory for advanced gene delivery studies, two different strategies of payload design were investigated, namely "bicistronic" delivery and "monocistronic" co-delivery. The side-by-side comparison of mRNA co-delivery via a bicistronic design (two genes, one mRNA) with a monocistronic design (two gene, two mRNAs) unexpectedly revealed that, despite the intrinsic equimolar nature of the bicistronic approach, it was outperformed by the monocistronic approach in terms of reliable co-expression when quantified on the single cell level. Overall, the incorporation of chemical modifications into IVT-mRNA by using respective building blocks, primarily with the aim to minimize immune activation as exemplified in this thesis, has the potential to facilitate the selection of the proper mRNA chemistry to address specific biological and clinical challenges. The technological aspects of gene delivery evaluated and validated by the quantitative methods allowed us to shed light on crucial process parameters and mRNA design criteria, required for reliable co-expression schemes of IVT-mRNA delivery.}, language = {en} } @phdthesis{Dahmani2021, author = {Dahmani, Ismail}, title = {Influenza A virus matrix protein M1}, doi = {10.25932/publishup-52740}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-527409}, school = {Universit{\"a}t Potsdam}, pages = {XI, 147}, year = {2021}, abstract = {Influenza A virus (IAV) is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. During the viral assembly process in the infected cells, the plasma membrane (PM) has to bend in localized regions into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. M1 is the most abundant protein in IAV particles. It plays an important role in virus assembly and budding at the PM. M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. However, the details of M1 interactions with the cellular PM, as well as M1-mediated membrane bending at the budozone, have not been clarified. In this work, we used several experimental approaches to analyze M1-lipids and M1-M1 interactions. By performing SPR analysis, we quantified membrane association for full-length M1 and different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region). This allowed us to obtain novel information on the protein regions mediating M1 binding to membranes. By using fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), and three-dimensional (3D) tomography (cryo-ET), we showed that M1 is indeed able to cause membrane deformation on vesicles containing negatively-charged lipids, in the absence of other viral components. Further, sFCS analysis proved that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required to alter the bilayer three-dimensional structure through the formation of a protein scaffold. Finally, to mimic the budding mechanism in cells that arise by the lateral organization of the virus membrane components on lipid raft domains, we created vesicles with lipid domains. Our results showed that local binding of M1 to spatial confined acidic lipids within membrane domains of vesicles led to local M1 inward curvature.}, language = {en} } @phdthesis{Krumbholz2021, author = {Krumbholz, Julia}, title = {Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme}, doi = {10.25932/publishup-54024}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-540240}, school = {Universit{\"a}t Potsdam}, pages = {xxiii, 187}, year = {2021}, abstract = {Specialized metabolites, so-called natural products, are produced by a variety of different organisms, including bacteria and fungi. Due to their wide range of different biological activities, including pharmaceutical relevant properties, microbial natural products are an important source for drug development. They are encoded by biosynthetic gene clusters (BGCs), which are a group of locally clustered genes. By screening genomic data for genes encoding typical core biosynthetic enzymes, modern bioinformatical approaches are able to predict a wide range of BGCs. To date, only a small fraction of the predicted BGCs have their associated products identified. The phylum of the cyanobacteria has been shown to be a prolific, but largely untapped source for natural products. Especially multicellular cyanobacterial genera, like Nostoc, harbor a high amount of BGCs in their genomes. A main goal of this study was to develop new concepts for the discovery of natural products in cyanobacteria. Due to its diverse setup of orphan BGCs and its amenability to genetic manipulation, Nostoc punctiforme PCC 73102 (N. punctiforme) appeared to be a promising candidate to be established as a model organism for natural product discovery in cyanobacteria. By utilizing a combination of genome-mining, bioactivity-screening, variations of culture conditions, as well as metabolic engineering, not only two new polyketides were discovered, but also first-time insights into the regulation of the specialized metabolism in N. punctiforme were gained during this study. The cultivation of N. punctiforme to very high densities by utilizing increasing light intensities and CO2 levels, led to an enhanced metabolite production, causing rather complex metabolite extracts. By utilizing a library of CFP reporter mutant strains, each strain reporting for one of the predicted BGCs, it was shown that eight out of 15 BGCs were upregulated under high density (HD) cultivation conditions. Furthermore, it could be demonstrated that the supernatant of an HD culture can increase the expression of four of the influenced BGCs, even under conventional cultivation conditions. This led to the hypothesis that a chemical mediator encoded by one of the affected BGCs is accumulating in the HD supernatant and is able to increase the expression of other BGCs as part of a cell-density dependent regulatory circuit. To identify which of the BGCs could be a main trigger of the presumed regulatory circuit, it was tried to activate four BGCs (pks1, pks2, ripp3, ripp4) selectively by overexpression of putative pathway-specific regulatory genes that were found inside the gene clusters. Transcriptional analysis of the mutants revealed that only the mutant strain targeting the pks1 BGC, called AraC_PKS1, was able to upregulate the expression of its associated BGC. From an RNA sequencing study of the AraC_PKS1 mutant strain, it was discovered that beside pks1, the orphan BGCs ripp3 and ripp4 were also upregulated in the mutant strain. Furthermore, it was observed that secondary metabolite production in the AraC_PKS1 mutant strain is further enhanced under high-light and high-CO2 cultivation conditions. The increased production of the pks1 regulator NvlA also had an impact on other regulatory factors, including sigma factors and the RNA chaperone Hfq. Analysis of the AraC_PKS1 cell and supernatant extracts led to the discovery of two novel polyketides, nostoclide and nostovalerolactone, both encoded by the pks1 BGC. Addition of the polyketides to N. punctiforme WT demonstrated that the pks1-derived compounds are able to partly reproduce the effects on secondary metabolite production found in the AraC_PKS1 mutant strain. This indicates that both compounds are acting as extracellular signaling factors as part of a regulatory network. Since not all transcriptional effects that were found in the AraC_PKS1 mutant strain could be reproduced by the pks1 products, it can be assumed that the regulator NvlA has a global effect and is not exclusively specific to the pks1 pathway. This study was the first to use a putative pathway specific regulator for the specific activation of BGC expression in cyanobacteria. This strategy did not only lead to the detection of two novel polyketides, it also gave first-time insights into the regulatory mechanism of the specialized metabolism in N. punctiforme. This study illustrates that understanding regulatory pathways can aid in the discovery of novel natural products. The findings of this study can guide the design of new screening strategies for bioactive compounds in cyanobacteria and help to develop high-titer production platforms for cyanobacterial natural products.}, language = {en} } @phdthesis{Sun2022, author = {Sun, Xianlei}, title = {Elasticity of fiber meshes derived from multiblock copolymers influences cell behaviors}, doi = {10.25932/publishup-53528}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-535285}, school = {Universit{\"a}t Potsdam}, pages = {96}, year = {2022}, abstract = {Objective: The behaviors of endothelial cells or mesenchymal stem cells are remarkably influenced by the mechanical properties of their surrounding microenvironments. Here, electrospun fiber meshes containing various mechanical characteristics were developed from polyetheresterurethane (PEEU) copolymers. The goal of this study was to explore how fiber mesh stiffness affected endothelial cell shape, growth, migration, and angiogenic potential of endothelial cells. Furthermore, the effects of the E-modulus of fiber meshes on human adipose-derived stem cells (hADSCs) osteogenic potential was investigated. Methods: Polyesteretherurethane (PEEU) polymers with various poly(p-dioxanone) (PPDO) to poly (ε-caprolactone) (PCL) weight percentages (40 wt.\%, 50 wt.\%, 60 wt.\%, and 70 wt.\%) were synthesized, termed PEEU40, PEEU50, PEEU60, and PEEU70, accordingly. The electrospinning method was used for the preparation of PEEU fiber meshes. The effects of PEEU fiber meshes with varying elasticities on the human umbilical vein endothelial cells (HUVECs) shape, growth, migration and angiogenic potential were characterized. To determine how the E-modulus of fiber meshes affects the osteogenic potential of hADSCs, the cellular and nuclear morphologies and osteogenic differentiation abilities were evaluated. Results: With the increasing stiffness of PEEU fiber meshes, the aspect ratios of HUVECs cultivated on PEEU materials increased. HUVECs cultivated on high stiffness fiber meshes (4.5 ± 0.8 MPa) displayed a considerably greater proliferation rate and migratory velocity, in addition demonstrating increased tube formation capability, compared with those of the cells cultivated on lower stiffness fiber meshes (2.6 ± 0.8 MPa). Furthermore, in comparison to those cultivated on lower stiffness fiber meshes, hADSCs adhered to the highest stiffness fiber meshes PEEU70 had an elongated shape. The hADSCs grown on the softer PEEU40 fiber meshes showed a reduced nuclear aspect ratio (width to height) than those cultivated on the stiffer fiber meshes. Culturing hADSCs on stiffer fibers improved their osteogenic differentiation potential. Compared with cells cultured on PEEU40, osteocalcin expression and alkaline phosphatase (ALP) activity increased by 73 ± 10\% and 43 ± 16\%, respectively, in cells cultured on PEEU70. Conclusion: The mechanical characteristics of the substrate are crucial in the modulation of cell behaviors. These findings indicate that adjusting the elasticity of fiber meshes might be a useful method for controlling the blood vessels development and regeneration. Furthermore, the mechanical characteristics of PEEU fiber meshes might be modified to control the osteogenic potential of hADSCs.}, language = {en} } @phdthesis{EsmaeeliMoghaddamTabalvandani2022, author = {Esmaeeli Moghaddam Tabalvandani, Mariam}, title = {ROS Generation in Human Aldehyde Oxidase And the Effects of ROS and Reactive Sulfhydryl on the Activity of the Enzyme}, doi = {10.25932/publishup-53460}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-534600}, school = {Universit{\"a}t Potsdam}, pages = {153}, year = {2022}, abstract = {Aldehyde oxidases (AOXs) (E.C. 1.2.3.1) are molybdoflavo-enzymes belonging to the xanthine oxidase (XO) family. AOXs in mammals contain one molybdenum cofactor (Moco), one flavin adenine dinucleotide (FAD) and two [2Fe-2S] clusters, the presence of which is essential for the activity of the enzyme. Human aldehyde oxidase (hAOX1) is a cytosolic enzyme mainly expressed in the liver. hAOX1is involved in the metabolism of xenobiotics. It oxidizes aldehydes to their corresponding carboxylic acids and hydroxylates N-heterocyclic compounds. Since these functional groups are widely present in therapeutics, understanding the behaviour of hAOX1 has important implications in medicine. During the catalytic cycle of hAOX1, the substrate is oxidized at Moco and electrons are internally transferred to FAD via the FeS clusters. An electron acceptor juxtaposed to the FAD receives the electrons and re-oxidizes the enzyme for the next catalytic cycle. Molecular oxygen is the endogenous electron acceptor of hAOX1 and in doing so it is reduced and produces reactive oxygen species (ROS) including hydrogen peroxide (H2O2) and superoxide (O2.-). The production of ROS has patho-physiological importance, as ROS can have a wide range of effects on cell components including the enzyme itself. In this thesis, we have shown that hAOX1 loses its activity over multiple cycles of catalysis due to endogenous ROS production and have identified a cysteine rich motif that protects hAOX1 from the ROS damaging effects. We have also shown that a sulfido ligand, which is bound at Moco and is essential for the catalytic activity of the enzyme, is vulnerable during turnover. The ROS produced during the course of the reaction are also able to remove this sulfido ligand from Moco. ROS, in addition, oxidize particular cysteine residues. The combined effects of ROS on the sulfido ligand and on specific cysteine residues in the enzyme result in its inactivation. Furthermore, we report that small reducing agents containing reactive sulfhydryl groups, in a selective manner, inactivate some of the mammalian AOXs by modifying the sulfido ligand at Moco. The mechanism of ROS production by hAOX1 is another scope that has been investigated as part of the work in this thesis. We have shown that the ratio of type of ROS, i.e. hydrogen peroxide (H2O2) and superoxide (O2.-), produced by hAOX1 is determined by a particular position on a flexible loop that locates in close proximity of FAD. The size of the cavity at the ROS producing site, i.e. the N5 position of the FAD isoalloxazine ring, kinetically affects the amount of each type of ROS generated by hAOX1. Taken together, hAOX1 is an enzyme with emerging importance in pharmacological and medical studies, not only due to its involvement in drug metabolism, but also due to ROS production which has physiological and pathological implications.}, language = {en} } @phdthesis{MorenoCurtidor2021, author = {Moreno Curtidor, Catalina}, title = {Elucidating the molecular basis of enhanced growth in the Arabidopsis thaliana accession Bur-0}, doi = {10.25932/publishup-52681}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-526814}, school = {Universit{\"a}t Potsdam}, pages = {136}, year = {2021}, abstract = {The life cycle of flowering plants is a dynamic process that involves successful passing through several developmental phases and tremendous progress has been made to reveal cellular and molecular regulatory mechanisms underlying these phases, morphogenesis, and growth. Although several key regulators of plant growth or developmental phase transitions have been identified in Arabidopsis, little is known about factors that become active during embryogenesis, seed development and also during further postembryonic growth. Much less is known about accession-specific factors that determine plant architecture and organ size. Bur-0 has been reported as a natural Arabidopsis thaliana accession with exceptionally big seeds and a large rosette; its phenotype makes it an interesting candidate to study growth and developmental aspects in plants, however, the molecular basis underlying this big phenotype remains to be elucidated. Thus, the general aim of this PhD project was to investigate and unravel the molecular mechanisms underlying the big phenotype in Bur-0. Several natural Arabidopsis accessions and late flowering mutant lines were analysed in this study, including Bur-0. Phenotypes were characterized by determining rosette size, seed size, flowering time, SAM size and growth in different photoperiods, during embryonic and postembryonic development. Our results demonstrate that Bur-0 stands out as an interesting accession with simultaneously larger rosettes, larger SAM, later flowering phenotype and larger seeds, but also larger embryos. Interestingly, inter-accession crosses (F1) resulted in bigger seeds than the parental self-crossed accessions, particularly when Bur-0 was used as the female parental genotype, suggesting parental effects on seed size that might be maternally controlled. Furthermore, developmental stage-based comparisons revealed that the large embryo size of Bur-0 is achieved during late embryogenesis and the large rosette size is achieved during late postembryonic growth. Interestingly, developmental phase progression analyses revealed that from germination onwards, the length of developmental phases during postembryonic growth is delayed in Bur-0, suggesting that in general, the mechanisms that regulate developmental phase progression are shared across developmental phases. On the other hand, a detailed physiological characterization in different tissues at different developmental stages revealed accession-specific physiological and metabolic traits that underlie accession-specific phenotypes and in particular, more carbon resources during embryonic and postembryonic development were found in Bur-0, suggesting an important role of carbohydrates in determination of the bigger Bur-0 phenotype. Additionally, differences in the cellular organization, nuclei DNA content, as well as ploidy level were analyzed in different tissues/cell types and we found that the large organ size in Bur-0 can be mainly attributed to its larger cells and also to higher cell proliferation in the SAM, but not to a different ploidy level. Furthermore, RNA-seq analysis of embryos at torpedo and mature stage, as well as SAMs at vegetative and floral transition stage from Bur-0 and Col-0 was conducted to identify accession-specific genetic determinants of plant phenotypes, shared across tissues and developmental stages during embryonic and postembryonic growth. Potential candidate genes were identified and further validation of transcriptome data by expression analyses of candidate genes as well as known key regulators of organ size and growth during embryonic and postembryonic development confirmed that the high confidence transcriptome datasets generated in this study are reliable for elucidation of molecular mechanisms regulating plant growth and accession-specific phenotypes in Arabidopsis. Taken together, this PhD project contributes to the plant development research field providing a detailed analysis of mechanisms underlying plant growth and development at different levels of biological organization, focusing on Arabidopsis accessions with remarkable phenotypical differences. For this, the natural accession Bur-0 was an ideal outlier candidate and different mechanisms at organ and tissue level, cell level, metabolism, transcript and gene expression level were identified, providing a better understanding of different factors involved in plant growth regulation and mechanisms underlying different growth patterns in nature.}, language = {en} } @phdthesis{Hasnat2021, author = {Hasnat, Muhammad Abrar}, title = {A-Type Carrier Proteins are involved in [4Fe-4S] Cluster insertion into the Radical S-adenosylmethionine (SAM) Protein MoaA and other molybdoenzymes}, doi = {10.25932/publishup-53079}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-530791}, school = {Universit{\"a}t Potsdam}, pages = {200}, year = {2021}, abstract = {Iron-sulfur clusters are essential enzyme cofactors. The most common and stable clusters are [2Fe-2S] and [4Fe-4S] that are found in nature. They are involved in crucial biological processes like respiration, gene regulation, protein translation, replication and DNA repair in prokaryotes and eukaryotes. In Escherichia coli, Fe-S clusters are essential for molybdenum cofactor (Moco) biosynthesis, which is a ubiquitous and highly conserved pathway. The first step of Moco biosynthesis is catalyzed by the MoaA protein to produce cyclic pyranopterin monophosphate (cPMP) from 5'GTP. MoaA is a [4Fe-4S] cluster containing radical S-adenosyl-L-methionine (SAM) enzyme. The focus of this study was to investigate Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions using E. coli as a model organism. Nitrate and TMAO respiration usually occur under anaerobic conditions, where oxygen is depleted. Under these conditions, E. coli uses nitrate and TMAO as terminal electron. Previous studies revealed that Fe-S cluster insertion is performed by Fe-S cluster carrier proteins. In E. coli, these proteins are known as A-type carrier proteins (ATC) by phylogenomic and genetic studies. So far, three of them have been characterized in detail in E. coli, namely IscA, SufA, and ErpA. This study shows that ErpA and IscA are involved in Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions. ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. SufA is not able to replace the functions of IscA or ErpA under nitrate respiratory conditions. Nitrate reductase is a molybdoenzyme that coordinates Moco and Fe-S clusters. Under nitrate respiratory conditions, the expression of nitrate reductase is significantly increased in E. coli. Nitrate reductase is encoded in narGHJI genes, the expression of which is regulated by the transcriptional regulator, fumarate and nitrate reduction (FNR). The activation of FNR under conditions of nitrate respiration requires one [4Fe-4S] cluster. In this part of the study, we analyzed the insertion of Fe-S cluster into FNR for the expression of narGHJI genes in E. coli. The results indicate that ErpA is essential for the FNR-dependent expression of the narGHJI genes, a role that can be replaced partially by IscA and SufA when they are produced sufficiently under the conditions tested. This observation suggests that ErpA is indirectly regulating nitrate reductase expression via inserting Fe-S clusters into FNR. Most molybdoenzymes are complex multi-subunit and multi-cofactor-containing enzymes that coordinate Fe-S clusters, which are functioning as electron transfer chains for catalysis. In E. coli, periplasmic aldehyde oxidoreductase (PaoAC) is a heterotrimeric molybdoenzyme that consists of flavin, two [2Fe-2S], one [4Fe-4S] cluster and Moco. In the last part of this study, we investigated the insertion of Fe-S clusters into E. coli periplasmic aldehyde oxidoreductase (PaoAC). The results show that SufA and ErpA are involved in inserting [4Fe-4S] and [2Fe-2S] clusters into PaoABC, respectively under aerobic respiratory conditions.