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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.
Arachidonsäurelipoxygenasen (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. Für jedes humane ALOX-Gen gibt es ein orthologes Mausgen. Obwohl sich die sechs humanen ALOX-Isoformen strukturell sehr ähnlich sind, unterscheiden sich ihre funktionellen Eigenschaften deutlich voneinander. In der vorliegenden Arbeit wurden vier unterschiedliche Fragestellungen zum Vorkommen, zur biologischen Rolle und zur Evolutionsabhängigkeit der enzymatischen Eigenschaften von Säugetier-ALOX-Isoformen untersucht:
1) Spitzhörnchen (Tupaiidae) sind evolutionär näher mit dem Menschen verwandt als Nagetiere und wurden deshalb als Alternativmodelle für die Untersuchung menschlicher Erkrankungen vorgeschlagen. In dieser Arbeit wurde erstmals der Arachidonsäurestoffwechsel von Spitzhörnchen untersucht. Dabei wurde festgestellt, dass im Genom von Tupaia belangeri vier unterschiedliche ALOX15-Gene vorkommen und die Enzyme sich hinsichtlich ihrer katalytischen Eigenschaften ähneln. Diese genomische Vielfalt, die weder beim Menschen noch bei Mäusen vorhanden ist, erschwert die funktionellen Untersuchungen zur biologischen Rolle des ALOX15-Weges. Damit scheint Tupaia belangeri kein geeigneteres Tiermodel für die Untersuchung des ALOX15-Weges des Menschen zu sein.
2) Entsprechend der Evolutionshypothese können Säugetier-ALOX15-Orthologe in Arachidonsäure-12-lipoxygenierende- und Arachidonsäure-15-lipoxygenierende Enzyme eingeteilt werden. Dabei exprimieren Säugetierspezies, die einen höheren Evolutionsgrad als Gibbons aufweisen, Arachidonsäure-15-lipoxygenierende ALOX15-Orthologe, während evolutionär weniger weit entwickelte Säugetiere Arachidonsäure-12 lipoxygenierende Enzyme besitzen. In dieser Arbeit wurden elf neue ALOX15-Orthologe als rekombinante Proteine exprimiert und funktionell charakterisiert. Die erhaltenen Ergebnisse fügen sich widerspruchsfrei in die Evolutionshypothese ein und verbreitern deren experimentelle Basis. Die experimentellen Daten bestätigen auch das Triadenkonzept.
3) Da humane und murine ALOX15B-Orthologe unterschiedliche funktionelle Eigenschaften aufweisen, können Ergebnisse aus murinen Krankheitsmodellen zur biologischen Rolle der ALOX15B nicht direkt auf den Menschen übertragen werden. Um die ALOX15B-Orthologen von Maus und Mensch funktionell einander anzugleichen, wurden im Rahmen der vorliegenden Arbeit Knock-in Mäuse durch die In vivo Mutagenese mittels CRISPR/Cas9-Technik hergestellt. Diese exprimieren eine humanisierte Mutante (Doppelmutation von Tyrosin603Asparaginsäure+Histidin604Valin) der murinen Alox15b. Diese Mäuse waren lebens- und fortpflanzungsfähig, zeigten aber geschlechtsspezifische Unterschiede zu ausgekreuzten Wildtyp-Kontrolltieren im Rahmen ihre Individualentwicklung.
4) In vorhergehenden Untersuchungen zur Rolle der ALOX15B in Rahmen der Entzündungsreaktion wurde eine antiinflammatorische Wirkung des Enzyms postuliert. In der vorliegenden Arbeit wurde untersucht, ob eine Humanisierung der murinen Alox15b die Entzündungsreaktion in zwei verschiedenen murinen Entzündungsmodellen beeinflusst. Eine Humanisierung der murinen Alox15b führte zu einer verstärkten Ausbildung von Entzündungssymptomen im induzierten Dextran-Natrium-Sulfat-Kolitismodell. Im Gegensatz dazu bewirkte die Humanisierung der Alox15b eine Abschwächung der Entzündungssymptome im Freund‘schen Adjuvans Pfotenödemmodell. Diese Daten deuten darauf hin, dass sich die Rolle der ALOX15B in verschiedenen Entzündungsmodellen unterscheidet.
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.
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.
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.