}, language = {en} } @phdthesis{Michelchen2021, author = {Michelchen, Sophia}, title = {Etablierung einer Antigen-spezifischen Aktivierung von B-Lymphozyten in vitro}, doi = {10.25932/publishup-53027}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-530272}, school = {Universit{\"a}t Potsdam}, pages = {XII,107}, year = {2021}, abstract = {Monoklonale Antik{\"o}rper sind essenzielle Werkzeuge in der modernen Laboranalytik sowie in der medizinischen Therapie und Diagnostik. Die Herstellung monoklonaler Antik{\"o}rper ist ein zeit- und arbeitsintensiver Prozess. Herk{\"o}mmliche Methoden beruhen auf der Immunisierung von Labortieren, die mitunter mehrere Monate in Anspruch nimmt. Anschließend werden die Antik{\"o}rper-produzierenden B-Lymphozyten bzw. deren Antik{\"o}rpergene isoliert und in Screening-Verfahren untersucht, um geeignete Binder zu identifizieren. Der Transfer der humoralen Immunantwort in eine in vitro Umgebung erlaubt eine Verk{\"u}rzung des Prozesses und umgeht die Notwendigkeit der in vivo Immunisierung. Das komplexe Zusammenspiel aller involvierten Immunzellen in vitro abzubilden, stellt sich allerdings als schwierig dar. Der Schwerpunkt dieser Arbeit war deshalb die Realisierung einer vereinfachten In vitro Immunisierung, die sich auf die Protagonisten der Antik{\"o}rper-Produktion konzentriert: die B-Lymphozyten. Dar{\"u}ber hinaus sollte eine permanente Zelllinie etabliert werden, die zur Antik{\"o}rper-Herstellung eingesetzt werden und die Verwendung von Prim{\"a}rzellen ersetzen w{\"u}rde. Im ersten Teil der Arbeit wurde ein Protokoll zur In vitro Immunisierung muriner BLymphozyten etabliert. In Vorversuchen wurden die optimalen Konditionen f{\"u}r die Antigenspezifische Aktivierung gereinigter Milz-B-Lymphozyten aus nicht-immunisierten M{\"a}usen determiniert. Dazu wurde der Einfluss verschiedener Stimuli auf die Produktion unspezifischer und spezifischer Antik{\"o}rper untersucht. Eine Kombination aus dem Modellantigen VP1 (Hamster Polyomavirus H{\"u}llprotein 1), einem Anti-CD40-Antik{\"o}rper, Interleukin 4 (IL 4) und Lipopolysaccharid (LPS) oder IL 7 induzierte nachweislich eine Antigen-spezifische Antik{\"o}rper-Antwort in vitro. Als Indikatoren einer erfolgreichen Aktivierung der B-Lymphozyten infolge der in vitro Stimulation wurden die rapide Proliferation und die Expression charakteristischer Aktivierungsmarker auf der Zelloberfl{\"a}che nachgewiesen. In einer Zeitreihe {\"u}ber zehn Tage wurde am zehnten Tag der In vitro Immunisierung die verh{\"a}ltnism{\"a}ßig h{\"o}chste Konzentration Antigen-spezifischer IgG-Antik{\"o}rper im Kultur{\"u}berstand der stimulierten Zellen nachgewiesen. Als n{\"a}chster Schritt sollte eine permanente Zelllinie hergestellt werden, die statt prim{\"a}rer BLymphozyten f{\"u}r die zuvor etablierte In vitro Immunisierung eingesetzt werden k{\"o}nnte. Zu diesem Zweck wurden retrovirale Vektoren hergestellt, die durch den Transfer verschiedener Onkogene in murine B-Lymphozyten bzw. deren Vorl{\"a}uferzellen das Proliferationsverhalten der Zellen manipulieren sollen. Es wurden Retroviren mit Doxycyclin-induzierbaren Expressionskassetten mit den Onkogenen cmyc, Bcl2, BclxL und dem Fusionsgen NUP98HOXB4 generiert. Eine Testzelllinie wurde erfolgreich mit den hergestellten Retroviren transduziert und die Funktionalit{\"a}t der hergestellten Viren anhand verschiedener Assays belegt. Die transferierten Gene konnten in der Testzelllinie auf DNAEbene nachgewiesen oder die {\"U}berexpression der entsprechenden Proteine im Western Blot detektiert werden. Es wurden schließlich B-Lymphozyten bzw. unreife Vorl{\"a}uferzellen derselben mit den generierten Retroviren transduziert und mit Knochenmark-{\"a}hnlichen Stromazellen co-kultiviert. Aus keinem der transduzierten Ans{\"a}tze konnte bisher eine Zelllinie oder eine Langzeit-Kultur etabliert werden. Im letzten Teil der Arbeit wurde die Effektivit{\"a}t und {\"U}bertragbarkeit des zuvor etablierten Protokolls zur In vitro Immunisierung muriner B-Lymphozyten anhand verschiedener Antigene gezeigt. Es konnten in vitro spezifische IgG-Antworten gegen VP1, Legionella pneumophila und das Protein Mip, von dem ein Peptid in das zur Immunisierung eingesetzte VP1 integriert wurde, induziert werden. Die stimulierten B-Lymphozyten wurden durch Fusion mit Myelomzellen in permanente Antik{\"o}rper-produzierende Zelllinien transformiert. Dabei konnten mehrere Hybridomzelllinien generiert werden, die spezifische IgGAntik{\"o}rper gegen VP1 oder Mip produzieren. Die generierten Antik{\"o}rper konnten sowohl im Western Blot als auch im ELISA (Enzyme-Linked Immunosorbent Assay) das entsprechende Antigen spezifisch binden. Die hier etablierte In vitro Immunisierung bietet eine effektive Alternative zu bisherigen Verfahren zur Herstellung spezifischer Antik{\"o}rper. Sie ersetzt die Immunisierung von Versuchstieren und reduziert den Zeitaufwand erheblich. In Kombination mit der Hybridomtechnologie k{\"o}nnen die in vitro immunisierten Zellen, wie hier demonstriert, zur Generation von Hybridomzelllinien und zur Herstellung monoklonaler Antik{\"o}rper genutzt werden. Um die Verwendung von Versuchstieren in dieser Methode durch eine ad{\"a}quate permanente Zelllinie zu ersetzen, muss die genetische Ver{\"a}nderung von B-Lymphozyten und unreifen h{\"a}matopoetischen Zellen optimiert werden. Die Ergebnisse bieten eine Basis f{\"u}r eine universelle, Spezies-unabh{\"a}ngige Methodik zur Antik{\"o}rperherstellung und f{\"u}r die Etablierung einer idealen, tierfreien In vitro Immunisierung.}, language = {de} } @phdthesis{Stark2021, author = {Stark, Markus}, title = {Implications of local and regional processes on the stability of metacommunities in diverse ecosystems}, doi = {10.25932/publishup-52639}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-526399}, school = {Universit{\"a}t Potsdam}, pages = {x, 167}, year = {2021}, abstract = {Anthropogenic activities such as continuous landscape changes threaten biodiversity at both local and regional scales. Metacommunity models attempt to combine these two scales and continuously contribute to a better mechanistic understanding of how spatial processes and constraints, such as fragmentation, affect biodiversity. There is a strong consensus that such structural changes of the landscape tend to negatively effect the stability of metacommunities. However, in particular the interplay of complex trophic communities and landscape structure is not yet fully understood. In this present dissertation, a metacommunity approach is used based on a dynamic and spatially explicit model that integrates population dynamics at the local scale and dispersal dynamics at the regional scale. This approach allows the assessment of complex spatial landscape components such as habitat clustering on complex species communities, as well as the analysis of population dynamics of a single species. In addition to the impact of a fixed landscape structure, periodic environmental disturbances are also considered, where a periodical change of habitat availability, temporally alters landscape structure, such as the seasonal drying of a water body. On the local scale, the model results suggest that large-bodied animal species, such as predator species at high trophic positions, are more prone to extinction in a state of large patch isolation than smaller species at lower trophic levels. Increased metabolic losses for species with a lower body mass lead to increased energy limitation for species on higher trophic levels and serves as an explanation for a predominant loss of these species. This effect is particularly pronounced for food webs, where species are more sensitive to increased metabolic losses through dispersal and a change in landscape structure. In addition to the impact of species composition in a food web for diversity, the strength of local foraging interactions likewise affect the synchronization of population dynamics. A reduced predation pressure leads to more asynchronous population dynamics, beneficial for the stability of population dynamics as it reduces the risk of correlated extinction events among habitats. On the regional scale, two landscape aspects, which are the mean patch isolation and the formation of local clusters of two patches, promote an increase in \$\beta\$-diversity. Yet, the individual composition and robustness of the local species community equally explain a large proportion of the observed diversity patterns. A combination of periodic environmental disturbance and patch isolation has a particular impact on population dynamics of a species. While the periodic disturbance has a synchronizing effect, it can even superimpose emerging asynchronous dynamics in a state of large patch isolation and unifies trends in synchronization between different species communities. In summary, the findings underline a large local impact of species composition and interactions on local diversity patterns of a metacommunity. In comparison, landscape structures such as fragmentation have a negligible effect on local diversity patterns, but increase their impact for regional diversity patterns. In contrast, at the level of population dynamics, regional characteristics such as periodic environmental disturbance and patch isolation have a particularly strong impact and contribute substantially to the understanding of the stability of population dynamics in a metacommunity. These studies demonstrate once again the complexity of our ecosystems and the need for further analysis for a better understanding of our surrounding environment and more targeted conservation of biodiversity.}, language = {en} } @phdthesis{Raffeiner2021, author = {Raffeiner, Margot}, title = {Funktionelle Charakterisierung des Xanthomonas Typ-III Effektorproteins XopS}, doi = {10.25932/publishup-52553}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-525532}, school = {Universit{\"a}t Potsdam}, pages = {IX, 185}, year = {2021}, abstract = {Angepasste Pathogene besitzen eine Reihe von Virulenzmechanismen, um pflanzliche Immunantworten unterhalb eines Schwellenwerts der effektiven Resistenz zu unterdr{\"u}cken. Dadurch sind sie in der Lage sich zu vermehren und Krankheiten auf einem bestimmten Wirt zu verursachen. Eine essentielle Virulenzstrategie Gram-negativer Bakterien ist die Translokation von sogenannten Typ-III Effektorproteinen (T3Es) direkt in die Wirtszelle. Dort st{\"o}ren diese die Immunantwort des Wirts oder f{\"o}rdern die Etablierung einer f{\"u}r das Pathogen g{\"u}nstigen Umgebung. Eine kritische Komponente der Pflanzenimmunit{\"a}t gegen eindringende Pathogene ist die schnelle transkriptionelle Umprogrammierung der angegriffenen Zelle. Viele adaptierte bakterielle Pflanzenpathogene verwenden T3Es, um die Induktion Abwehr-assoziierter Gene zu st{\"o}ren. Die Aufkl{\"a}rung von Effektor-Funktionen, sowie die Identifikation ihrer pflanzlichen Zielproteine sind f{\"u}r das Verst{\"a}ndnis der bakteriellen Pathogenese essentiell. Im Rahmen dieser Arbeit sollte das Typ-III Effektorprotein XopS aus Xanthomonas campestris pv. vesicatoria (Xcv) funktionell charakterisiert werden. Zudem lag hier ein besonderer Fokus auf der Untersuchung der Wechselwirkung zwischen XopS und seinem in Vorarbeiten identifizierten pflanzlichen Interaktionspartner WRKY40, einem transkriptionellen Regulator der Abwehr-assoziierten Genexpression. Es konnte gezeigt werden, dass XopS ein essentieller Virulenzfaktor des Phytopathogens Xcv w{\"a}hrend der pr{\"a}invasiven Immunantwort ist. So zeigten xopS-defiziente Xcv Bakterien bei einer Inokulation der Blattoberfl{\"a}che suszeptibler Paprika Pflanzen eine deutlich reduzierte Virulenz im Vergleich zum Xcv Wildtyp. Die Translokation von XopS durch Xcv, sowie die ektopische Expression von XopS in Arabidopsis oder N. benthamiana verhinderte das Schließen von Stomata als Reaktion auf Bakterien bzw. einem Pathogen-assoziierten Stimulus, wobei zudem gezeigt werden konnte, dass dies in einer WRKY40-abh{\"a}ngigen Weise geschieht. Weiter konnte gezeigt werden, dass XopS in der Lage ist, die Expression Abwehr-assoziierter Gene zu manipulieren. Dies deutet darauf hin, dass XopS sowohl in die pr{\"a}-als auch in die postinvasive, apoplastische Abwehr eingreift. Phytohormon-Signalnetzwerke spielen w{\"a}hrend des Aufbaus einer effizienten pflanzlichen Immunantwort eine wichtige Rolle. Hier konnte gezeigt werden, dass XopS mit genau diesen Signalnetzwerken zu interferieren scheint. Eine ektopische Expression des Effektors in Arabidopsis f{\"u}hrte beispielsweise zu einer signifikanten Induktion des Phytohormons Jasmons{\"a}ure (JA), w{\"a}hrend eine Infektion von suszeptiblen Paprika Pflanzen mit einem xopS-defizienten Xcv Stamm zu einer ebenfalls signifikanten Akkumulation des Salicyls{\"a}ure (SA)-Gehalts f{\"u}hrte. So kann zu diesem Zeitpunkt vermutet werden, dass XopS die Virulenz von Xcv f{\"o}rdert, indem JA-abh{\"a}ngige Signalwege induziert werden und es gleichzeitig zur Unterdr{\"u}ckung SA-abh{\"a}ngiger Signalwege kommt. Die Virus-induzierte Genstilllegung des XopS Interaktionspartners WRKY40a in Paprika erh{\"o}hte die Toleranz der Pflanze gegen{\"u}ber einer Xcv Infektion, was darauf hindeutet, dass es sich bei diesem Protein um einen transkriptionellen Repressor pflanzlicher Immunantworten handelt. Die Hypothese, dass WRKY40 die Abwehr-assoziierte Genexpression reprimiert, konnte hier {\"u}ber verschiedene experimentelle Ans{\"a}tze bekr{\"a}ftigt werden. So wurde beispielsweise gezeigt, dass die Expression von verschiedenen Abwehrgenen einschließlich des SA-abh{\"a}ngigen Gens PR1 und die des Negativregulators des JA-Signalwegs JAZ8 von WRKY40 gehemmt wird. Um bei einem Pathogenangriff die Abwehr-assoziierte Genexpression zu gew{\"a}hrleisten, muss WRKY40 als Negativregulator abgebaut werden. Vorarbeiten zeigten, dass WRKY40 {\"u}ber das 26S Proteasom abgebaut wird. In der hier vorliegenden Studie konnte weiter best{\"a}tigt, dass der T3E XopS zu einer Stabilisierung des WRKY40 Proteins f{\"u}hrt, indem er auf bislang ungekl{\"a}rte Weise dessen Abbau {\"u}ber das 26S Proteasom verhindert. Die Ergebnisse aus der hier vorliegenden Arbeit lassen die Vermutung zu, dass die Stabilisierung des Negativregulators der Immunantwort WRKY40 seitens XopS dazu f{\"u}hrt, dass eine dar{\"u}ber vermittelte Manipulation der Abwehr-assoziierten Genexpression, sowie eine Umsteuerung phytohormoneller Wechselwirkungen die Ausbreitung von Xcv auf suszeptiblen Paprikapflanzen f{\"o}rdert. Ein weiteres Ziel dieser Arbeit war es, weitere potentielle in planta Interaktionspartner von XopS zu identifizieren die f{\"u}r seine Interaktion mit WRKY40 bzw. f{\"u}r die Aufschl{\"u}sselung seines Wirkmechanismus relevant sein k{\"o}nnten. So konnte die Deubiquitinase UBP12 als weiterer pflanzlicher Interaktionspartner sowohl von XopS als auch von WRKY40 gefunden werden. Dieses Enzym ist in der Lage, die Ubiquitinierung von Substratproteinen zu modifizieren und seine Funktion k{\"o}nnte somit ein Bindeglied zwischen XopS und dessen Interferenz mit dem proteasomalen Abbau von WRKY40 sein. W{\"a}hrend einer kompatiblen Xcv-Wirtsinteraktion f{\"u}hrte die Virus-induzierte Genstilllegung von UBP12 zu einer reduzierten Resistenz der Pflanze gegen{\"u}ber des Pathogens Xcv, was auf dessen positiv-regulatorische Wirkung w{\"a}hrend der Immunantwort hindeutet. Zudem zeigten Western Blot Analysen, dass das Protein WRKY40 bei einer Herunterregulierung von UBP12 akkumuliert und dass diese Akkumulation von der Anwesenheit des T3Es XopS zus{\"a}tzlich verst{\"a}rkt wird. Weiterf{\"u}hrende Analysen zur biochemischen Charakterisierung der XopS/WRKY40/UBP12 Interaktion sollten in Zukunft durchgef{\"u}hrt werden, um den genauen Wirkmechanismus des XopS T3Es weiter aufzuschl{\"u}sseln.}, language = {de} } @phdthesis{Borghi2021, author = {Borghi, Gian Luca}, title = {Evolution and diversity of photosynthetic metabolism in C3, C3-C4 intermediate and C4 plants}, doi = {10.25932/publishup-52220}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-522200}, school = {Universit{\"a}t Potsdam}, pages = {163}, year = {2021}, abstract = {In C3 plants, CO2 diffuses into the leaf and is assimilated by the Calvin-Benson cycle in the mesophyll cells. It leaves Rubisco open to its side reaction with O2, resulting in a wasteful cycle known as photorespiration. A sharp fall in atmospheric CO2 levels about 30 million years ago have further increased the side reaction with O2. The pressure to reduce photorespiration led, in over 60 plant genera, to the evolution of a CO2-concentrating mechanism called C4 photosynthesis; in this mode, CO2 is initially incorporated into 4-carbon organic acids, which diffuse to the bundle sheath and are decarboxylated to provide CO2 to Rubisco. Some genera, like Flaveria, contain several species that represent different steps in this complex evolutionary process. However, the majority of terrestrial plant species did not evolve a CO2-concentrating mechanism and perform C3 photosynthesis. This thesis compares photosynthetic metabolism in several species with C3, C4 and intermediate modes of photosynthesis. Metabolite profiling and stable isotope labelling were performed to detect inter-specific differences changes in metabolite profile and, hence, how a pathway operates. The results obtained were subjected to integrative data analyses like hierarchical clustering and principal component analysis, and were deepened by correlation analyses to uncover specific metabolic features and reaction steps that were conserved or differed between species. The main findings are that Calvin-Benson cycle metabolite profiles differ between C3 and C4 species and between different C3 species, including a very different response to rising irradiance in Arabidopsis and rice. These findings confirm Calvin-Benson cycle operation diverged between C3 and C4 species and, most unexpectedly, even between different C3 species. Moreover, primary metabolic profiles supported the current C4 evolutionary model in the genus Flaveria and also provided new insights and opened up new questions. Metabolite profiles also point toward a progressive adjustment of the Calvin-Benson cycle during the evolution of C4 photosynthesis. Overall, this thesis point out the importance of a metabolite-centric approach to uncover underlying differences in species apparently sharing the same photosynthetic routes and as a valid method to investigate evolutionary transition between C3 and C4 photosynthesis.}, language = {en} } @phdthesis{Woehlecke2021, author = {Woehlecke, Sandra}, title = {Das erweiterte Fachwissen f{\"u}r den schulischen Kontext als Leitlinie f{\"u}r eine additive fachliche Lehrveranstaltung im Lehramtsstudium Biologie}, doi = {10.25932/publishup-52120}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-521209}, school = {Universit{\"a}t Potsdam}, pages = {304}, year = {2021}, abstract = {Das Fachwissen von Lehrkr{\"a}ften weist f{\"u}r die Auspr{\"a}gung fachdidaktischer Expertise eine hohe Bedeutung auf. Welche Merkmale universit{\"a}re Lehrveranstaltungen aufweisen sollten, um Lehramtsstudierenden ein berufsspezifisches Fachwissen zu vermitteln, ist jedoch {\"u}berwiegend noch unklar. Innerhalb des Projekts PSI-Potsdam wurde auf theoretischer Grundlage das fach{\"u}bergreifende Modell des erweiterten Fachwissens f{\"u}r den schulischen Kontext entwickelt. Als Ansatz zur Verbesserung des Biologie-Lehramtsstudiums diente dieses Modell als Konzeptionsgrundlage f{\"u}r eine additive Lehrveranstaltung. Hierbei werden Lerngelegenheiten geboten, um das universit{\"a}r erworbene Fachwissen {\"u}ber zellbiologische Inhalte auf schulische Kontexte anzuwenden, z.B. durch die Dekonstruktion und anschließende Rekonstruktion von schulischen Lerntexten. Die Wirkung des Seminars wurde in mehreren Zyklen im Forschungsformat der Fachdidaktischen Entwicklungsforschung beforscht. Eine der zentralen Forschungsfragen lautet dabei: Wie kann eine Lerngelegenheit f{\"u}r Lehramtsstudierende der Biologie gestaltet sein, um ein erweitertes Fachwissen f{\"u}r den schulischen Kontext f{\"u}r den zellbiologischen Themenbereich „Struktur und Funktion der Biomembran" zu f{\"o}rdern? Anhand fall{\"u}bergreifender Analysen (n = 29) wird im empirischen Teil aufgezeigt, welche Einstellungen zum Lehramtsstudium in der Stichprobe bestehen. Als ein wichtiges Ergebnis kann hierbei herausgestellt werden, dass sich das Fachinteresse hinsichtlich schulisch und universit{\"a}r vermittelter Inhalte bei den untersuchten Studierenden auffallend unterscheidet, wobei dem Schulwissen ein deutlich h{\"o}heres Interesse entgegengebracht wird. Die Berufsrelevanz fachlicher Inhalte wird seitens der Studierenden h{\"a}ufig am Schulwissen festgemacht. Innerhalb konkreter Einzelfallanalysen (n = 6) wird anhand von Lernpfaden dargestellt, wie sich {\"u}ber mehrere Design-Experimente hinweg fachliche Konzepte entwickelt haben. Bei der Beschreibung wird vor allem auf Schl{\"u}sselstellen und H{\"u}rden im Lernprozess fokussiert. Aus diesen Ergebnissen folgend werden vorgenommene Iterationen f{\"u}r die einzelnen Zyklen beschrieben, die ebenfalls anhand der iterativen Entwicklung der Design-Prinzipien dargelegt werden. Es konnte gezeigt werden, dass die Schl{\"u}sselstellen sehr individuell aufgrund der subjektiv fokussierten Inhalte zu Tage treten. Meist treten sie jedoch im Zusammenhang mit der Verkn{\"u}pfung verschiedener fachlicher Konzepte oder durch kooperative Aufschl{\"u}sselungen von Konzepten auf. Fachliche H{\"u}rden konnten hingegen in Form von fachlich unangemessenen Vorstellungen fall{\"u}bergreifend identifiziert werden. Dies betrifft unter anderem die Vorstellung der Biomembran als Wand, die mit den Vorstellungen einer Schutzfunktion und einer formgebenden Funktion der Biomembran einhergeht. Weiterhin wird beleuchtet, wie das erweiterte Fachwissen f{\"u}r den schulischen Kontext zur Bearbeitung der Lernaufgaben angewendet wurde. Es hat sich gezeigt, dass sich bestimmte Lerngelegenheiten eigenen, um bestimmte Facetten des erweiterten Fachwissens zu f{\"o}rdern. Insgesamt scheint das Modell des erweiterten Fachwissens f{\"u}r den schulischen Kontext {\"a}ußerst geeignet zu sein, um anhand der Facetten und deren Beschreibungen Lerngelegenheiten oder Gestaltungsprinzipien f{\"u}r diese zu konzipieren. F{\"u}r das untersuchte Lehr-Lernarrangement haben sich kleinere Adaptationen des Modells als sinnvoll erwiesen. Hinsichtlich der Methodologie konnten Ableitungen f{\"u}r die Anwendung der fachdidaktischen Entwicklungsforschung f{\"u}r additive fachliche Lehrveranstaltungen dieser Art herausgestellt werden. Um den Professionsbezug der fachwissenschaftlichen Anteile im Lehramtsstudium zu verbessern, ist der weitere Einbezug des erweiterten Fachwissens f{\"u}r den schulischen Kontext in die fachwissenschaftlichen Studienanteile {\"u}beraus w{\"u}nschenswert.}, language = {de} } @phdthesis{Raatz2021, author = {Raatz, Larissa}, title = {Boon and bane}, doi = {10.25932/publishup-51965}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-519653}, school = {Universit{\"a}t Potsdam}, pages = {130}, year = {2021}, abstract = {Semi-natural habitats (SNHs) in agricultural landscapes represent important refugia for biodiversity including organisms providing ecosystem services. Their spill-over into agricultural fields may lead to the provision of regulating ecosystem services such as biological pest control ultimately affecting agricultural yield. Still, it remains largely unexplored, how different habitat types and their distributions in the surrounding landscape shape this provision of ecosystem services within arable fields. Hence, in this thesis I investigated the effect of SNHs on biodiversity-driven ecosystem services and disservices affecting wheat production with an emphasis on the role and interplay of habitat type, distance to the habitat and landscape complexity. I established transects from the field border into the wheat field, starting either from a field-to-field border, a hedgerow, or a kettle hole, and assessed beneficial and detrimental organisms and their ecosystem functions as well as wheat yield at several in-field distances. Using this study design, I conducted three studies where I aimed to relate the impacts of SNHs at the field and at the landscape scale on ecosystem service providers to crop production. In the first study, I observed yield losses close to SNHs for all transect types. Woody habitats, such as hedgerows, reduced yields stronger than kettle holes, most likely due to shading from the tall vegetation structure. In order to find the biotic drivers of these yield losses close to SNHs, I measured pest infestation by selected wheat pests as potential ecosystem disservices to crop production in the second study. Besides relating their damage rates to wheat yield of experimental plots, I studied the effect of SNHs on these pest rates at the field and at the landscape scale. Only weed cover could be associated to yield losses, having their strongest impact on wheat yield close to the SNH. While fungal seed infection rates did not respond to SNHs, fungal leaf infection and herbivory rates of cereal leaf beetle larvae were positively influenced by kettle holes. The latter even increased at kettle holes with increasing landscape complexity suggesting a release of natural enemies at isolated habitats within the field interior. In the third study, I found that also ecosystem service providers benefit from the presence of kettle holes. The distance to a SNH decreased species richness of ecosystem service providers, whereby the spatial range depended on species mobility, i.e. arable weeds diminished rapidly while carabids were less affected by the distance to a SNH. Contrarily, weed seed predation increased with distance suggesting that a higher food availability at field borders might have diluted the predation on experimental seeds. Intriguingly, responses to landscape complexity were rather mixed: While weed species richness was generally elevated with increasing landscape complexity, carabids followed a hump-shaped curve with highest species numbers and activity-density in simple landscapes. The latter might give a hint that carabids profit from a minimum endowment of SNHs, while a further increase impedes their mobility. Weed seed predation was affected differently by landscape complexity depending on weed species displayed. However, in habitat-rich landscapes seed predation of the different weed species converged to similar rates, emphasising that landscape complexity can stabilize the provision of ecosystem services. Lastly, I could relate a higher weed seed predation to an increase in wheat yield even though seed predation did not diminish weed cover. The exact mechanisms of the provision of weed control to crop production remain to be investigated in future studies. In conclusion, I found habitat-specific responses of ecosystem (dis)service providers and their functions emphasizing the need to evaluate the effect of different habitat types on the provision of ecosystem services not only at the field scale, but also at the landscape scale. My findings confirm that besides identifying species richness of ecosystem (dis)service providers the assessment of their functions is indispensable to relate the actual delivery of ecosystem (dis)services to crop production.}, language = {en} } @phdthesis{Leiser2021, author = {Leiser, Rico}, title = {Biogeochemical processes governing microplastic transport in freshwater reservoirs}, doi = {10.25932/publishup-52024}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-520240}, school = {Universit{\"a}t Potsdam}, pages = {ix, 143}, year = {2021}, abstract = {The presented study investigated the influence of microbial and biogeochemical processes on the physical transport related properties and the fate of microplastics in freshwater reservoirs. The overarching goal was to elucidate the mechanisms leading to sedimentation and deposition of microplastics in such environments. This is of importance, as large amounts of initially buoyant microplastics are found in reservoir sediments worldwide. However, the transport processes which lead to microplastics accumulation in sediments, were up to now understudied. The impact of biofilm formation on the density and subsequent sedimentation of microplastics was investigated in the eutrophic Bautzen reservoirs (Chapter 2). Biofilms are complex microbial communities fixed to submerged surfaces through a slimy organic film. The mineral calcite was detected in the biofilms, which led to the sinking of the overgrown microplastic particles. The calcite was of biogenic origin, most likely precipitated by sessile cyanobacteria within the biofilms. Biofilm formation was also studied in the mesotrophic Malter reservoir. Unlike in Bautzen reservoir, biofilm formation did not govern the sedimentation of different microplastics in Malter reservoir (Chapter 3). Instead autumnal lake mixing led to the formation of sinking aggregates of microplastics and iron colloids. Such colloids form when anoxic, iron-rich water from the hypolimnion mixes with the oxygenated epilimnetic waters. The colloids bind organic material from the lake water, which leads to the formation of large and sinking iron-organo flocs. Hence, iron-organo floc formation and their influence on the buoyancy or burial of microplastics into sediments of Bautzen reservoir was studied in laboratory experiments (Chapter 4). Microplastics of different shapes (fiber, fragment, sphere) and sizes were readily incorporated into sinking iron-organo flocs. By this initially buoyant polyethylene microplastics were transported on top of sediments from Bautzen reservoir. Shortly after deposition, the microplastic bearing flocs started to subside and transported the pollutants into deeper sediment layers. The microplastics were not released from the sediments within two months of laboratory incubation. The stability of floc microplastic deposition was further investigated employing experiments with the iron reducing model organism Shewanella oneidensis (Chapter 5). It was shown, that reduction or re-mineralization of the iron minerals did not affect the integrity of the iron-organo flocs. The organic matrix was stable under iron reducing conditions. Hence, no incorporated microplastics were released from the flocs. As similar processes are likely to take place in natural sediments, this might explain the previous described low microplastic release from the sediments. This thesis introduced different mechanisms leading to the sedimentation of initially buoyant microplastics and to their subsequent deposition in freshwater reservoirs. Novel processes such as the aggregation with iron-organo flocs were identified and the understudied issue of biofilm densification through biogenic mineral formation was further investigated. The findings might have implications for the fate of microplastics within the river-reservoir system and outline the role of freshwater reservoirs as important accumulation zone for microplastics. Microplastics deposited in the sediments of reservoirs might not be transported further by through flowing river. Hence the study might contribute to better risk assessment and transport balances of these anthropogenic contaminants.}, language = {en} } @phdthesis{Bartholomaeus2021, author = {Bartholom{\"a}us, Lisa}, title = {Impact of growth-related genes on petal size in Arabidopsis thaliana and the formation of two distinct floral morphs in Amsinckia spectabilis}, doi = {10.25932/publishup-51986}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-519861}, school = {Universit{\"a}t Potsdam}, pages = {158}, year = {2021}, abstract = {Der Lebenszyklus von Pflanzen ist gepr{\"a}gt von sich wiederholenden Wachstums- und Entwicklungsphasen, die auf wiederkehrenden Abl{\"a}ufen, bestehend aus Zellteilung, Zellvergr{\"o}ßerung und Zelldifferenzierung, basieren. Diese Dissertation ist aus zwei Projekten aufgebaut, die sich beide mit unterschiedlichen Blickwinkeln des Zellwachstums besch{\"a}ftigen. Im ersten steht die Charakterisierung einer Arabidopsis thaliana Mutante, die eine generelle Zellvergr{\"o}ßerung aufweist, im Vordergrund. Das zweite fokussiert sich auf zwei nat{\"u}rlich vorkommende Bl{\"u}tenmorphologien in Amsinckia spectabilis (Boraginaceae), die sich, aufgrund von Zelll{\"a}ngenunterschieden, in Griffell{\"a}nge und H{\"o}he der Staubblattposition unterscheiden. Es wurde gezeigt, dass die EMS-Mutante eop1 durch gr{\"o}ßere Zellen 26\% gr{\"o}ßere Bl{\"u}tenbl{\"a}tter aufweist. Außerdem wurden weitere Ph{\"a}notypen beschrieben, wie zum Beispiel, vergr{\"o}ßerte Kotyledonen, (ebenfalls aufgrund von Zellvergr{\"o}ßerung), Fruchtbl{\"a}tter, Kelchbl{\"a}tter, Rosettenbl{\"a}tter und Pollen. Die Gesamtwuchsh{\"o}he der Mutante zeigte sich ebenfalls erh{\"o}ht und zus{\"a}tzliche Trichom{\"a}ste erkl{\"a}rten den haarigen Ph{\"a}notyp. Feinkartierung enth{\"u}llte eine C zu T Transition des letzten Nukleotids des Introns 7 des INCURVATA 11 (ICU11) Gens, einer 2-oxoglutarat/Fe(II)-abh{\"a}ngigen Dioxygenase, als urs{\"a}chlichen SNP, welcher missgespleißte mRNA verursacht. Zwei T-DNA Insertionslinien (icu11-2 \& icu11-4), ebenfalls mit vergr{\"o}ßerten Bl{\"u}tenbl{\"a}ttern, best{\"a}tigten ICU11 als kausales Gen, und erlaubten somit die Analyse von drei verschiedenen icu11 Allelen. Ein Vergleich der verursachten molekularen Ver{\"a}nderung durch die jeweiligen Mutationen ermittelte Unterschiede in den drei Mutanten, wie zum Beispiel {\"U}berexpression von ICU11, als auch die Modifikation von ICU11 mRNA. Zusammen bildete das die Grundlage f{\"u}r die Untersuchung des molekularen Mechanismus, der f{\"u}r den beobachteten Ph{\"a}notyp verantwortlich ist. Verschiedene Ans{\"a}tze ermittelten widerspr{\"u}chliche Ergebnisse hinsichtlich der Proteinfunktion von ICU11 in den drei Mutanten. So zeigte eine Komplementierungsanalyse, dass alle drei Mutationen austauschbar sind, was, zusammen mit der Beobachtung, dass eine ICU11 {\"U}berexpression im Wildtyp zu einem icu11-{\"a}hnlichen Ph{\"a}notyp zeigte, dazu f{\"u}hrte, dass die icu11 Mutanten als gain-of-function Mutationen eingeordnet wurden. Im Widerspruch dazu stand die Entdeckung, dass sich icu11-4 durch ein genomisches ICU11 Transgen retten ließ. So wurde ein Model, basierend auf der Annahme, dass eine ICU11 {\"U}berexpression die Proteinfunktion ebenso hemmt wie ein nichtfunktionales Protein, vorgeschlagen. Außerdem wurde eine erh{\"o}hte Resistenz der icu11-3 (eop1) gegen{\"u}ber Paclobutrazol, einem Gibberellin (GA)-Inhibitor, und die Aktivierung der Expression von AtGA20ox2, einem Haupt-GA-Biosynthese-Gen, festgestellt. Zus{\"a}tzlich wurde eine zytoplasmatische Lokalisation von ICU11 detektiert, sodass ein Einfluss von ICU11 auf die GA- Biosynthese und somit auf das Gesamt-GA-Level angenommen wird, der den beobachteten (GA-{\"u}berdosierten) Ph{\"a}notyp erkl{\"a}ren k{\"o}nnte. Das zweite Projekt strebte die Identifizierung der genetischen Grundlage des S-Locus in Amsinckia spectabilis an, da die Gattung Amsinckia einige untypische Charakteristiken f{\"u}r eine heterostyle Art, wie zum Beispiel das Fehlen einer offensichtlichen Selbstinkompatibilit{\"a}t (SI), sowie die mehrmalige Entwicklung zu Homostyly und 100\% autonomem Selbsten, aufweist. Die Analyse basierte auf drei Amsinckia spectabilis Varianten: einer heterostylen Form, bestehend aus zwei Bl{\"u}tenmorphologien mit gegens{\"a}tzlich positionierten Sexualorganen (S-Morph: hohe Staubblattposition und kurzer Griffel und L-Morph: niedrige Staubblattans{\"a}tze und langer Griffel), und zwei homostylen Formen, einer großbl{\"u}tigen teilweise selbstenden und einer kleinbl{\"u}tigen voll selbstenden. Nat{\"u}rliche Populationen weisen ungef{\"a}hr ein 1:1 S:L Morph-Verh{\"a}ltnis auf, welches sich durch vorherrschend disassortative Paarung beider Morphs erkl{\"a}ren lasst. Dadurch kann das dominante S-Allel ausschließlich heterozygot auftreten (heterozygot (Ss) im S-morph und homozygot rezessiv (ss) im L-morph). Die Suche nach Morph-spezifischen Ph{\"a}notypen offenbarte 56\% l{\"a}ngere L-Morph Griffel und 58\% h{\"o}here S-Morph Staubblattans{\"a}tze. Zus{\"a}tzlich wurden 21\% gr{\"o}ßere S-Morph Pollen, sowie das Fehlen einer offensichtlichen SI gefunden. Dies war die Grundlage f{\"u}r die Annahme, dass der Amsinckia spec. S-Locus mindestens aus G- (Griffel), A- (Staubblatt) und P- (Pollen) Locus besteht. Vergleichende Transkriptom-Analyse beider Morphs offenbarte 22 unterschiedlich exprimierte Marker, die in 2 Contigs der PacBio Genom-Assemblierung eines SS-Individuums lokalisiert werden konnten. Dies erlaubte die genetische Einengung des S-Locus auf einen Bereich von circa 23 Mb. Gegens{\"a}tzlich zu bisher aufgekl{\"a}rten S-Loci in anderen Pflanzenarten konnte kein Hinweis auf eine hemizygote Region gefunden werden, die die supprimierte Rekombination am S-Locus erkl{\"a}ren k{\"o}nnte, sodass eine Inversion als Ursache dieser vermutet wurde.}, language = {en} } @phdthesis{Schuster2020, author = {Schuster, Maja}, title = {High resolution decoding of the tobacco chloroplast translatome and its dynamics during light-intensity acclimation}, doi = {10.25932/publishup-51268}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-512680}, school = {Universit{\"a}t Potsdam}, pages = {xvii, 155}, year = {2020}, abstract = {Chloroplasts are the photosynthetic organelles in plant and algae cells that enable photoautotrophic growth. Due to their prokaryotic origin, modern-day chloroplast genomes harbor 100 to 200 genes. These genes encode for core components of the photosynthetic complexes and the chloroplast gene expression machinery, making most of them essential for the viability of the organism. The regulation of those genes is predominated by translational adjustments. The powerful technique of ribosome profiling was successfully used to generate highly resolved pictures of the translational landscape of Arabidopsis thaliana cytosol, identifying translation of upstream open reading frames and long non-coding transcripts. In addition, differences in plastidial translation and ribosomal pausing sites were addressed with this method. However, a highly resolved picture of the chloroplast translatome is missing. Here, with the use of chloroplast isolation and targeted ribosome affinity purification, I generated highly enriched ribosome profiling datasets of the chloroplasts translatome for Nicotiana tabacum in the dark and light. Chloroplast isolation was found unsuitable for the unbiased analysis of translation in the chloroplast but adequate to identify potential co-translational import. Affinity purification was performed for the small and large ribosomal subunit independently. The enriched datasets mirrored the results obtained from whole-cell ribosome profiling. Enhanced translational activity was detected for psbA in the light. An alternative translation initiation mechanism was not identified by selective enrichment of small ribosomal subunit footprints. In sum, this is the first study that used enrichment strategies to obtain high-depth ribosome profiling datasets of chloroplasts to study ribosome subunit distribution and chloroplast associated translation. Ever-changing light intensities are challenging the photosynthetic capacity of photosynthetic organism. Increased light intensities may lead to over-excitation of photosynthetic reaction centers resulting in damage of the photosystem core subunits. Additional to an expensive repair mechanism for the photosystem II core protein D1, photosynthetic organisms developed various features to reduce or prevent photodamage. In the long-term, photosynthetic complex contents are adjusted for the efficient use of experienced irradiation. However, the contribution of chloroplastic gene expression in the acclimation process remained largely unknown. Here, comparative transcriptome and ribosome profiling was performed for the early time points of high-light acclimation in Nicotiana tabacum chloroplasts in a genome-wide scale. The time- course data revealed stable transcript level and only minor changes in translational activity of specific chloroplast genes during high-light acclimation. Yet, psbA translation was increased by two-fold in the high light from shortly after the shift until the end of the experiment. A stress-inducing shift from low- to high light exhibited increased translation only of psbA. This study indicate that acclimation fails to start in the observed time frame and only short-term responses to reduce photoinhibition were observed.}, language = {en} } @phdthesis{Schaarschmidt2021, author = {Schaarschmidt, Stephanie}, title = {Evaluation and application of omics approaches to characterize molecular responses to abiotic stresses in plants}, doi = {10.25932/publishup-50963}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-509630}, school = {Universit{\"a}t Potsdam}, pages = {viii, 117}, year = {2021}, abstract = {Aufgrund des globalen Klimawandels ist die Gew{\"a}hrleistung der Ern{\"a}hrungssicherheit f{\"u}r eine wachsende Weltbev{\"o}lkerung eine große Herausforderung. Insbesondere abiotische Stressoren wirken sich negativ auf Ernteertr{\"a}ge aus. Um klimaangepasste Nutzpflanzen zu entwickeln, ist ein umfassendes Verst{\"a}ndnis molekularer Ver{\"a}nderungen in der Reaktion auf unterschiedlich starke Umweltbelastungen erforderlich. Hochdurchsatz- oder "Omics"-Technologien k{\"o}nnen dazu beitragen, Schl{\"u}sselregulatoren und Wege abiotischer Stressreaktionen zu identifizieren. Zus{\"a}tzlich zur Gewinnung von Omics-Daten m{\"u}ssen auch Programme und statistische Analysen entwickelt und evaluiert werden, um zuverl{\"a}ssige biologische Ergebnisse zu erhalten. Ich habe diese Problemstellung in drei verschiedenen Studien behandelt und daf{\"u}r zwei Omics-Technologien benutzt. In der ersten Studie wurden Transkript-Daten von den beiden polymorphen Arabidopsis thaliana Akzessionen Col-0 und N14 verwendet, um sieben Programme hinsichtlich ihrer F{\"a}higkeit zur Positionierung und Quantifizierung von Illumina RNA Sequenz-Fragmenten („Reads") zu evaluieren. Zwischen 92\% und 99\% der Reads konnten an die Referenzsequenz positioniert werden und die ermittelten Verteilungen waren hoch korreliert f{\"u}r alle Programme. Bei der Durchf{\"u}hrung einer differentiellen Genexpressionsanalyse zwischen Pflanzen, die bei 20 °C oder 4 °C (K{\"a}lteakklimatisierung) exponiert wurden, ergab sich eine große paarweise {\"U}berlappung zwischen den Programmen. In der zweiten Studie habe ich die Transkriptome von zehn verschiedenen Oryza sativa (Reis) Kultivaren sequenziert. Daf{\"u}r wurde die PacBio Isoform Sequenzierungstechnologie benutzt. Die de novo Referenztranskriptome hatten zwischen 38.900 bis 54.500 hoch qualitative Isoformen pro Sorte. Die Isoformen wurden kollabiert, um die Sequenzredundanz zu verringern und danach evaluiert z.B. hinsichtlich des Vollst{\"a}ndigkeitsgrades (BUSCO), der Transkriptl{\"a}nge und der Anzahl einzigartiger Transkripte pro Genloci. F{\"u}r die hitze- und trockenheitstolerante Sorte N22 wurden ca. 650 einzigartige und neue Transkripte identifiziert, von denen 56 signifikant unterschiedlich in sich entwickelnden Samen unter kombiniertem Trocken- und Hitzestress exprimiert wurden. In der letzten Studie habe ich die Ver{\"a}nderungen in Metabolitprofilen von acht Reissorten gemessen und analysiert, die dem Stress hoher Nachttemperaturen (HNT) ausgesetzt waren und w{\"a}hrend der Trocken- und Regenzeit im Feld auf den Philippinen angebaut wurden. Es wurden jahreszeitlich bedingte Ver{\"a}nderungen im Metabolitspiegel sowie f{\"u}r agronomische Parameter identifiziert und m{\"o}gliche Stoffwechselwege, die einen Ertragsr{\"u}ckgang unter HNT-Bedingungen verursachen, vorgeschlagen. Zusammenfassend konnte ich zeigen, dass der Vergleich der RNA-seq Programme den Pflanzenwissenschaftler*innen helfen kann, sich f{\"u}r das richtige Werkzeug f{\"u}r ihre Daten zu entscheiden. Die de novo Transkriptom-Rekonstruktion von Reissorten ohne Genomsequenz bietet einen gezielten, kosteneffizienten Ansatz zur Identifizierung neuer Gene, die durch verschiedene Stressbedingungen reguliert werden unabh{\"a}ngig vom Organismus. Mit dem Metabolomik-Ansatz f{\"u}r HNT-Stress in Reis habe ich stress- und jahreszeitenspezifische Metabolite identifiziert, die in Zukunft als molekulare Marker f{\"u}r die Verbesserung von Nutzpflanzen verwendet werden k{\"o}nnten.}, language = {en} } @phdthesis{Moga2021, author = {Moga, Akanksha}, title = {Reconstitution of molybdenum cofactor biosynthesis in giant vesicles}, doi = {10.25932/publishup-51016}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-510167}, school = {Universit{\"a}t Potsdam}, pages = {XIV, 148}, year = {2021}, abstract = {Bottom-up synthetic biology is used for the understanding of how a cell works. It is achieved through developing techniques to produce lipid-based vesicular structures as cellular mimics. The most common techniques used to produce cellular mimics or synthetic cells is through electroformation and swelling method. However, the abovementioned techniques cannot efficiently encapsulate macromolecules such as proteins, enzymes, DNA and even liposomes as synthetic organelles. This urges the need to develop new techniques that can circumvent this issue and make the artificial cell a reality where it is possible to imitate a eukaryotic cell through encapsulating macromolecules. In this thesis, the aim to construct a cell system using giant unilamellar vesicles (GUVs) to reconstitute the mitochondrial molybdenum cofactor biosynthetic pathway. This pathway is highly conserved among all life forms, and therefore is known for its biological significance in disorders induced through its malfunctioning. Furthermore, the pathway itself is a multi-step enzymatic reaction that takes place in different compartments. Initially, GTP in the mitochondrial matrix is converted to cPMP in the presence of cPMP synthase. Further, produced cPMP is transported across the membrane to the cytosol, to be converted by MPT synthase into MPT. This pathway provides a possibility to address the general challenges faced in the development of a synthetic cell, to encapsulate large biomolecules with good efficiency and greater control and to evaluate the enzymatic reactions involved in the process. For this purpose, the emulsion-based technique was developed and optimised to allow rapid production of GUVs (~18 min) with high encapsulation efficiency (80\%). This was made possible by optimizing various parameters such as density, type of oil, the impact of centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume. Furthermore, the method was optimised in microtiter plates for direct experimentation and visualization after the GUV formation. Using this technique, the two steps - formation of cPMP from GTP and the formation of MPT from cPMP were encapsulated in different sets of GUVs to mimic the two compartments. Two independent fluorescence-based detection systems were established to confirm the successful encapsulation and conversion of the reactants. Alternatively, the enzymes produced using bacterial expression and measured. Following the successful encapsulation and evaluation of enzymatic reactions, cPMP transport across mitochondrial membrane has been mimicked using GUVs using a complex mitochondrial lipid composition. It was found that the cPMP interaction with the lipid bilayer results in transient pore-formation and leakage of internal contents. Overall, it can be concluded that in this thesis a novel technique has been optimised for fast production of functional synthetic cells. The individual enzymatic steps of the Moco biosynthetic pathway have successfully implemented and quantified within these cellular mimics.}, language = {en} } @phdthesis{Barchewitz2021, author = {Barchewitz, Tino}, title = {Impact of microcystin on the non-canonical localization of RubisCO in the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC7806}, doi = {10.25932/publishup-50829}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-508299}, school = {Universit{\"a}t Potsdam}, pages = {vii, 106}, year = {2021}, abstract = {Cyanobacteria are an abundant bacterial group and are found in a variety of ecological niches all around the globe. They can serve as a real threat for fish or mammals and can restrict the use of lakes or rivers for recreational purposes or as a source of drinking water, when they form blooms. One of the most abundant bloom-forming cyanobacteria is Microcystis aeruginosa. In the first part of the study, the role and possible dynamics of RubisCO in M. aeruginosa during high-light irradiation were examined. Its response was analyzed on the protein and peptide level via immunoblotting, immunofluorescence microscopy and with high performance liquid chromatography (HPLC). It was revealed that large amounts of RubisCO were located outside of carboxysomes under the applied high light stress. RubisCO aggregated mainly underneath the cytoplasmic membrane. There it forms a putative Calvin-Benson-Bassham (CBB) super complex together with other enzymes of photosynthesis. This complex could be part of an alternative carbon-concentrating mechanism (CCM) in M. aeruginosa, which enables a faster, and energy saving adaptation to high light stress of the whole bloom. Furthermore, the re-localization of RubisCO was delayed in the microcystin-deficient mutant ΔmcyB and RubisCO was more evenly distributed over the cell in comparison to the wild type. Since ΔmcyB is not harmed in its growth, possibly other produced cyanopeptides as aeruginosin or cyanopeptolin also play a role in the stabilization of RubisCO and the putative CBB complex, especially in the microcystin-free mutant. In the second part of this work, the possible role of microcystin as an extracellular signaling peptide during the diurnal cycle was studied. HPLC analysis showed a strong increase of extracellular microcystin in the wild type when the population entered nighttime and it resumed into the next day as well. Together with the increase of extracellular microcystin, a strong decrease of protein-bound intracellular microcystin was observed via immunoblot analysis. Interestingly, the signal of the large subunit of RubisCO (RbcL) also diminished when high amounts of microcystin were present in the surrounding medium. Microcystin addition experiments to M. aeruginosa WT and ΔmcyB cultures support this observation, since the immunoblot signal of both subunits of RubisCO and CcmK, a shell protein of carboxysomes, diminished after the addition of microcystin. In addition, the fluctuation of cyanopeptolin during the diurnal cycle indicates a more prominent role of other cyanopeptides besides microcystin as a signaling peptide, intracellularly as well as extracellularly.}, language = {en} } @phdthesis{Gibert2021, author = {Gibert, Arthur}, title = {Influence of Amyloid Aggregates on the Trafficking and Signaling of GPCRs}, doi = {10.25932/publishup-50665}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-506659}, school = {Universit{\"a}t Potsdam}, pages = {100}, year = {2021}, abstract = {The prevalence of diseases associated with misfolded proteins increases with age. When cellular defense mechanisms become limited, misfolded proteins form aggregates and may also develop more stable cross-β structures ultimately forming amyloid aggregates. Amyloid aggregates are associated with neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. The formation of amyloid deposits, their toxicity and cellular defense mechanisms have been intensively studied. However, surprisingly little is known about the effects of protein aggregates on cellular signal transduction. It is also not understood whether the presence of aggregation-prone, but still soluble proteins affect signal transduction. In this study, the still soluble aggregation-prone HttExon1Q74 and its amyloid aggregates were used to analyze the effect of amyloid aggregates on internalization and receptor activation of G protein-coupled receptors (GPCRs), the largest protein family of mammalian cell surface receptors involved in signal transduction. The aggregated HttExon1Q74, but not its soluble form, could inhibit ligand-induced clathrin-mediated endocytosis (CME) of various GPCRs. Most likely this inhibitory effect is based on a terminal sequestration of the HSC70 chaperone to the aggregates which is necessary for CME. Using the vasopressinV1a receptor (V1aR) and the corticotropin-releasing factor receptor 1 (CRF1R) as a model, it could be shown that the presence of HttExon1Q74 aggregates and the inhibition of ligand-induced CME leads to an accumulation of desensitized receptors at the plasma membrane. In turn, this disrupts Gq-mediated Ca2+ signaling and Gs-mediated cAMP signaling of the V1aR and the CRF1R respectively. In contrast to HttExon1Q74 amyloid aggregates, soluble HttExon1Q74 as well as amorphous aggregates did not inhibit GPCR internalization and signaling demonstrating that cellular signal transduction mechanisms are specifically impaired in response to the formation of amyloid aggregates. In addition, preliminary experiments could show that HttExon1Q74 aggregates provoke an increase in membrane expression of a protein from a structurally and functionally unrelated membrane protein family, namely the serotonin transporter SERT. As SERT is the main pharmacological target to treat depression this could shed light on this commonly occurring comorbidity in neurodegenerative diseases, in particular in early disease states.}, language = {en} } @phdthesis{Spinti2021, author = {Spinti, Daniela}, title = {Proteasomal protein turnover during defense priming in Arabidopsis}, doi = {10.25932/publishup-50590}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-505909}, school = {Universit{\"a}t Potsdam}, pages = {x, 164}, year = {2021}, abstract = {The ubiquitin-proteasome-system (UPS) is a cellular cascade involving three enzymatic steps for protein ubiquitination to target them to the 26S proteasome for proteolytic degradation. Several components of the UPS have been shown to be central for regulation of defense responses during infections with phytopathogenic bacteria. Upon recognition of the pathogen, local defense is induced which also primes the plant to acquire systemic resistance (SAR) for enhanced immune responses upon challenging infections. Here, ubiquitinated proteins were shown to accumulate locally and systemically during infections with Psm and after treatment with the SAR-inducing metabolites salicylic acid (SA) and pipecolic acid (Pip). The role of the 26S proteasome in local defense has been described in several studies, but the potential role during SAR remains elusive and was therefore investigated in this project by characterizing the Arabidopsis proteasome mutants rpt2a-2 and rpn12a-1 during priming and infections with Pseudomonas. Bacterial replication assays reveal decreased basal and systemic immunity in both mutants which was verified on molecular level showing impaired activation of defense- and SAR-genes. rpt2a-2 and rpn12a-1 accumulate wild type like levels of camalexin but less SA. Endogenous SA treatment restores local PR gene expression but does not rescue the SAR-phenotype. An RNAseq experiment of Col-0 and rpt2a-2 reveal weak or absent induction of defense genes in the proteasome mutant during priming. Thus, a functional 26S proteasome was found to be required for induction of SAR while compensatory mechanisms can still be initiated. E3-ubiquitin ligases conduct the last step of substrate ubiquitination and thereby convey specificity to proteasomal protein turnover. Using RNAseq, 11 E3-ligases were found to be differentially expressed during priming in Col-0 of which plant U-box 54 (PUB54) and ariadne 12 (ARI12) were further investigated to gain deeper understanding of their potential role during priming. PUB54 was shown to be expressed during priming and /or triggering with virulent Pseudomonas. pub54 I and pub54-II mutants display local and systemic defense comparable to Col-0. The heavy-metal associated protein 35 (HMP35) was identified as potential substrate of PUB54 in yeast which was verified in vitro and in vivo. PUB54 was shown to be an active E3-ligase exhibiting auto-ubiquitination activity and performing ubiquitination of HMP35. Proteasomal turnover of HMP35 was observed indicating that PUB54 targets HMP35 for ubiquitination and subsequent proteasomal degradation. Furthermore, hmp35-I benefits from increased resistance in bacterial replication assays. Thus, HMP35 is potentially a negative regulator of defense which is targeted and ubiquitinated by PUB54 to regulate downstream defense signaling. ARI12 is transcriptionally activated during priming or triggering and hyperinduced during priming and triggering. Gene expression is not inducible by the defense related hormone salicylic acid (SA) and is dampened in npr1 and fmo1 mutants consequently depending on functional SA- and Pip-pathways, respectively. ARI12 accumulates systemically after priming with SA, Pip or Pseudomonas. ari12 mutants are not altered in resistance but stable overexpression leads to increased resistance in local and systemic tissue. During priming and triggering, unbalanced ARI12 levels (i.e. knock out or overexpression) leads to enhanced FMO1 activation indicating a role of ARI12 in Pip-mediated SAR. ARI12 was shown to be an active E3-ligase with auto-ubiquitination activity likely required for activation with an identified ubiquitination site at K474. Mass spectrometrically identified potential substrates were not verified by additional experiments yet but suggest involvement of ARI12 in regulation of ROS in turn regulating Pip-dependent SAR pathways. Thus, data from this project provide strong indications about the involvement of the 26S proteasome in SAR and identified a central role of the two so far barely described E3-ubiquitin ligases PUB54 and ARI12 as novel components of plant defense.}, language = {en} } @phdthesis{Moeser2021, author = {M{\"o}ser, Christin}, title = {Modular DNA constructs for oligovalent bio-enhancement and functional screening}, doi = {10.25932/publishup-50728}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-507289}, school = {Universit{\"a}t Potsdam}, pages = {XIV, 148}, year = {2021}, abstract = {Deoxyribonucleic acid (DNA) nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. This dissertation covers three main projects. All of them use variations of functionalized DNA nanostructures that act as platform for oligovalent presentation of ligands. The purpose of this work was to evaluate the ability of DNA nanostructures to precisely display different types of functional molecules and to consequently enhance their efficacy according to the concept of multivalency. Moreover, functionalized DNA structures were examined for their suitability in functional screening assays. The developed DNA-based compound ligands were used to target structures in different biological systems. One part of this dissertation attempted to bind pathogens with small modified DNA nanostructures. Pathogens like viruses and bacteria are known for their multivalent attachment to host cells membranes. By blocking their receptors for recognition and/or fusion with their targeted host in an oligovalent manner, the objective was to impede their ability to adhere to and invade cells. For influenza A, only enhanced binding of oligovalent peptide-DNA constructs compared to the monovalent peptide could be observed, whereas in the case of respiratory syncytial virus (RSV), binding as well as blocking of the target receptors led to an increased inhibition of infection in vitro. In the final part, the ability of chimeric DNA-peptide constructs to bind to and activate signaling receptors on the surface of cells was investigated. Specific binding of DNA trimers, conjugated with up to three peptides, to EphA2 receptor expressing cells was evaluated in flow cytometry experiments. Subsequently, their ability to activate these receptors via phosphorylation was assessed. EphA2 phosphorylation was significantly increased by DNA trimers carrying three peptides compared to monovalent peptide. As a result of activation, cells underwent characteristic morphological changes, where they "round up" and retract their periphery. The results obtained in this work comprehensively prove the capability of DNA nanostructures to serve as stable, biocompatible, controllable platforms for the oligovalent presentation of functional ligands. Functionalized DNA nanostructures were used to enhance biological effects and as tool for functional screening of bio-activity. This work demonstrates that modified DNA structures have the potential to improve drug development and to unravel the activation of signaling pathways.}, language = {en} } @phdthesis{Ceulemans2021, author = {Ceulemans, Ruben}, title = {Diversity effects on ecosystem functions of tritrophic food webs}, doi = {10.25932/publishup-50325}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-503259}, school = {Universit{\"a}t Potsdam}, pages = {vii, 166}, year = {2021}, abstract = {There is a general consensus that diverse ecological communities are better equipped to adapt to changes in their environment, but our understanding of the mechanisms by which they do so remains incomplete. Accurately predicting how the global biodiversity crisis affects the functioning of ecosystems, and the services they provide, requires extensive knowledge about these mechanisms. Mathematical models of food webs have been successful in uncovering many aspects of the link between diversity and ecosystem functioning in small food web modules, containing at most two adaptive trophic levels. Meaningful extrapolation of this understanding to the functioning of natural food webs remains difficult, due to the presence of complex interactions that are not always accurately captured by bitrophic descriptions of food webs. In this dissertation, we expand this approach to tritrophic food web models by including the third trophic level. Using a functional trait approach, coexistence of all species is ensured using fitness-balancing trade-offs. For example, the defense-growth trade-off implies that species may be defended against predation, but this defense comes at the cost of a lower maximal growth rate. In these food webs, the functional diversity on a given trophic level can be varied by modifying the trait differences between the species on that level. In the first project, we find that functional diversity promotes high biomass on the top level, which, in turn, leads to a reduction in the temporal variability due to compensatory dynamical patterns governed by the top level. Next, these results are generalized by investigating the average behavior of tritrophic food webs, for wide intervals of all parameters describing species interactions in the food web. We find that the diversity on the top level is most important for determining the biomass and temporal variability of all other trophic levels, and show how biomass is only transferred efficiently to the top level when diversity is high everywhere in the food web. In the third project, we compare the response of a simple food chain against a nutrient pulse perturbation, to that of a food web with diversity on every trophic level. By joint consideration of the resistance, resilience, and elasticity, we uncover that the response is efficiently buffered when biomass on the top level is high, which is facilitated by functional diversity on every trophic level in the food web. Finally, in the fourth project, we show that even in a simple consumer-resource model without any diversity, top-down control on the intermediate level frequently causes the phase difference between the intermediate and basal level to deviate from the quarter-cycle lag rule. By adding a top predator, we show that these deviations become even more likely, and anti-phase cycles are often observed. The combined results of these projects show how the properties of the top trophic level, including its functional diversity, have a decisive influence on the functioning of tritrophic food webs from a mechanistic perspective. Because top species are often among the most vulnerable to extinction, our results emphasize the importance of their conservation in ecosystem management and restoration strategies.}, language = {en} } @phdthesis{Siemiatkowska2020, author = {Siemiatkowska, Beata}, title = {Redox signalling in plants}, doi = {10.25932/publishup-48911}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-489119}, school = {Universit{\"a}t Potsdam}, pages = {127}, year = {2020}, abstract = {Once proteins are synthesized, they can additionally be modified by post-translational modifications (PTMs). Proteins containing reactive cysteine thiols, stabilized in their deprotonated form due to their local environment as thiolates (RS-), serve as redox sensors by undergoing a multitude of oxidative PTMs (Ox-PTMs). Ox-PTMs such as S-nitrosylation or formation of inter- or intra-disulfide bridges induce functional changes in these proteins. Proteins containing cysteines, whose thiol oxidation state regulates their functions, belong to the so-called redoxome. Such Ox-PTMs are controlled by site-specific cellular events that play a crucial role in protein regulation, affecting enzyme catalytic sites, ligand binding affinity, protein-protein interactions or protein stability. Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in all photosynthetic organisms. Therefore, studying PTMs will remain crucial for understanding plant adaptation to external stimuli like fluctuating light conditions. Optimizing methods suitable for studying plants Ox-PTMs is of high importance for elucidation of the redoxome in plants. This study focusses on thiol modifications occurring in plant and provides novel insight into in vivo redoxome of Arabidopsis thaliana in response to light vs. dark. This was achieved by utilizing a resin-assisted thiol enrichment approach. Furthermore, confirmation of candidates on the single protein level was carried out by a differential labelling approach. The thiols and disulfides were differentially labelled, and the protein levels were detected using immunoblot analysis. Further analysis was focused on light-reduced proteins. By the enrichment approach many well studied redox-regulated proteins were identified. Amongst those were fructose 1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) which have previously been described as thioredoxin system targeted enzymes. The redox regulated proteins identified in the current study were compared to several published, independent results showing redox regulated proteins in Arabidopsis leaves, root, mitochondria and specifically S-nitrosylated proteins. These proteins were excluded as potential new candidates but remain as a proof-of-concept to the enrichment experiments to be effective. Additionally, CSP41A and CSP41B proteins, which emerged from this study as potential targets of redox-regulation, were analyzed by Ribo-Seq. The active translatome study of csp41a mutant vs. wild-type showed most of the significant changes at end of the night, similarly as csp41b. Yet, in both mutants only several chloroplast-encoded genes were altered. Further studies of CSP41A and CSP41B proteins are needed to reveal their functions and elucidate the role of redox regulation of these proteins.}, language = {en} } @phdthesis{Egli2021, author = {Egli, Lukas}, title = {Stabilizing agricultural systems through diversity}, doi = {10.25932/publishup-49684}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-496848}, school = {Universit{\"a}t Potsdam}, pages = {VII, 125}, year = {2021}, abstract = {In the light of climate change, rising demands for agricultural products and the intensification and specialization of agricultural systems, ensuring an adequate and reliable supply of food is fundamental for food security. Maintaining diversity and redundancy has been postulated as one generic principle to increase the resilience of agricultural production and other ecosystem services. For example, if one crop fails due to climate instability and extreme events, others can compensate the losses. Crop diversity might be particularly important if different crops show asynchronous production trends. Furthermore, spatial heterogeneity has been suggested to increase stability at larger scales as production losses in some areas can be buffered by surpluses in undisturbed ones. Besides systematically investigating the mechanisms underlying stability, identifying transformative pathways that foster them is important. In my thesis, I aim at answering the following questions: (i) How does yield stability differ between nations, regions and farms, and what is the effect of crop diversity on yield stability in relation to agricultural inputs, climate heterogeneity, climate instability and time at the national, regional or farm level? (ii) Is asynchrony between crops a better predictor of production stability than crop diversity? (iii) What is the effect of asynchrony between and within crops on stability and how is it related to crop diversity and space, respectively? (iv) What is the state of the art and what are knowledge gaps in exploring resilience and its multidimensionality in ecological and social-ecological systems with agent-based models and what are potential ways forward? In the first chapter, I provide the theoretical background for the subsequent analyses. I stress the need to better understand the resilience of social-ecological systems and particularly the stability of agricultural production. Moreover, I introduce diversity and spatial heterogeneity as two prominently discussed resilience mechanisms and describe approaches to assess resilience. In the second chapter, I combined agriculture and climate data at three levels of organization and spatial extents to investigate yield stability patterns and their relation to crop diversity, fertilizer, irrigation, climate heterogeneity and instability and time of nations globally, regions in Europe and farms in Germany using statistical analyses. Yield stability decreased from the national to the farm level. Several nations and regions substantially contributed to larger-scale stability. Crop diversity was positively associated with yield stability across all three levels of organization. This effect was typically more profound at smaller scales and in variable climates. In addition to crop diversity, climate heterogeneity was an important stabilizing mechanism especially at larger scales. These results confirm the stabilizing effect of crop diversity and spatial heterogeneity, yet their importance depends on the scale and agricultural management. Building on the findings of the second chapter, I deepened in the third chapter my research on the effect of crop diversity at the national level. In particular, I tested if asynchrony between crops, i.e. between the temporal production patterns of different crops, better predicts agricultural production stability than crop diversity. The stabilizing effect of asynchrony was multiple times higher than the effect of crop diversity, i.e. asynchrony is one important property that can explain why a higher diversity supports the stability of national food production. Therefore, strategies to stabilize agricultural production through crop diversification also need to account for the asynchrony of the crops considered. The previous chapters suggest that both asynchrony between crops and spatial heterogeneity are important stabilizing mechanisms. In the fourth chapter, I therefore aimed at better understanding the relative importance of asynchrony between and within crops, i.e. between the temporal production patterns of different crops and between the temporal production patterns of different cultivation areas of the same crop. Better understanding their relative importance is important to inform agricultural management decisions, but so far this has been hardly assessed. To address this, I used crop production data to study the effect of asynchrony between and within crops on the stability of agricultural production in regions in Germany and nations in Europe. Both asynchrony between and within crops consistently stabilized agricultural production. Adding crops increased asynchrony between crops, yet this effect levelled off after eight crops in regions in Germany and after four crops in nations in Europe. Combining already ten farms within a region led to high asynchrony within crops, indicating distinct production patters, while this effect was weaker when combining multiple regions within a nation. The results suggest, that both mechanisms need to be considered in agricultural management strategies that strive for more resilient farming systems. The analyses in the foregoing chapters focused at different levels of organization, scales and factors potentially influencing agricultural stability. However, these statistical analyses are restricted by data availability and investigate correlative relationships, thus they cannot provide a mechanistic understanding of the actual processes underlying resilience. In this regard, agent-based models (ABM) are a promising tool. Besides their ability to measure different properties and to integrate multiple situations through extensive manipulation in a fully controlled system, they can capture the emergence of system resilience from individual interactions and feedbacks across different levels of organization. In the fifth chapter, I therefore reviewed the state of the art and potential knowledge gaps in exploring resilience and its multidimensionality in ecological and social-ecological systems with ABMs. Next, I derived recommendations for a more effective use of ABMs in resilience research. The review suggests that the potential of ABMs is not utilized in most models as they typically focus on a single dimension of resilience and are mostly limited to one reference state, disturbance type and scale. Moreover, only few studies explicitly test the ability of different mechanisms to support resilience. To solve real-world problems related to the resilience of complex systems, ABMs need to assess multiple stability properties for different situations and under consideration of the mechanisms that are hypothesized to render a system resilient. In the sixth chapter, I discuss the major conclusions that can be drawn from the previous chapters. Moreover, I showcase the use of simulation models to identify management strategies to enhance asynchrony and thus stability, and the potential of ABMs to identify pathways to implement such strategies. The results of my thesis confirm the stabilizing effect of crop diversity, yet its importance depends on the scale, agricultural management and climate. Moreover, strategies to stabilize agricultural production through crop diversification also need to account for the asynchrony of the crops considered. As spatial heterogeneity and particularly asynchrony within crops strongly enhances stability, integrated management approaches are needed that simultaneously address multiple resilience mechanisms at different levels of organization, scales and time horizons. For example, the simulation suggests that only increasing the number of crops at both the pixel and landscape level avoids trade-offs between asynchrony between and within crops. If their potential is better exploited, agent-based models have the capacity to systematically assess resilience and to identify comprehensive pathways towards resilient farming systems.}, language = {en} } @phdthesis{Šustr2020, author = {Šustr, David}, title = {Molecular diffusion in polyelectrolyte multilayers}, doi = {10.25932/publishup-48903}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-489038}, school = {Universit{\"a}t Potsdam}, pages = {106}, year = {2020}, abstract = {Research on novel and advanced biomaterials is an indispensable step towards their applications in desirable fields such as tissue engineering, regenerative medicine, cell culture, or biotechnology. The work presented here focuses on such a promising material: polyelectrolyte multilayer (PEM) composed of hyaluronic acid (HA) and poly(L-lysine) (PLL). This gel-like polymer surface coating is able to accumulate (bio-)molecules such as proteins or drugs and release them in a controlled manner. It serves as a mimic of the extracellular matrix (ECM) in composition and intrinsic properties. These qualities make the HA/PLL multilayers a promising candidate for multiple bio-applications such as those mentioned above. The work presented aims at the development of a straightforward approach for assessment of multi-fractional diffusion in multilayers (first part) and at control of local molecular transport into or from the multilayers by laser light trigger (second part). The mechanism of the loading and release is governed by the interaction of bioactives with the multilayer constituents and by the diffusion phenomenon overall. The diffusion of a molecule in HA/PLL multilayers shows multiple fractions of different diffusion rate. Approaches, that are able to assess the mobility of molecules in such a complex system, are limited. This shortcoming motivated the design of a novel evaluation tool presented here. The tool employs a simulation-based approach for evaluation of the data acquired by fluorescence recovery after photobleaching (FRAP) method. In this approach, possible fluorescence recovery scenarios are primarily simulated and afterwards compared with the data acquired while optimizing parameters of a model until a sufficient match is achieved. Fluorescent latex particles of different sizes and fluorescein in an aqueous medium are utilized as test samples validating the analysis results. The diffusion of protein cytochrome c in HA/PLL multilayers is evaluated as well. This tool significantly broadens the possibilities of analysis of spatiotemporal FRAP data, which originate from multi-fractional diffusion, while striving to be widely applicable. This tool has the potential to elucidate the mechanisms of molecular transport and empower rational engineering of the drug release systems. The second part of the work focuses on the fabrication of such a spatiotemporarily-controlled drug release system employing the HA/PLL multilayer. This release system comprises different layers of various functionalities that together form a sandwich structure. The bottom layer, which serves as a reservoir, is formed by HA/PLL PEM deposited on a planar glass substrate. On top of the PEM, a layer of so-called hybrids is deposited. The hybrids consist of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) -based hydrogel microparticles with surface-attached gold nanorods. The layer of hybrids is intended to serve as a gate that controls the local molecular transport through the PEM-solution-interface. The possibility of stimulating the molecular transport by near-infrared (NIR) laser irradiation is being explored. From several tested approaches for the deposition of hybrids onto the PEM surface, the drying-based approach was identified as optimal. Experiments, that examine the functionality of the fabricated sandwich at elevated temperature, document the reversible volume phase transition of the PEM-attached hybrids while sustaining the sandwich stability. Further, the gold nanorods were shown to effectively absorb light radiation in the tissue- and cell-friendly NIR spectral region while transducing the energy of light into heat. The rapid and reversible shrinkage of the PEM-attached hybrids was thereby achieved. Finally, dextran was employed as a model transport molecule. It loads into the PEM reservoir in a few seconds with the partition constant of 2.4, while it spontaneously releases in a slower, sustained manner. The local laser irradiation of the sandwich, which contains the fluorescein isothiocyanate tagged dextran, leads to a gradual reduction of fluorescence intensity in the irradiated region. The release system fabricated employs renowned photoresponsivity of the hybrids in an innovative setting. The results of the research are a step towards a spatially-controlled on-demand drug release system that paves the way to spatiotemporally controlled drug release. The approaches developed in this work have the potential to elucidate the molecular dynamics in ECM and to foster engineering of multilayers with properties tuned to mimic the ECM. The work aims at spatiotemporal control over the diffusion of bioactives and their presentation to the cells.}, language = {en} } @phdthesis{Banerjee2020, author = {Banerjee, Pallavi}, title = {Glycosylphosphatidylinositols (GPIs) and GPI-anchored proteins tethered to lipid bilayers}, doi = {10.25932/publishup-48956}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-489561}, school = {Universit{\"a}t Potsdam}, pages = {xv, 141}, year = {2020}, abstract = {Glycosylphosphatidylinositols (GPIs) are highly complex glycolipids that serve as membrane anchors to a large variety of eukaryotic proteins. These are covalently attached to a group of peripheral proteins called GPI-anchored proteins (GPI-APs) through a post-translational modification in the endoplasmic reticulum. The GPI anchor is a unique structure composed of a glycan, with phospholipid tail at one end and a phosphoethanolamine linker at the other where the protein attaches. The glycan part of the GPI comprises a conserved pseudopentasaccharide core that could branch out to carry additional glycosyl or phosphoethanolamine units. GPI-APs are involved in a diverse range of cellular processes, few of which are signal transduction, protein trafficking, pathogenesis by protozoan parasites like the malaria- causing parasite Plasmodium falciparum. GPIs can also exist freely on the membrane surface without an attached protein such as those found in parasites like Toxoplasma gondii, the causative agent of Toxoplasmosis. These molecules are both structurally and functionally diverse, however, their structure-function relationship is still poorly understood. This is mainly because no clear picture exists regarding how the protein and the glycan arrange with respect to the lipid layer. Direct experimental evidence is rather scarce, due to which inconclusive pictures have emerged, especially regarding the orientation of GPIs and GPI-APs on membrane surfaces and the role of GPIs in membrane organization. It appears that computational modelling through molecular dynamics simulations would be a useful method to make progress. In this thesis, we attempt to explore characteristics of GPI anchors and GPI-APs embedded in lipid bilayers by constructing molecular models at two different resolutions - all-atom and coarse-grained. First, we show how to construct a modular molecular model of GPIs and GPI-anchored proteins that can be readily extended to a broad variety of systems, addressing the micro-heterogeneity of GPIs. We do so by creating a hybrid link to which GPIs of diverse branching and lipid tails of varying saturation with their optimized force fields, GLYCAM06 and Lipid14 respectively, can be attached. Using microsecond simulations, we demonstrate that GPI prefers to "flop-down" on the membrane, thereby, strongly interacting with the lipid heads, over standing upright like a "lollipop". Secondly, we extend the model of the GPI core to carry out a systematic study of the structural aspects of GPIs carrying different side chains (parasitic and human GPI variants) inserted in lipid bilayers. Our results demonstrate the importance of the side branch residues as these are the most accessible, and thereby, recognizable epitopes. This finding qualitatively agrees with experimental observations that highlight the role of the side branches in immunogenicity of GPIs and the specificity thereof. The overall flop-down orientation of the GPIs with respect to the bilayer surface presents the side chain residues to face the solvent. Upon attaching the green fluorescent protein (GFP) to the GPI, it is seen to lie in close proximity to the bilayer, interacting both with the lipid heads and glycan part of the GPI. However the orientation of GFP is sensitive to the type of GPI it is attached to. Finally, we construct a coarse-grained model of the GPI and GPI-anchored GFP using a modified version of the MARTINI force-field, using which the timescale is enhanced by at least an order of magnitude compared to the atomistic system. This study provides a theoretical perspective on the conformational behavior of the GPI core and some of its branched variations in presence of lipid bilayers, as well as draws comparisons with experimental observations. Our modular atomistic model of GPI can be further employed to study GPIs of variable branching, and thereby, aid in designing future experiments especially in the area of vaccines and drug therapies. Our coarse-grained model can be used to study dynamic aspects of GPIs and GPI-APs w.r.t plasma membrane organization. Furthermore, the backmapping technique of converting coarse-grained trajectory back to the atomistic model would enable in-depth structural analysis with ample conformational sampling.}, language = {en} } @phdthesis{Fontana2020, author = {Fontana, Federica}, title = {Antagonistic activities of Vegfr3/Flt4 and Notch1b fine-tune mechanosensitive signaling during zebrafish cardiac valvulogenesis}, doi = {10.25932/publishup-48751}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-487517}, school = {Universit{\"a}t Potsdam}, pages = {III, 110}, year = {2020}, abstract = {Cardiac valves are essential for the continuous and unidirectional flow of blood throughout the body. During embryonic development, their formation is strictly connected to the mechanical forces exerted by blood flow. The endocardium that lines the interior of the heart is a specialized endothelial tissue and is highly sensitive to fluid shear stress. Endocardial cells harbor a signal transduction machinery required for the translation of these forces into biochemical signaling, which strongly impacts cardiac morphogenesis and physiology. To date, we lack a solid understanding on the mechanisms by which endocardial cells sense the dynamic mechanical stimuli and how they trigger different cellular responses. In the zebrafish embryo, endocardial cells at the atrioventricular canal respond to blood flow by rearranging from a monolayer to a double-layer, composed of a luminal cell population subjected to blood flow and an abluminal one that is not exposed to it. These early morphological changes lead to the formation of an immature valve leaflet. While previous studies mainly focused on genes that are positively regulated by shear stress, the mechanisms regulating cell behaviors and fates in cells that lack the stimulus of blood flow are largely unknown. One key discovery of my work is that the flow-sensitive Notch receptor and Kr{\"u}ppel-like factor (Klf) 2, one of the best characterized flow-regulated transcriptional factors, are activated by shear stress but that they function in two parallel signal transduction pathways. Each of these two pathways is essential for the rearrangement of atrioventricular cells into an immature double-layered valve leaflets. A second key discovery of my study is the finding that both Notch and Klf2 signaling negatively regulate the expression of the angiogenesis receptor Vegfr3/Flt4, which becomes restricted to abluminal endocardial cells of the valve leaflet. Within these cells, Flt4 downregulates the expressions of the cell adhesion proteins Alcam and VE-cadherin. A loss of Flt4 causes abluminal endocardial cells to ectopically express Notch, which is normally restricted to luminal cells, and impairs valve morphology. My study suggests that abluminal endocardial cells that do not experience mechanical stimuli loose Notch expression and this triggers expression of Flt4. In turn, Flt4 negatively regulates Notch on the abluminal side of the valve leaflet. These antagonistic signaling activities and fine-tuned gene regulatory mechanisms ultimately shape cardiac valve leaflets by inducing unique differences in the fates of endocardial cells.}, language = {en} } @phdthesis{Aneley2020, author = {Aneley, Gedif Mulugeta}, title = {Drought tolerance prediction of potato by automatic phenotyping of morphological and physiological traits}, doi = {10.25932/publishup-48683}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-486836}, school = {Universit{\"a}t Potsdam}, pages = {xi, 176}, year = {2020}, abstract = {Potato is the 4th most important food crop in the world. Especially in tropical and sub-tropical potato production, drought is a yield limiting factor. Potato is sensitive to water stress. Potato yield loss under water stress could be reduced by using tolerant varieties and adjusted agronomic practices. Direct selection for yield under water-stressed conditions requires long selection cycles. Thus, identification of markers for marker-assisted selection may speed up breeding. The objective of this thesis is to identify morphological markers for drought tolerance by continuously monitoring plant growth and canopy temperature with an automatic phenotyping system. The phenotyping was performed in drought-stress experiments that were conducted in population A with 64 genotypes and population B with 21 genotypes in the screenhouse in 2015 and 2016 (population A) and in 2017 and 2018 (population B). Drought tolerance was quantified as deviation of the relative tuber starch yield from the experimental median (DRYM) and parent median (DRYMp). Relative tuber starch yield is starch yield under drought stress relative to the average starch yield of the respective cultivar under control conditions in the same experiment. The specific DRYM value was calculated based on the yield data of the same experiment or the global DRYM that was calculated from yield data derived from data combined over yeas of respective population or across multiple experiments including VALDIS and TROST experiments (2011-2016). Analysis of variance found a significant effect of genotype on DRYM indicating that the tolerance variation required for marker identification was given in both populations. Canopy growth was monitored continuously six times a day over five to ten weeks by a laser scanner system and yielded information on leaf area, plant height and leaf angle for population A and additionally on leaf inclination and light penetration depth for population B. Canopy temperature was measured 48 times a day over six to seven weeks by infrared thermometry in population B. From the continuous IRT surface temperature data set, the canopy temperature for each plant was selected by matching the time stamp of the IRT data with laser scanner data. Mean, maximum, range and growth rate values were calculated from continuous laser scanner measurements of respective canopy parameters. Among the canopy parameters, the maximum and mean values in long-term stress conditions showed better correlation with DRYM values calculated in the same experiment than growth rate and diurnal range values. Therefore, drought tolerance index prediction was done from maximum and mean values of canopy parameters. The tolerance index in specific experiment condition was linearly predicted by simple regression model from different single canopy parameters under long-term stress condition in population A (2016) and population B (2017 and 2018). Among the canopy parameters maximum light penetration depth (2017), mean leaf angle (2017, 2018, and 2016), mean leaf inclination or mean canopy temperature depression (2017 and 2018), maximum plant height (2017) were selected as tolerance predictors. However, no single parameters were sufficient to predict DRYM. Therefore, several independent parameters were integrated in a multiple regression model. In multiple regression model, specific experiment DRYM values in population A was predicted from mean leaf angle (2016). In population B, specific tolerance could be predicted from maximum light penetration depth and mean leaf inclination (2017) and mean leaf inclination (2018) or mean canopy temperature depression and mean leaf angle (2018). In data combined over season of population A, the multiple linear regression model selected maximum plant height and mean leaf angle as tolerance predictor. In Population B, mean leaf inclination was selected as tolerance predictor. However, in population A, the variation explained by the final model was too low. Furthermore, the average tolerances respective to parent median (2011-2018) across FGH plants or all plants (FGH and field) were predicted from maximum plant height (population A) and maximum plant height and mean leaf inclination (population B). Altogether, canopy parameters could be used as markers for drought tolerance. Therefore, water stress breeding in potato could be speed up through using leaf inclination, light penetration depth, plant height and canopy temperature depression as markers for drought tolerance, especially in long-term stress conditions.}, language = {en} } @phdthesis{ALRawi2020, author = {AL-Rawi, Shadha}, title = {Biochemical studies to determine the role of Early Starvation 1 (ESV1) protein and its homologue Like-Early Starvation 1 (LESV) during starch degradation}, doi = {10.25932/publishup-48395}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-483956}, school = {Universit{\"a}t Potsdam}, pages = {215}, year = {2020}, abstract = {Depending on the biochemical and biotechnical approach, the aim of this work was to understand the mechanism of protein-glucan interactions in regulation and control of starch degradation. Although starch degradation starts with the phosphorylation process, the mechanisms by which this process is controlling and adjusting starch degradation are not yet fully understood. Phosphorylation is a major process performed by the two dikinases enzymes α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). GWD and PWD enzymes phosphorylate the starch granule surface; thereby stimulate starch degradation by hydrolytic enzymes. Despite these important roles for GWD and PWD, so far the biochemical processes by which these enzymes are able to regulate and adjust the rate of phosphate incorporation into starch during the degradation process haven't been understood. Recently, some proteins were found associated with the starch granule. Two of these proteins are named Early Starvation Protein 1 (ESV1) and its homologue Like-Early Starvation Protein 1 (LESV). It was supposed that both are involved in the control of starch degradation, but their function has not been clearly known until now. To understand how ESV1 and LESV-glucan interactions are regulated and affect the starch breakdown, it was analyzed the influence of ESV1 and LESV proteins on the phosphorylating enzyme GWD and PWD and hydrolysing enzymes ISA, BAM, and AMY. However, the analysis determined the location of LESV and ESV1 in the chloroplast stroma of Arabidopsis. Mass spectrometry data predicted ESV1and LESV proteins as a product of the At1g42430 and At3g55760 genes with a predicted mass of ~50 kDa and ~66 kDa, respectively. The ChloroP program predicted that ESV1 lacks the chloroplast transit peptide, but it predicted the first 56 amino acids N-terminal region as a chloroplast transit peptide for LESV. Usually, the transit peptide is processed during transport of the proteins into plastids. Given that this processing is critical, two forms of each ESV1 and LESV were generated and purified, a full-length form and a truncated form that lacks the transit peptide, namely, (ESV1and tESV1) and (LESV and tLESV), respectively. Both protein forms were included in the analysis assays, but only slight differences in glucan binding and protein action between ESV1 and tESV1 were observed, while no differences in the glucan binding and effect on the GWD and PWD action were observed between LESV and tLESV. The results revealed that the presence of the N-terminal is not massively altering the action of ESV1 or LESV. Therefore, it was only used the ESV1 and tLESV forms data to explain the function of both proteins. However, the analysis of the results revealed that LESV and ESV1 proteins bind strongly at the starch granule surface. Furthermore, not all of both proteins were released after their incubation with starches after washing the granules with 2\% [w/v] SDS indicates to their binding to the deeper layers of the granule surface. Supporting of this finding comes after the binding of both proteins to starches after removing the free glucans chains from the surface by the action of ISA and BAM. Although both proteins are capable of binding to the starch structure, only LESV showed binding to amylose, while in ESV1, binding was not observed. The alteration of glucan structures at the starch granule surface is essential for the incorporation of phosphate into starch granule while the phosphorylation of starch by GWD and PWD increased after removing the free glucan chains by ISA. Furthermore, PWD showed the possibility of starch phosphorylation without prephosphorylation by GWD. Biochemical studies on protein-glucan interactions between LESV or ESV1 with different types of starch showed a potentially important mechanism of regulating and adjusting the phosphorylation process while the binding of LESV and ESV1 leads to altering the glucan structures of starches, hence, render the effect of the action of dikinases enzymes (GWD and PWD) more able to control the rate of starch degradation. Despite the presence of ESV1 which revealed an antagonistic effect on the PWD action as the PWD action was decreased without prephosphorylation by GWD and increased after prephosphorylation by GWD (Chapter 4), PWD showed a significant reduction in its action with or without prephosphorylation by GWD in the presence of ESV1 whether separately or together with LESV (Chapter 5). However, the presence of LESV and ESV1 together revealed the same effect compared to the effect of each one alone on the phosphorylation process, therefore it is difficult to distinguish the specific function between them. However, non-interactions were detected between LESV and ESV1 or between each of them with GWD and PWD or between GWD and PWD indicating the independent work for these proteins. It was also observed that the alteration of the starch structure by LESV and ESV1 plays a role in adjusting starch degradation rates not only by affecting the dikinases but also by affecting some of the hydrolysing enzymes since it was found that the presence of LESV and ESV1leads to the reduction of the action of BAM, but does not abolish it.}, language = {en} } @phdthesis{Ghandour2020, author = {Ghandour, Rabea}, title = {Identification of chloroplast translational feedback regulation and establishment of aptamer based mRNA purification to unravel involved regulatory factors}, doi = {10.25932/publishup-48289}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-482896}, school = {Universit{\"a}t Potsdam}, pages = {XIII, 173}, year = {2020}, abstract = {After endosymbiosis, chloroplasts lost most of their genome. Many former endosymbiotic genes are now nucleus-encoded and the products are re-imported post-translationally. Consequently, photosynthetic complexes are built of nucleus- and plastid-encoded subunits in a well-defined stoichiometry. In Chlamydomonas, the translation of chloroplast-encoded photosynthetic core subunits is feedback-regulated by the assembly state of the complexes they reside in. This process is called Control by Epistasy of Synthesis (CES) and enables the efficient production of photosynthetic core subunits in stoichiometric amounts. In chloroplasts of embryophytes, only Rubisco subunits have been shown to be feedback-regulated. That opens the question if there is additional CES regulation in embryophytes. I analyzed chloroplast gene expression in tobacco and Arabidopsis mutants with assembly defects for each photosynthetic complex to broadly answer this question. My results (i) confirmed CES within Rubisco and hint to potential translational feedback regulation in the synthesis of (ii) cytochrome b6f (Cyt b6f) and (iii) photosystem II (PSII) subunits. This work suggests a CES network in PSII that links psbD, psbA, psbB, psbE, and potentially psbH expression by a feedback mechanism that at least partially differs from that described in Chlamydomonas. Intriguingly, in the Cyt b6f complex, a positive feedback regulation that coordinates the synthesis of PetA and PetB was observed, which was not previously reported in Chlamydomonas. No evidence for CES interactions was found in the expression of NDH and ATP synthase subunits of embryophytes. Altogether, this work provides solid evidence for novel assembly-dependent feedback regulation mechanisms controlling the expression of photosynthetic genes in chloroplasts of embryophytes. In order to obtain a comprehensive inventory of the rbcL and psbA RNA-binding proteomes (including factors that regulate their expression, especially factors involved in CES), an aptamer based affinity purification method was adapted and refined for the specific purification these transcripts from tobacco chloroplasts. To this end, three different aptamers (MS2, Sephadex ,and streptavidin binding) were stably introduced into the 3' UTRs of psbA and rbcL by chloroplast transformation. RNA aptamer based purification and subsequent chip analysis (RAP Chip) demonstrated a strong enrichment of psbA and rbcL transcripts and currently, ongoing mass spectrometry analyses shall reveal potential regulatory factors. Furthermore, the suborganellar localization of MS2 tagged psbA and rbcL transcripts was analyzed by a combined affinity, immunology, and electron microscopy approach and demonstrated the potential of aptamer tags for the examination of the spatial distribution of chloroplast transcripts.}, language = {en} } @phdthesis{Crawford2020, author = {Crawford, Michael Scott}, title = {Using individual-based modeling to understand grassland diversity and resilience in the Anthropocene}, doi = {10.25932/publishup-47941}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-479414}, school = {Universit{\"a}t Potsdam}, pages = {174}, year = {2020}, abstract = {The world's grassland systems are increasingly threatened by anthropogenic change. Susceptible to a variety of different stressors, from land-use intensification to climate change, understanding the mechanisms driving the maintenance of these systems' biodiversity and stability, and how these mechanisms may shift under human-mediated disturbance, is thus critical for successfully navigating the next century. Within this dissertation, I use an individual-based and spatially-explicit model of grassland community assembly (IBC-grass) to examine several processes, thought key to understanding their biodiversity and stability and how it changes under stress. In the first chapter of my thesis, I examine the conditions under which intraspecific trait variation influences the diversity of simulated grassland communities. In the second and third chapters of my thesis, I shift focus towards understanding how belowground herbivores influence the stability of these grassland systems to either a disturbance that results in increased, stochastic, plant mortality, or eutrophication. Intraspecific trait variation (ITV), or variation in trait values between individuals of the same species, is fundamental to the structure of ecological communities. However, because it has historically been difficult to incorporate into theoretical and statistical models, it has remained largely overlooked in community-level analyses. This reality is quickly shifting, however, as a consensus of research suggests that it may compose a sizeable proportion of the total variation within an ecological community and that it may play a critical role in determining if species coexist. Despite this increasing awareness that ITV matters, there is little consensus of the magnitude and direction of its influence. Therefore, to better understand how ITV changes the assembly of grassland communities, in the first chapter of my thesis, I incorporate it into an established, individual-based grassland community model, simulating both pairwise invasion experiments as well as the assembly of communities with varying initial diversities. By varying the amount of ITV in these species' functional traits, I examine the magnitude and direction of ITV's influence on pairwise invasibility and community coexistence. During pairwise invasion, ITV enables the weakest species to more frequently invade the competitively superior species, however, this influence does not generally scale to the community level. Indeed, unless the community has low alpha- and beta- diversity, there will be little effect of ITV in bolstering diversity. In these situations, since the trait axis is sparsely filled, the competitively inferior may suffer less competition and therefore ITV may buffer the persistence and abundance of these species for some time. In the second and third chapters of my thesis, I model how one of the most ubiquitous trophic interactions within grasslands, herbivory belowground, influences their diversity and stability. Until recently, the fundamental difficulty in studying a process within the soil has left belowground herbivory "out of sight, out of mind." This dilemma presents an opportunity for simulation models to explore how this understudied process may alter community dynamics. In the second chapter of my thesis, I implement belowground herbivory - represented by the weekly removal of plant biomass - into IBC-grass. Then, by introducing a pulse disturbance, modelled as the stochastic mortality of some percentage of the plant community, I observe how the presence of belowground herbivores influences the resistance and recovery of Shannon diversity in these communities. I find that high resource, low diversity, communities are significantly more destabilized by the presence of belowground herbivores after disturbance. Depending on the timing of the disturbance and whether the grassland's seed bank persists for more than one season, the impact of the disturbance - and subsequently the influence of the herbivores - can be greatly reduced. However, because human-mediated eutrophication increases the amount of resources in the soil, thus pressuring grassland systems, our results suggest that the influence of these herbivores may become more important over time. In the third chapter of my thesis, I delve further into understanding the mechanistic underpinnings of belowground herbivores on the diversity of grasslands by replicating an empirical mesocosm experiment that crosses the presence of herbivores above- and below-ground with eutrophication. I show that while aboveground herbivory, as predicted by theory and frequently observed in experiments, mitigates the impact of eutrophication on species diversity, belowground herbivores counterintuitively reduce biodiversity. Indeed, this influence positively interacts with the eutrophication process, amplifying its negative impact on diversity. I discovered the mechanism underlying this surprising pattern to be that, as the herbivores consume roots, they increase the proportion of root resources to root biomass. Because root competition is often symmetric, herbivory fails to mitigate any asymmetries in the plants' competitive dynamics. However, since the remaining roots have more abundant access to resources, the plants' competition shifts aboveground, towards asymmetric competition for light. This leads the community towards a low-diversity state, composed of mostly high-performance, large plant species. We further argue that this pattern will emerge unless the plants' root competition is asymmetric, in which case, like its counterpart aboveground, belowground herbivory may buffer diversity by reducing this asymmetry between the competitively superior and inferior plants. I conclude my dissertation by discussing the implications of my research on the state of the art in intraspecific trait variation and belowground herbivory, with emphasis on the necessity of more diverse theory development in the study of these fundamental interactions. My results suggest that the influence of these processes on the biodiversity and stability of grassland systems is underappreciated and multidimensional, and must be thoroughly explored if researchers wish to predict how the world's grasslands will respond to anthropogenic change. Further, should researchers myopically focus on understanding central ecological interactions through only mathematically tractable analyses, they may miss entire suites of potential coexistence mechanisms that can increase the coviability of species, potentially leading to coexistence over ecologically-significant timespans. Individual-based modelling, therefore, with its focus on individual interactions, will prove a critical tool in the coming decades for understanding how local interactions scale to larger contexts, and how these interactions shape ecological communities and further predicting how these systems will change under human-mediated stress.}, language = {en} } @phdthesis{Dehm2020, author = {Dehm, Daniel}, title = {Development of concepts for the genomic mining of novel secondary metabolites in symbiotic cyanobacteria}, doi = {10.25932/publishup-47834}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-478342}, school = {Universit{\"a}t Potsdam}, pages = {VIII, 122, XII}, year = {2020}, abstract = {Naturstoffe sind seit der goldenen {\"A}ra der Antibiotika von immer gr{\"o}ßerem Interesse, sowohl f{\"u}r die Grundlagenforschung als auch die Angewandten Wissenschaften, da sie die Hauptquelle f{\"u}r neuartige Pharmazeutika mit starken antibiotischen, anti-entz{\"u}ndlichen und Antitumor-Aktivit{\"a}ten darstellen. Neben den technologischen Fortschritten im Bereich der Hochdurchsatz Genomsequenzierung und dem verbesserten Verst{\"a}ndnis des modularen Aufbaus der Biosynthesewege von Sekund{\"a}rmetaboliten, kam es auch zu einem Wechsel vom labor-gest{\"u}tzten Screening aktiver Zellextrakte hin zum Algorithmen-basierten in silico Screening nach neuen Naturstoff-Biosyntheseclustern. Obwohl die steigende Zahl verf{\"u}gbarer Genomsequenzen zeigte, dass nicht-ribosomale Peptid-Synthetasen (NRPS), Polyketid-Synthasen (PKS), und ribosomal synthetisierte und posttranslational modifizierte Peptide (RiPPs) ubiquit{\"a}r in allen Sparten des Lebens gefunden werden k{\"o}nnen, so zeigen einige Phyla wie Actinobakterien oder Cyanobakterien eine besonders hohe Dichte an Sekund{\"a}rmetabolitclustern. Der fakultativ symbiotische, N2-fixierende Modellorganismus N. punctiforme PCC73102 ist ein terrestrisches typ-IV Cyanobakterium, welches nicht nur einen besonders hohen Anteil seines Genoms der Produktion von Sekund{\"a}rmetaboliten widmet, sondern zus{\"a}tzlich noch genetisch modifizierbar ist. Eine AntiSMASH Analyse des Genoms zeigte, dass N. punctiforme insgesamt sechzehn potentielle Sekund{\"a}rmetabolitcluster besitzt, von denen aber bis heute nur zweien ein spezifisches Produkt zugewiesen werden konnte. Das macht N. punctiforme zu einem perfekten Testorganismus f{\"u}r die Entwicklung eines neuartigen kombinatorischen Genomic Mining Ansatzes zur Detektion von bislang unbeschriebenen Naturstoffen. Der neuartige Ansatz, der im Rahmen dieser Studie entwickelt wurde, stellt eine Kombination aus Genomic Mining, unabh{\"a}ngigen Monitoring-Techniken sowie modifizierten Kultivierungsbedingungen dar und f{\"u}hrte nicht nur zu neuen Erkenntnissen im Bereich cyanobakterieller Naturstoffsynthese, sondern letztlich auch zur Entdeckung eines neuen, von N. punctiforme produzierten, Naturstoffs. Die Herstellung und Untersuchung einer Reporterstamm Bibliothek, bestehend aus je einem CFP-produzierenden Transkriptionsreporter f{\"u}r jedes der sechzehn Sekund{\"a}rmetabolitcluster von N. punctiforme, zeigte, dass im Gegensatz zur Erwartung nicht alle Biosynthesecluster f{\"u}r die man kein Produkt nachweisen kann auch nicht exprimiert werden. Stattdessen konnten klar definierbare Expressionsmuster beschrieben werden, was deutlich machte, dass die Naturstoffproduktion einer engen Regulation unterliegt und nur ein kleiner Teil der Biosynthesecluster unter Standardbedingungen tats{\"a}chlich still sind. Dar{\"u}ber hinaus f{\"u}hrte die Erh{\"o}hung der Lichtintensit{\"a}t sowie der Kohlenstoffdioxid-Verf{\"u}gbarkeit zusammen mit der Kultivierung von N. punctiforme zu extrem hohen Zelldichten zu einer starken Erh{\"o}hung der gesamten metabolischen Aktivit{\"a}t des Organismus. N{\"a}here Untersuchungen der Zellextrakte dieser hoch-dichte Kultivierungen f{\"u}hrten letztlich zur Entdeckung einer neuartigen Gruppe von Microviridinen mit verl{\"a}ngerter Peptidsequenz, welche Microviridin N3-N9 genannt wurden. Sowohl die Kultivierung der Transkriptionsreporter als auch die RTqPCR-basierte Untersuchung der Transkriptionslevel der verschiedenen Biosynthesecluster zeigten, dass die hoch-Zelldichte Kultivierung von N. punctiforme zu einer Aktivierung von 50\% der vorhandenen Sekund{\"a}rmetabolitcluster f{\"u}hrt. Im Gegensatz zu dieser sehr breit-gef{\"a}cherten Aktivierung, f{\"u}hrt die Co-Kultivierung von N. punctiforme in chemischen oder physischen Kontakt zu einer N-gehungerten Wirtspflanze (Blasia pusilla) zu einer sehr spezifischen Aktivierung der RIPP4 und RiPP3 Biosynthesecluster. Obwohl dieser Effekt mittels verschiedener unabh{\"a}ngiger Methoden best{\"a}tigt werden konnte und trotz intensiver Analysebem{\"u}hungen, konnte jedoch keinem der beiden Cluster ein Produkt zugeordnet werden. Diese Studie stellt die erste weitreichende, systematische Analyse eines cyanobakteriellen Sekund{\"a}rmetaboloms durch einen kombinatorischen Ansatz aus Genomic Mining und unabh{\"a}ngigen Monitoring-Techniken dar und kann als neue strategische Herangehensweise f{\"u}r die Untersuchung anderer Organismen hinsichtlich ihrer Sekund{\"a}rmetabolit-Produktion dienen. Obwohl es bereits gut beschriebene einzelne Sekund{\"a}rmetabolite gibt, wie beispielweise den Zelldifferenzierungsfaktor PatS in Anabaena sp. PCC7120, so ist der Grad an Regulation der in dieser Studie gezeigt werden konnte bislang beispiellos und die Entschl{\"u}sselung dieser Mechanismen k{\"o}nnte die Entdeckung neuer Naturstoffe stark beschleunigen. Daneben lassen die Ergebnisse aber auch darauf schließen, dass die Induktion der Biosynthesewege nicht das eigentliche Problem darstellt, sondern vielmehr die verl{\"a}ssliche Detektion deren Produkte. Die Erarbeitung neuer Analytik-Strategien k{\"o}nnte somit auch einen deutlichen Einfluss auf die Geschwindigkeit der Entdeckung neuer Naturstoffe haben.}, language = {en} } @phdthesis{Folkertsma2020, author = {Folkertsma, Remco}, title = {Evolutionary adaptation to climate in microtine mammals}, doi = {10.25932/publishup-47680}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476807}, school = {Universit{\"a}t Potsdam}, pages = {135}, year = {2020}, abstract = {Understanding how organisms adapt to their local environment is a major focus of evolutionary biology. Local adaptation occurs when the forces of divergent natural selection are strong enough compared to the action of other evolutionary forces. An improved understanding of the genetic basis of local adaptation can inform about the evolutionary processes in populations and is of major importance because of its relevance to altered selection pressures due to climate change. So far, most insights have been gained by studying model organisms, but our understanding about the genetic basis of local adaptation in wild populations of species with little genomic resources is still limited. With the work presented in this thesis I therefore set out to provide insights into the genetic basis of local adaptation in populations of two voles species: the common vole (Microtus arvalis) and the bank vole (Myodes glareolus). Both voles species are small mammals, they have a high evolutionary potential compared to their dispersal capabilities and are thus likely to show genetic responses to local conditions, moreover, they have a wide distribution in which they experience a broad range of different environmental conditions, this makes them an ideal species to study local adaptation. The first study focused on producing a novel mitochondrial genome to facilitate further research in M. arvalis. To this end, I generated the first mitochondrial genome of M. arvalis using shotgun sequencing and an iterative mapping approach. This was subsequently used in a phylogenetic analysis that produced novel insights into the phylogenetic relationships of the Arvicolinae. The following two studies then focused on the genetic basis of local adaptation using ddRAD-sequencing data and genome scan methods. The first of these involved sequencing the genomic DNA of individuals from three low-altitude and three high-altitude M. arvalis study sites in the Swiss Alps. High-altitude environments with their low temperatures and low levels of oxygen (hypoxia) pose considerable challenges for small mammals. With their small body size and proportional large body surface they have to sustain high rates of aerobic metabolism to support thermogenesis and locomotion, which can be restricted with only limited levels of oxygen available. To generate insights into high-altitude adaptation I identified a large number of single nucleotide polymorphisms (SNPs). These data were first used to identify high levels of differentiation between study sites and a clear pattern of population structure, in line with a signal of isolation by distance. Using genome scan methods, I then identified signals of selection associated with differences in altitude in genes with functions related to oxygen transport into tissue and genes related to aerobic metabolic pathways. This indicates that hypoxia is an important selection pressure driving local adaptation at high altitude in M. arvalis. A number of these genes were linked with high-altitude adaptation in other species before, which lead to the suggestion that high-altitude populations of several species have evolved in a similar manner as a response to the unique conditions at high altitude The next study also involved the genetic basis of local adaptation, here I provided insights into climate-related adaptation in M. glareolus across its European distribution. Climate is an important environmental factor affecting the physiology of all organisms. In this study I identified a large number of SNPs in individuals from twelve M. glareolus populations distributed across Europe. I used these, to first establish that populations are highly differentiated and found a strong pattern of population structure with signal of isolation by distance. I then employed genome scan methods to identify candidate loci showing signals of selection associated with climate, with a particular emphasis on polygenic loci. A multivariate analysis was used to determine that temperature was the most important climate variable responsible for adaptive genetic variation among all variables tested. By using novel methods and genome annotation of related species I identified the function of genes of candidate loci. This showed that genes under selection have functions related to energy homeostasis and immune processes. Suggesting that M. glareolus populations have evolved in response to local temperature and specific local pathogenic selection pressures. The studies presented in this thesis provide evidence for the genetic basis of local adaptation in two vole species across different environmental gradients, suggesting that the identified genes are involved in local adaptation. This demonstrates that with the help of novel methods the study of wild populations, which often have little genomic resources available, can provide unique insights into evolutionary processes.}, language = {en} } @phdthesis{Zhang2020, author = {Zhang, Jianrui}, title = {Completely water-based emulsions as compartmentalized systems via pickering stabilization}, doi = {10.25932/publishup-47654}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476542}, school = {Universit{\"a}t Potsdam}, pages = {II, 119}, year = {2020}, abstract = {Completely water-based systems are of interest for the development of novel material for various reasons: On one hand, they provide benign environment for biological systems and on the other hand they facilitate effective molecular transport in a membrane-free environment. In order to investigate the general potential of aqueous two-phase systems (ATPSs) for biomaterials and compartmentalized systems, various solid particles were applied to stabilize all-aqueous emulsion droplets. The target ATPS to be investigated should be prepared via mixing of two aqueous solutions of water-soluble polymers, which turn biphasic when exceeding a critical polymer concentration. Hydrophilic polymers with a wide range of molar mass such as dextran/poly(ethylene glycol) (PEG) can therefore be applied. Solid particles adsorbed at the interfaces can be exceptionally efficient stabilizers forming so-called Pickering emulsions, and nanoparticles can bridge the correlation length of polymer solutions and are thereby the best option for water-in-water emulsions. The first approach towards the investigation of ATPS was conducted with all aqueous dextran-PEG emulsions in the presence of poly(dopamine) particles (PDP) in Chapter 4. The water-in-water emulsions were formed with a PEG/dextran system via utilizing PDP as stabilizers. Studies of the formed emulsions were performed via laser scanning confocal microscope (CLSM), optical microscope (OM), cryo-scanning electron microscope (SEM) and tensiometry. The stable emulsions (at least 16 weeks) were demulsified easily via dilution or surfactant addition. Furthermore, the solid PDP at the water-water interface were crosslinked in order to inhibit demulsification of the Pickering emulsion. Transmission electron microscope (TEM) and scanning electron microscope (SEM) were used to visualize the morphology of PDP before and after crosslinking. PDP stabilized water-in-water emulsions were utilized in the following Chapter 5 to form supramolecular compartmentalized hydrogels. Here, hydrogels were prepared in pre-formed water-in-water emulsions and gelled via α-cyclodextrin-PEG (α-CD-PEG) inclusion complex formation. Studies of the formed complexes were performed via X-ray powder diffraction (XRD) and the mechanical properties of the hydrogels were measured with oscillatory shear rheology. In order to verify the compartmentalized state and its triggered decomposition, hydrogels and emulsions were assessed via OM, SEM and CLSM. The last chapter broadens the investigations from the previous two systems by utilizing various carbon nitrides (CN) as different stabilizers in ATPS. CN introduces another way to trigger demulsification, namely irradiation with visible light. Therefore, emulsification and demulsification with various triggers were probed. The investigated all aqueous multi-phase systems will act as model for future fabrication of biocompatible materials, cell micropatterning as well as separation of compartmentalized systems.}, language = {en} } @phdthesis{Tunn2020, author = {Tunn, Isabell}, title = {From single molecules to bulk materials: tuning the viscoelastic properties of coiled coil cross-linked hydrogels}, doi = {10.25932/publishup-47595}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475955}, school = {Universit{\"a}t Potsdam}, pages = {XVI, 140}, year = {2020}, abstract = {The development of bioinspired self-assembling materials, such as hydrogels, with promising applications in cell culture, tissue engineering and drug delivery is a current focus in material science. Biogenic or bioinspired proteins and peptides are frequently used as versatile building blocks for extracellular matrix (ECM) mimicking hydrogels. However, precisely controlling and reversibly tuning the properties of these building blocks and the resulting hydrogels remains challenging. Precise control over the viscoelastic properties and self-healing abilities of hydrogels are key factors for developing intelligent materials to investigate cell matrix interactions. Thus, there is a need to develop building blocks that are self-healing, tunable and self-reporting. This thesis aims at the development of α-helical peptide building blocks, called coiled coils (CCs), which integrate these desired properties. Self-healing is a direct result of the fast self-assembly of these building blocks when used as material cross-links. Tunability is realized by means of reversible histidine (His)-metal coordination bonds. Lastly, implementing a fluorescent readout, which indicates the CC assembly state, self-reporting hydrogels are obtained. Coiled coils are abundant protein folding motifs in Nature, which often have mechanical function, such as in myosin or fibrin. Coiled coils are superhelices made up of two or more α-helices wound around each other. The assembly of CCs is based on their repetitive sequence of seven amino acids, so-called heptads (abcdefg). Hydrophobic amino acids in the a and d position of each heptad form the core of the CC, while charged amino acids in the e and g position form ionic interactions. The solvent-exposed positions b, c and f are excellent targets for modifications since they are more variable. His-metal coordination bonds are strong, yet reversible interactions formed between the amino acid histidine and transition metal ions (e.g. Ni2+, Cu2+ or Zn2+). His-metal coordination bonds essentially contribute to the mechanical stability of various high-performance proteinaceous materials, such as spider fangs, Nereis worm jaws and mussel byssal threads. Therefore, I bioengineered reversible His-metal coordination sites into a well-characterized heterodimeric CC that served as tunable material cross-link. Specifically, I took two distinct approaches facilitating either intramolecular (Chapter 4.2) and/or intermolecular (Chapter 4.3) His-metal coordination. Previous research suggested that force-induced CC unfolding in shear geometry starts from the points of force application. In order to tune the stability of a heterodimeric CC in shear geometry, I inserted His in the b and f position at the termini of force application (Chapter 4.2). The spacing of His is such that intra-CC His-metal coordination bonds can form to bridge one helical turn within the same helix, but also inter-CC coordination bonds are not generally excluded. Starting with Ni2+ ions, Raman spectroscopy showed that the CC maintained its helical structure and the His residues were able to coordinate Ni2+. Circular dichroism (CD) spectroscopy revealed that the melting temperature of the CC increased by 4 °C in the presence of Ni2+. Using atomic force microscope (AFM)-based single molecule force spectroscopy, the energy landscape parameters of the CC were characterized in the absence and the presence of Ni2+. His-Ni2+ coordination increased the rupture force by ~10 pN, accompanied by a decrease of the dissociation rate constant. To test if this stabilizing effect can be transferred from the single molecule level to the bulk viscoelastic material properties, the CC building block was used as a non-covalent cross-link for star-shaped poly(ethylene glycol) (star-PEG) hydrogels. Shear rheology revealed a 3-fold higher relaxation time in His-Ni2+ coordinating hydrogels compared to the hydrogel without metal ions. This stabilizing effect was fully reversible when using an excess of the metal chelator ethylenediaminetetraacetate (EDTA). The hydrogel properties were further investigated using different metal ions, i.e. Cu2+, Co2+ and Zn2+. Overall, these results suggest that Ni2+, Cu2+ and Co2+ primarily form intra-CC coordination bonds while Zn2+ also participates in inter-CC coordination bonds. This may be a direct result of its different coordination geometry. Intermolecular His-metal coordination bonds in the terminal regions of the protein building blocks of mussel byssal threads are primarily formed by Zn2+ and were found to be intimately linked to higher-order assembly and self-healing of the thread. In the above example, the contribution of intra-CC and inter-CC His-Zn2+ cannot be disentangled. In Chapter 4.3, I redesigned the CC to prohibit the formation of intra-CC His-Zn2+ coordination bonds, focusing only on inter-CC interactions. Specifically, I inserted His in the solvent-exposed f positions of the CC to focus on the effect of metal-induced higher-order assembly of CC cross-links. Raman and CD spectroscopy revealed that this CC building block forms α-helical Zn2+ cross-linked aggregates. Using this CC as a cross-link for star-PEG hydrogels, I showed that the material properties can be switched from viscoelastic in the absence of Zn2+ to elastic-like in the presence of Zn2+. Moreover, the relaxation time of the hydrogel was tunable over three orders of magnitude when using different Zn2+:His ratios. This tunability is attributed to a progressive transformation of single CC cross-links into His-Zn2+ cross-linked aggregates, with inter-CC His-Zn2+ coordination bonds serving as an additional, cross-linking mode. Rheological characterization of the hydrogels with inter-CC His-Zn2+ coordination raised the question whether the His-Zn2+ coordination bonds between CCs or also the CCs themselves rupture when shear strain is applied. In general, the amount of CC cross-links initially formed in the hydrogel as well as the amount of CC cross-links breaking under force remains to be elucidated. In order to more deeply probe these questions and monitor the state of the CC cross-links when force is applied, a fluorescent reporter system based on F{\"o}rster resonance energy transfer (FRET) was introduced into the CC (Chapter 4.4). For this purpose, the donor-acceptor pair carboxyfluorescein and tetramethylrhodamine was used. The resulting self-reporting CC showed a FRET efficiency of 77 \% in solution. Using this fluorescently labeled CC as a self-reporting, reversible cross-link in an otherwise covalently cross-linked star-PEG hydrogel enabled the detection of the FRET efficiency change under compression force. This proof-of-principle result sets the stage for implementing the fluorescently labeled CCs as molecular force sensors in non-covalently cross-linked hydrogels. In summary, this thesis highlights that rationally designed CCs are excellent reversibly tunable, self-healing and self-reporting hydrogel cross-links with high application potential in bioengineering and biomedicine. For the first time, I demonstrated that His-metal coordination-based stabilization can be transferred from the single CC level to the bulk material with clear viscoelastic consequences. Insertion of His in specific sequence positions was used to implement a second non-covalent cross-linking mode via intermolecular His-metal coordination. This His-metal binding induced aggregation of the CCs enabled for reversibly tuning the hydrogel properties from viscoelastic to elastic-like. As a proof-of-principle to establish self-reporting CCs as material cross-links, I labeled a CC with a FRET pair. The fluorescently labelled CC acts as a molecular force sensor and first preliminary results suggest that the CC enables the detection of hydrogel cross-link failure under compression force. In the future, fluorescently labeled CC force sensors will likely not only be used as intelligent cross-links to study the failure of hydrogels but also to investigate cell-matrix interactions in 3D down to the single molecule level.}, language = {en} } @phdthesis{Alirezaeizanjani2020, author = {Alirezaeizanjani, Zahra}, title = {Movement strategies of a multi-mode bacterial swimmer}, doi = {10.25932/publishup-47580}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475806}, school = {Universit{\"a}t Potsdam}, pages = {xix, 111}, year = {2020}, abstract = {Bacteria are one of the most widespread kinds of microorganisms that play essential roles in many biological and ecological processes. Bacteria live either as independent individuals or in organized communities. At the level of single cells, interactions between bacteria, their neighbors, and the surrounding physical and chemical environment are the foundations of microbial processes. Modern microscopy imaging techniques provide attractive and promising means to study the impact of these interactions on the dynamics of bacteria. The aim of this dissertation is to deepen our understanding four fundamental bacterial processes - single-cell motility, chemotaxis, bacterial interactions with environmental constraints, and their communication with neighbors - through a live cell imaging technique. By exploring these processes, we expanded our knowledge on so far unexplained mechanisms of bacterial interactions. Firstly, we studied the motility of the soil bacterium Pseudomonas putida (P. putida), which swims through flagella propulsion, and has a complex, multi-mode swimming tactic. It was recently reported that P. putida exhibits several distinct swimming modes - the flagella can push and pull the cell body or wrap around it. Using a new combined phase-contrast and fluorescence imaging set-up, the swimming mode (push, pull, or wrapped) of each run phase was automatically recorded, which provided the full swimming statistics of the multi-mode swimmer. Furthermore, the investigation of cell interactions with a solid boundary illustrated an asymmetry for the different swimming modes; in contrast to the push and pull modes, the curvature of runs in wrapped mode was not affected by the solid boundary. This finding suggested that having a multi-mode swimming strategy may provide further versatility to react to environmental constraints. Then we determined how P. putida navigates toward chemoattractants, i.e. its chemotaxis strategies. We found that individual run modes show distinct chemotactic responses in nutrition gradients. In particular, P. putida cells exhibited an asymmetry in their chemotactic responsiveness; the wrapped mode (slow swimming mode) was affected by the chemoattractant, whereas the push mode (fast swimming mode) was not. These results can be seen as a starting point to understand more complex chemotaxis strategies of multi-mode swimmers going beyond the well-known paradigm of Escherichia coli, that exhibits only one swimming mode. Finally we considered the cell dynamics in a dense population. Besides physical interactions with their neighbors, cells communicate their activities and orchestrate their population behaviors via quorum-sensing. Molecules that are secreted to the surrounding by the bacterial cells, act as signals and regulate the cell population behaviour. We studied P. putida's motility in a dense population by exposing the cells to environments with different concentrations of chemical signals. We found that higher amounts of chemical signals in the surrounding influenced the single-cell behaviourr, suggesting that cell-cell communications may also affect the flagellar dynamics. In summary, this dissertation studies the dynamics of a bacterium with a multi-mode swimming tactic and how it is affected by the surrounding environment using microscopy imaging. The detailed description of the bacterial motility in fundamental bacterial processes can provide new insights into the ecology of microorganisms.}, language = {en} } @phdthesis{Liu2020, author = {Liu, Qi}, title = {Influence of CO2 degassing on microbial community distribution and activity in the Hartoušov degassing system, western Eger Rift (Czech Republic)}, doi = {10.25932/publishup-47534}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-475341}, school = {Universit{\"a}t Potsdam}, pages = {146}, year = {2020}, abstract = {The Cheb Basin (CZ) is a shallow Neogene intracontinental basin located in the western Eger Rift. The Cheb Basin is characterized by active seismicity and diffuse degassing of mantle-derived CO2 in mofette fields. Within the Cheb Basin, the Hartoušov mofette field shows a daily CO2 flux of 23-97 tons. More than 99\% of CO2 released over an area of 0.35 km2. Seismic active periods have been observed in 2000 and 2014 in the Hartoušov mofette field. Due to the active geodynamic processes, the Cheb Basin is considered to be an ideal region for the continental deep biosphere research focussing on the interaction of biological processes with geological processes. To study the influence of CO2 degassing on microbial community in the surface and subsurface environments, two 3-m shallow drillings and a 108.5-m deep scientific drilling were conducted in 2015 and 2016 respectively. Additionally, the fluid retrieved from the deep drilling borehole was also recovered. The different ecosystems were compared regarding their geochemical properties, microbial abundances, and microbial community structures. The geochemistry of the mofette is characterized by low pH, high TOC, and sulfate contents while the subsurface environment shows a neutral pH, and various TOC and sulfate contents in different lithological settings. Striking differences in the microbial community highlight the substantial impact of elevated CO2 concentrations and high saline groundwater on microbial processes. In general, the microorganisms had low abundance in the deep subsurface sediment compared with the shallow mofette. However, within the mofette and the deep subsurface sediment, the abundance of microbes does not show a typical decrease with depth, indicating that the uprising CO2-rich groundwater has a strong influence on the microbial communities via providing sufficient substrate for anaerobic chemolithoautotrophic microorganisms. Illumina MiSeq sequencing of the 16S rRNA genes and multivariate statistics reveals that the pH strongly influences the microbial community composition in the mofette, while the subsurface microbial community is significantly influenced by the groundwater which motivated by the degassing CO2. Acidophilic microorganisms show a much higher relative abundance in the mofette. Meanwhile, the OTUs assigned to family Comamonadaceae are the dominant taxa which characterize the subsurface communities. Additionally, taxa involved in sulfur cycling characterizing the microbial communities in both mofette and CO2 dominated subsurface environments. Another investigated important geo-bio interaction is the influence of the seismic activity. During seismic events, released H2 may serve as the electron donor for microbial hydrogenotrophic processes, such as methanogenesis. To determine whether the seismic events can potentially trigger methanogenesis by the elevated geogenic H2 concentration, we performed laboratory simulation experiments with sediments retrieved from the drillings. The simulation results indicate that after the addition of hydrogen, substantial amounts of methane were produced in incubated mofette sediments and deep subsurface sediments. The methanogenic hydrogenotrophic genera Methanobacterium was highly enriched during the incubation. The modeling of the in-situ observation of the earthquake swarm period in 2000 at the Novy Kostel focal area/Czech Republic and our laboratory simulation experiments reveals a close relation between seismic activities and microbial methane production via earthquake-induced H2 release. We thus conclude that H2 - which is released during seismic activity - can potentially trigger methanogenic activity in the deep subsurface. Based on this conclusion, we further hypothesize that the hydrogenotrophic early life on Earth was boosted by the Late Heavy Bombardment induced seismic activity in approximately 4.2 to 3.8 Ga.}, language = {en} } @phdthesis{He2019, author = {He, Hai}, title = {Exploring and engineering formaldehyde assimilation}, doi = {10.25932/publishup-47386}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-473867}, school = {Universit{\"a}t Potsdam}, pages = {vi, 105}, year = {2019}, abstract = {Increasing concerns regarding the environmental impact of our chemical production have shifted attention towards possibilities for sustainable biotechnology. One-carbon (C1) compounds, including methane, methanol, formate and CO, are promising feedstocks for future bioindustry. CO2 is another interesting feedstock, as it can also be transformed using renewable energy to other C1 feedstocks for use. While formaldehyde is not suitable as a feedstock due to its high toxicity, it is a central intermediate in the process of C1 assimilation. This thesis explores formaldehyde metabolism and aims to engineer formaldehyde assimilation in the model organism Escherichia coli for the future C1-based bioindustry. The first chapter of the thesis aims to establish growth of E. coli on formaldehyde via the most efficient naturally occurring route, the ribulose monophosphate pathway. Linear variants of the pathway were constructed in multiple-gene knockouts strains, coupling E. coli growth to the activities of the key enzymes of the pathway. Formaldehyde-dependent growth was achieved in rationally designed strains. In the final strain, the synthetic pathway provides the cell with almost all biomass and energy requirements. In the second chapter, taking advantage of the unique feature of its reactivity, formaldehyde assimilation via condensation with glycine and pyruvate by two promiscuous aldolases was explored. Facilitated by these two reactions, the newly designed homoserine cycle is expected to support higher yields of a wide array of products than its counterparts. By dividing the pathway into segments and coupling them to the growth of dedicated strains, all pathway reactions were demonstrated to be sufficiently active. The work paves a way for future implementation of a highly efficient route for C1 feedstocks into commodity chemicals. In the third chapter, the in vivo rate of the spontaneous formaldehyde tetrahydrofolate condensation to methylene-tetrahydrofolate was assessed in order to evaluate its applicability as a biotechnological process. Tested within an E. coli strain deleted in essential genes for native methylene-tetrahydrofolate biosynthesis, the reaction was shown to support the production of this essential intermediate. However, only low growth rates were observed and only at high formaldehyde concentrations. Computational analysis dependent on in vivo evidence from this strain deduced the slow rate of this spontaneous reaction, thus ruling out its substantial contribution to growth on C1 feedstocks. The reactivity of formaldehyde makes it highly toxic. In the last chapter, the formation of thioproline, the condensation product of cysteine and formaldehyde, was confirmed to contribute this toxicity effect. Xaa-Pro aminopeptidase (PepP), which genetically links with folate metabolism, was shown to hydrolyze thioproline-containing peptides. Deleting pepP increased strain sensitivity to formaldehyde, pointing towards the toxicity of thioproline-containing peptides and the importance of their removal. The characterization in this study could be useful in handling this toxic intermediate. Overall, this thesis identified challenges related to formaldehyde metabolism and provided novel solutions towards a future bioindustry based on sustainable C1 feedstocks in which formaldehyde serves as a key intermediate.}, language = {en} } @phdthesis{Irmscher2020, author = {Irmscher, Tobias}, title = {Enzymatic remodelling of the exopolysaccharide stewartan network}, doi = {10.25932/publishup-47248}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-472486}, school = {Universit{\"a}t Potsdam}, pages = {xiv, 170}, year = {2020}, abstract = {In nature, bacteria are found to reside in multicellular communities encased in self-produced extracellular matrices. Indeed, biofilms are the default lifestyle of the bacteria which cause persistent infections in humans. The biofilm assembly protects bacterial cells from desiccation and limits the effectiveness of antimicrobial treatments. A myriad of biomolecules in the extracellular matrix, including proteins, exopolysaccharides, lipids, extracellular DNA and other, form a dense and viscoelastic three dimensional network. Many studies emphasized that a destabilization of the mechanical integrity of biofilm architectures potentially eliminates the protective shield and renders bacteria more susceptible to the immune system and antibiotics. Pantoea stewartii is a plant pathogen which infects monocotyledons such as maize and sweet corn. These bacteria produce dense biofilms in the xylem of infected plants which cause wilting of plants and crops. Stewartan is an exopolysaccharide which is produced by Pantoea stewartii and secreted as the major component to the extracellular matrix. It consists of heptasaccharide repeating units with a high degree of polymerization (2-4 MDa). In this work, the physicochemical properties of stewartan were investigated to understand the contributions of this exopolysaccharide to the mechanical integrity and cohesiveness of Pantoea stewartii biofilms. Therefore, a coarse-grained model of stewartan was developed with computational techniques to obtain a model for its three dimensional structural features. Here, coarse-grained molecular dynamic simulations revealed that the exopolysaccharide forms a hydrogel in which the exopolysaccharide chains arrange into a three dimensional mesh-like network. Simulations at different concentrations were used to investigate the influence of the water content on the network formation. Stewartan was further purified from 72 h grown Pantoea stewartii biofilms and the diffusion of bacteriophage and differently-sized nanoparticles (which ranged from 1.1 to 193 nm diameter) was analyzed in reconstituted stewartan solutions. Fluorescence correlation spectroscopy and single-particle tracking revealed that the stewartan network impeded the mobility of a set of differently-sized fluorescent particles in a size-dependent manner. Diffusion of these particles became more anomalous, as characterized by fitting the diffusion data to an anomalous diffusion model, with increasing stewartan concentrations. Further bulk and microrheological experiments were used to analyze the transitions in stewartan fluid behavior and stewartan chain entanglements were described. Moreover, it was noticed, that a small fraction of bacteriophage particles was trapped in small-sized pores deviating from classical random walks which highlighted the structural heterogeneity of the stewartan network. Additionally, the mobility of fluorescent particles also depended on the charge of the stewartan exopolysaccharide and a model of a molecular sieve for the stewartan network was proposed. The here reported structural features of the stewartan polymers were used to provide a detailed description of the mechanical properties of typically glycan-based biofilms such as the one from Pantoea stewartii. In addition, the mechanical properties of the biofilm architecture are permanently sensed by the embedded bacteria and enzymatic modifications of the extracellular matrix take place to address environmental cues. Hence, in this work the influence of enzymatic degradation of the stewartan exopolysaccharides on the overall exopolysaccharide network structure was analyzed to describe relevant physiological processes in Pantoea stewartii biofilms. Here, the stewartan hydrolysis kinetics of the tailspike protein from the ΦEa1h bacteriophage, which is naturally found to infect Pantoea stewartii cells, was compared to WceF. The latter protein is expressed from the Pantoea stewartii stewartan biosynthesis gene cluster wce I-III. The degradation of stewartan by the ΦEa1h tailspike protein was shown to be much faster than the hydrolysis kinetics of WceF, although both enzymes cleaved the β D GalIII(1→3)-α-D-GalI glycosidic linkage from the stewartan backbone. Oligosaccharide fragments which were produced during the stewartan cleavage, were analyzed in size-exclusion chromatography and capillary electrophoresis. Bioinformatic studies and the analysis of a WceF crystal structure revealed a remarkably high structural similarity of both proteins thus unveiling WceF as a bacterial tailspike-like protein. As a consequence, WceF might play a role in stewartan chain length control in Pantoea stewartii biofilms.}, language = {en} } @phdthesis{Mitzscherling2020, author = {Mitzscherling, Julia}, title = {Microbial communities in submarine permafrost and their response to permafrost degradation and warming}, doi = {10.25932/publishup-47124}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-471240}, school = {Universit{\"a}t Potsdam}, pages = {I, 231}, year = {2020}, abstract = {The Arctic region is especially impacted by global warming as temperatures in high latitude regions have increased and are predicted to further rise at levels above the global average. This is crucial to Arctic soils and the shallow shelves of the Arctic Ocean as they are underlain by permafrost. Perennially frozen ground is a habitat for a large number and great diversity of viable microorganisms, which can remain active even under freezing conditions. Warming and thawing of permafrost makes trapped soil organic carbon more accessible to microorganisms. They can transform it to the greenhouse gases carbon dioxide, methane and nitrous oxide. On the other hand, it is assumed that thawing of the frozen ground stimulates microbial activity and carbon turnover. This can lead to a positive feedback loop of warming and greenhouse gas release. Submarine permafrost covers most areas of the Siberian Arctic Shelf and contains a large though unquantified carbon pool. However, submarine permafrost is not only affected by changes in the thermal regime but by drastic changes in the geochemical composition as it formed under terrestrial conditions and was inundated by Holocene sea level rise and coastal erosion. Seawater infiltration into permafrost sediments resulted in an increase of the pore water salinity and, thus, in thawing of permafrost in the upper sediment layers even at subzero temperatures. The permafrost below, which was not affected by seawater, remained ice-bonded, but warmed through seawater heat fluxes. The objective of this thesis was to study microbial communities in submarine permafrost with a focus on their response to seawater influence and long-term warming using a combined approach of molecular biological and physicochemical analyses. The microbial abundance, community composition and structure as well as the diversity were investigated in drill cores from two locations in the Laptev Sea, which were subjected to submarine conditions for centuries to millennia. The microbial abundance was measured through total cell counts and copy numbers of the 16S rRNA gene and of functional genes. The latter comprised genes which are indicative for methane production (mcrA) and sulfate reduction (dsrB). The microbial community was characterized by high-throughput-sequencing of the 16S rRNA gene. Physicochemical analyses included the determination of the pore water geochemical and stable isotopic composition, which were used to describe the degree of seawater influence. One major outcome of the thesis is that the submarine permafrost stratified into different so-called pore water units centuries as well as millennia after inundation: (i) sediments that were mixed with seafloor sediments, (ii) sediments that were infiltrated with seawater, and (iii) sediments that were unaffected by seawater. This stratification was reflected in the submarine permafrost microbial community composition only millennia after inundation but not on time-scales of centuries. Changes in the community composition as well as abundance were used as a measure for microbial activity and the microbial response to changing thermal and geochemical conditions. The results were discussed in the context of permafrost temperature, pore water composition, paleo-climatic proxies and sediment age. The combination of permafrost warming and increasing salinity as well as permafrost warming alone resulted in a disturbance of the microbial communities at least on time-scales of centuries. This was expressed by a loss of microbial abundance and bacterial diversity. At the same time, the bacterial community of seawater unaffected but warmed permafrost was mainly determined by environmental and climatic conditions at the time of sediment deposition. A stimulating effect of warming was observed only in seawater unaffected permafrost after millennia-scale inundation, visible through increased microbial abundance and reduced amounts of substrate. Despite submarine exposure for centuries to millennia, the community of bacteria in submarine permafrost still generally resembled the community of terrestrial permafrost. It was dominated by phyla like Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes and Proteobacteria, which can be active under freezing conditions. Moreover, the archaeal communities of both study sites were found to harbor high abundances of marine and terrestrial anaerobic methane oxidizing archaea (ANME). Results also suggested ANME populations to be active under in situ conditions at subzero temperatures. Modeling showed that potential anaerobic oxidation of methane (AOM) could mitigate the release of almost all stored or microbially produced methane from thawing submarine permafrost. Based on the findings presented in this thesis, permafrost warming and thawing under submarine conditions as well as permafrost warming without thaw are supposed to have marginal effects on the microbial abundance and community composition, and therefore likely also on carbon mobilization and the formation of methane. Thawing under submarine conditions even stimulates AOM and thus mitigates the release of methane.}, language = {en} } @phdthesis{Wen2020, author = {Wen, Xi}, title = {Distribution patterns and environmental drivers of methane-cycling microorganisms in natural environments and restored wetlands}, doi = {10.25932/publishup-47177}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-471770}, school = {Universit{\"a}t Potsdam}, pages = {VIII, iii, 152}, year = {2020}, abstract = {Methane is an important greenhouse gas contributing to global climate change. Natural environments and restored wetlands contribute a large proportion to the global methane budget. Methanogenic archaea (methanogens) and methane oxidizing bacteria (methanotrophs), the biogenic producers and consumers of methane, play key roles in the methane cycle in those environments. A large number of studies revealed the distribution, diversity and composition of these microorganisms in individual habitats. However, uncertainties exist in predicting the response and feedback of methane-cycling microorganisms to future climate changes and related environmental changes due to the limited spatial scales considered so far, and due to a poor recognition of the biogeography of these important microorganisms combining global and local scales. With the aim of improving our understanding about whether and how methane-cycling microbial communities will be affected by a series of dynamic environmental factors in response to climate change, this PhD thesis investigates the biogeographic patterns of methane-cycling communities, and the driving factors which define these patterns at different spatial scales. At the global scale, a meta-analysis was performed by implementing 94 globally distributed public datasets together with environmental data from various natural environments including soils, lake sediments, estuaries, marine sediments, hydrothermal sediments and mud volcanos. In combination with a global biogeographic map of methanogenic archaea from multiple natural environments, this thesis revealed that biogeographic patterns of methanogens exist. The terrestrial habitats showed higher alpha diversities than marine environments. Methanoculleus and Methanosaeta (Methanothrix) are the most frequently detected taxa in marine habitats, while Methanoregula prevails in terrestrial habitats. Estuary ecosystems, the transition zones between marine and terrestrial/limnic ecosystems, have the highest methanogenic richness but comparably low methane emission rates. At the local scale, this study compared two rewetted fens with known high methane emissions in northeastern Germany, a coastal brackish fen (H{\"u}telmoor) and a freshwater riparian fen (Polder Zarnekow). Consistent with different geochemical conditions and land-use history, the two rewetted fens exhibit dissimilar methanogenic and, especially, methanotrophic community compositions. The methanotrophic community was generally under-represented among the prokaryotic communities and both fens show similarly low ratios of methanotrophic to methanogenic abundances. Since few studies have characterized methane-cycling microorganisms in rewetted fens, this study provides first evidence that the rapid and well re-established methanogenic community in combination with the low and incomplete re-establishment of the methanotrophic community after rewetting contributes to elevated sustained methane fluxes following rewetting. Finally, this thesis demonstrates that dispersal limitation only slightly regulates the biogeographic distribution patterns of methanogenic microorganisms in natural environments and restored wetlands. Instead, their existence, adaption and establishment are more associated with the selective pressures under different environmental conditions. Salinity, pH and temperature are identified as the most important factors in shaping microbial community structure at different spatial scales (global versus terrestrial environments). Predicted changes in climate, such as increasing temperature, changes in precipitation patterns and increasing frequency of flooding events, are likely to induce a series of environmental alterations, which will either directly or indirectly affect the driving environmental forces of methanogenic communities, leading to changes in their community composition and thus potentially also in methane emission patterns in the future.}, language = {en} } @phdthesis{Guislain2019, author = {Guislain, Alexis}, title = {Eco-physiological consequences of fluctuating light on phytoplankton}, doi = {10.25932/publishup-46927}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-469272}, school = {Universit{\"a}t Potsdam}, pages = {161}, year = {2019}, abstract = {Phytoplankton growth depends not only on the mean intensity but also on the dynamics of the light supply. The nonlinear light-dependency of growth is characterized by a small number of basic parameters: the compensation light intensity PARcompμ, where production and losses are balanced, the growth efficiency at sub-saturating light αµ, and the maximum growth rate at saturating light µmax. In surface mixed layers, phytoplankton may rapidly move between high light intensities and almost darkness. Because of the different frequency distribution of light and/or acclimation processes, the light-dependency of growth may differ between constant and fluctuating light. Very few studies measured growth under fluctuating light at a sufficient number of mean light intensities to estimate the parameters of the growth-irradiance relationship. Hence, the influence of light dynamics on µmax, αµ and PARcompμ are still largely unknown. By extension, accurate modelling predictions of phytoplankton development under fluctuating light exposure remain difficult to make. This PhD thesis does not intend to directly extrapolate few experimental results to aquatic systems - but rather improving the mechanistic understanding of the variation of the light-dependency of growth under light fluctuations and effects on phytoplankton development. In Lake TaiHu and at the Three Gorges Reservoir (China), we incubated phytoplankton communities in bottles placed either at fixed depths or moved vertically through the water column to mimic vertical mixing. Phytoplankton at fixed depths received only the diurnal changes in light (defined as constant light regime), while phytoplankton received rapidly fluctuating light by superimposing the vertical light gradient on the natural sinusoidal diurnal sunlight. The vertically moved samples followed a circular movement with 20 min per revolution, replicating to some extent the full overturn of typical Langmuir cells. Growth, photosynthesis, oxygen production and respiration of communities (at Lake TaiHu) were measured. To complete these investigations, a physiological experiment was performed in the laboratory on a toxic strain of Microcystis aeruginosa (FACBH 1322) incubated under 20 min period fluctuating light. Here, we measured electron transport rates and net oxygen production at a much higher time resolution (single minute timescale). The present PhD thesis provides evidence for substantial effects of fluctuating light on the eco-physiology of phytoplankton. Both experiments performed under semi-natural conditions in Lake TaiHu and at the Three Gorges Reservoir gave similar results. The significant decline in community growth efficiencies αµ under fluctuating light was caused for a great share by different frequency distribution of light intensities that shortened the effective daylength for production. The remaining gap in community αµ was attributed to species-specific photoacclimation mechanisms and to light-dependent respiratory losses. In contrast, community maximal growth rates µmax were similar between incubations at constant and fluctuating light. At daily growth saturating light supply, differences in losses for biosynthesis between the two light regimes were observed. Phytoplankton experiencing constant light suffered photo-inhibition - leading to photosynthesis foregone and additional respiratory costs for photosystems repair. On the contrary, intermittent exposure to low and high light intensities prevented photo-inhibition of mixed algae but forced them to develop alternative light strategy. They better harvested and exploited surface irradiance by enhancing their photosynthesis. In the laboratory, we showed that Microcystis aeruginosa increased its oxygen consumption by dark respiration in the light few minutes only after exposure to increasing light intensities. More, we proved that within a simulated Langmuir cell, the net production at saturating light and the compensation light intensity for production at limiting light are positively related. These results are best explained by an accumulation of photosynthetic products at increasing irradiance and mobilization of these fresh resources by rapid enhancement of dark respiration for maintenance and biosynthesis at decreasing irradiance. At the daily timescale, we showed that the enhancement of photosynthesis at high irradiance for biosynthesis of species increased their maintenance respiratory costs at limiting light. Species-specific growth at saturating light µmax and compensation light intensity for growth PARcompμ of species incubated in Lake TaiHu were positively related. Because of this species-specific physiological tradeoff, species displayed different light affinities to limiting and saturating light - thereby exhibiting a gleaner-opportunist tradeoff. In Lake TaiHu, we showed that inter-specific differences in light acquisition traits (µmax and PARcompμ) allowed coexis¬tence of species on a gradient of constant light while avoiding competitive exclusion. More interestingly we demonstrated for the first time that vertical mixing (inducing fluctuating light supply for phytoplankton) may alter or even reverse the light utilization strategies of species within couple of days. The intra-specific variation in traits under fluctuating light increased the niche space for acclimated species, precluding competitive exclusion. Overall, this PhD thesis contributes to a better understanding of phytoplankton eco-physiology under fluctuating light supply. This work could enhance the quality of predictions of phytoplankton development under certain weather conditions or climate change scenarios.}, language = {en} } @phdthesis{Zemella2019, author = {Zemella, Anne}, title = {Fluoreszenzmarkierung und Modifizierung von komplexen Proteinen in eukaryotischen zellfreien Systemen durch die Etablierung von orthogonalen tRNA/Aminoacyl-tRNA-Synthetase-Paaren}, doi = {10.25932/publishup-44236}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-442361}, school = {Universit{\"a}t Potsdam}, pages = {XI, 141}, year = {2019}, abstract = {Die funktionelle Charakterisierung von therapeutisch relevanten Proteinen kann bereits durch die Bereitstellung des Zielproteins in ad{\"a}quaten Mengen limitierend sein. Dies trifft besonders auf Membranproteine zu, die aufgrund von zytotoxischen Effekten auf die Produktionszelllinie und der Tendenz Aggregate zu bilden, in niedrigen Ausbeuten an aktivem Protein resultieren k{\"o}nnen. Der lebende Organismus kann durch die Verwendung von translationsaktiven Zelllysaten umgangen werden- die Grundlage der zellfreien Proteinsynthese. Zu Beginn der Arbeit wurde die ATP-abh{\"a}ngige Translation eines Lysates auf der Basis von kultivierten Insektenzellen (Sf21) analysiert. F{\"u}r diesen Zweck wurde ein ATP-bindendes Aptamer eingesetzt, durch welches die Translation der Nanoluziferase reguliert werden konnte. Durch die dargestellte Applizierung von Aptameren, k{\"o}nnten diese zuk{\"u}nftig in zellfreien Systemen f{\"u}r die Visualisierung der Transkription und Translation eingesetzt werden, wodurch zum Beispiel komplexe Prozesse validiert werden k{\"o}nnen. Neben der reinen Proteinherstellung k{\"o}nnen Faktoren wie posttranslationale Modifikationen sowie eine Integration in eine lipidische Membran essentiell f{\"u}r die Funktionalit{\"a}t des Membranproteins sein. Im zweiten Abschnitt konnte, im zellfreien Sf21-System, f{\"u}r den G-Protein-gekoppelten Rezeptor Endothelin B sowohl eine Integration in die endogen vorhandenen Endoplasmatisch Retikulum-basierten Membranstrukturen als auch Glykosylierungen, identifiziert werden. Auf der Grundlage der erfolgreichen Synthese des ET-B-Rezeptors wurden verschiedene Methoden zur Fluoreszenzmarkierung des Adenosin-Rezeptors A2a (Adora2a) angewandt und optimiert. Im dritten Abschnitt wurde der Adora2a mit Hilfe einer vorbeladenen tRNA, welche an eine fluoreszierende Aminos{\"a}ure gekoppelt war, im zellfreien Chinesischen Zwerghamster Ovarien (CHO)-System markiert. Zus{\"a}tzlich konnte durch den Einsatz eines modifizierten tRNA/Aminoacyl-tRNA-Synthetase-Paares eine nicht-kanonische Aminos{\"a}ure an Position eines integrierten Amber-Stopcodon in die Polypeptidkette eingebaut und die funktionelle Gruppe im Anschluss an einen Fluoreszenzfarbstoff gekoppelt werden. Aufgrund des offenen Charakters eignen sich zellfreie Proteinsynthesesysteme besonders f{\"u}r eine Integration von exogenen Komponenten in den Translationsprozess. Mit Hilfe der Fluoreszenzmarkierung wurde eine ligandvermittelte Konformations{\"a}nderung im Adora2a {\"u}ber einen Biolumineszenz-Resonanzenergietransfer detektiert. Durch die Etablierung der Amber-Suppression wurde dar{\"u}ber hinaus das Hormon Erythropoetin pegyliert, wodurch Eigenschaften wie Stabilit{\"a}t und Halbwertszeit des Proteins ver{\"a}ndert wurden. Zu guter Letzt wurde ein neues tRNA/Aminoacyl-tRNA-Synthetase-Paar auf Basis der Methanosarcina mazei Pyrrolysin-Synthetase etabliert, um das Repertoire an nicht-kanonischen Aminos{\"a}uren und den damit verbundenen Kopplungsreaktionen zu erweitern. Zusammenfassend wurden die Potenziale zellfreier Systeme in Bezug auf der Herstellung von komplexen Membranproteinen und der Charakterisierung dieser durch die Einbringung einer positionsspezifischen Fluoreszenzmarkierung verdeutlicht, wodurch neue M{\"o}glichkeiten f{\"u}r die Analyse und Funktionalisierung von komplexen Proteinen geschaffen wurden.}, language = {de} } @phdthesis{Hoang2019, author = {Hoang, Yen}, title = {De novo binning strategy to analyze and visualize multi-dimensional cytometric data}, doi = {10.25932/publishup-44307}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-443078}, school = {Universit{\"a}t Potsdam}, pages = {vii, 81, xxxii}, year = {2019}, abstract = {Since half a century, cytometry has been a major scientific discipline in the field of cytomics - the study of system's biology at single cell level. It enables the investigation of physiological processes, functional characteristics and rare events with proteins by analysing multiple parameters on an individual cell basis. In the last decade, mass cytometry has been established which increased the parallel measurement to up to 50 proteins. This has shifted the analysis strategy from conventional consecutive manual gates towards multi-dimensional data processing. Novel algorithms have been developed to tackle these high-dimensional protein combinations in the data. They are mainly based on clustering or non-linear dimension reduction techniques, or both, often combined with an upstream downsampling procedure. However, these tools have obstacles either in comprehensible interpretability, reproducibility, computational complexity or in comparability between samples and groups. To address this bottleneck, a reproducible, semi-automated cytometric data mining workflow PRI (pattern recognition of immune cells) is proposed which combines three main steps: i) data preparation and storage; ii) bin-based combinatorial variable engineering of three protein markers, the so called triploTs, and subsequent sectioning of these triploTs in four parts; and iii) deployment of a data-driven supervised learning algorithm, the cross-validated elastic-net regularized logistic regression, with these triploT sections as input variables. As a result, the selected variables from the models are ranked by their prevalence, which potentially have discriminative value. The purpose is to significantly facilitate the identification of meaningful subpopulations, which are most distinguish between two groups. The proposed workflow PRI is exemplified by a recently published public mass cytometry data set. The authors found a T cell subpopulation which is discriminative between effective and ineffective treatment of breast carcinomas in mice. With PRI, that subpopulation was not only validated, but was further narrowed down as a particular Th1 cell population. Moreover, additional insights of combinatorial protein expressions are revealed in a traceable manner. An essential element in the workflow is the reproducible variable engineering. These variables serve as basis for a clearly interpretable visualization, for a structured variable exploration and as input layers in neural network constructs. PRI facilitates the determination of marker levels in a semi-continuous manner. Jointly with the combinatorial display, it allows a straightforward observation of correlating patterns, and thus, the dominant expressed markers and cell hierarchies. Furthermore, it enables the identification and complex characterization of discriminating subpopulations due to its reproducible and pseudo-multi-parametric pattern presentation. This endorses its applicability as a tool for unbiased investigations on cell subsets within multi-dimensional cytometric data sets.}, language = {en} }