Development of a CRISPR/Cas gene editing technique for the coccolithophore Chrysotila carterae
(2024)
The African weakly electric fish genus Campylomormyrus includes 15 described species mostly native to the Congo River and its tributaries. They are considered sympatric species, because their distribution area overlaps. These species generate species-specific electric organ discharges (EODs) varying in waveform characteristics, including duration, polarity, and phase number. They exhibit also pronounced divergence in their snout, i.e. the length, thickness, and curvature. The diversifications in these two phenotypical traits (EOD and snout) have been proposed as key factors promoting adaptive radiation in Campylomormyrus. The role of EODs as a pre-zygotic isolation mechanism driving sympatric speciation by promoting assortative mating has been examined using behavioral, genetical, and histological approaches. However, the evolutionary effects of the snout morphology and its link to species divergence have not been closely examined. Hence, the main objective of this study is to investigate the effect of snout morphology diversification and its correlated EOD to better understand their sympatric speciation and evolutionary drivers. Moreover, I aim to utilize the intragenus and intergenus hybrids of Campylomormyrus to better understand trait divergence as well as underlying molecular/genetic mechanisms involved in the radiation scenario. To this end, I utilized three different approaches: feeding behavior analysis, diet assessment, and geometric morphometrics analysis. I performed feeding behavior experiments to evaluate the concept of the phenotype-environment correlation by testing whether Campylomormyrus species show substrate preferences. The behavioral experiments showed that the short snout species exhibits preference to sandy substrate, the long snout species prefers a stone substrate, and the species with intermediate snout size does not exhibit any substrate preference. The experiments suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to their microhabitats. I also performed diet assessments of sympatric Campylomormyrus species and a sister genus species (Gnathonemus petersii) with markedly different snout morphologies and EOD using NGS-based DNA metabarcoding of their stomach contents. The diet of each species was documented showing that aquatic insects such as dipterans, coleopterans and trichopterans represent the major diet component. The results showed also that all species are able to exploit diverse food niches in their habitats. However, comparing the diet overlap indices showed that different snout morphologies and the associated divergence in the EOD translated into different prey spectra. These results further support the idea that the EOD could be a ‘magic trait’ triggering both adaptation and reproductive isolation. Geometric morphometrics method was also used to compare the phenotypical shape traits of the F1 intragenus (Campylomormyrus) and intergenus (Campylomormyrus species and Gnathonemus petersii) hybrids relative to their parents. The hybrids of these species were well separated based on the morphological traits, however the hybrid phenotypic traits were closer to the short-snouted species. In addition, the likelihood that the short snout expressed in the hybrids increases with increasing the genetic distance of the parental species. The results confirmed that additive effects produce intermediate phenotypes in F1-hybrids. It seems, therefore, that morphological shape traits in hybrids, unlike the physiological traits, were not expressed straightforward.
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.
Background: The role of fatty acid (FA) intake and metabolism in type 2 diabetes (T2D) incidence is controversial. Some FAs are not synthesised endogenously and, therefore, these circulating FAs reflect dietary intake, for example, the trans fatty acids (TFAs), saturated odd chain fatty acids (OCFAs), and linoleic acid, an n-6 polyunsaturated fatty acids (PUFA). It remains unclear if intake of TFA influence T2D risk and whether industrial TFAs (iTFAs) and ruminant TFAs (rTFAs) exert the same effect. Unlike even chain saturated FAs, the OCFAs have been inversely associated with T2D risk, but this association is poorly understood. Furthermore, the associations of n-6 PUFAs intake with T2D risk are still debated, while delta-5 desaturase (D5D), a key enzyme in the metabolism of PUFAs, has been consistently related to T2D risk. To better understand these relationships, the FA composition in circulating lipid fractions can be used as biomarkers of dietary intake and metabolism. The exploration of TFAs subtypes in plasma phospholipids and OCFAs and n-6 PUFAs within a wide range of lipid classes may give insights into the pathophysiology of T2D.
Aim: This thesis aimed mainly to analyse the association of TFAs, OCFAs and n-6 PUFAs with self-reported dietary intake and prospective T2D risk, using seven types of TFAs in plasma phospholipids and deep lipidomics profiling data from fifteen lipid classes.
Methods: A prospective case-cohort study was designed within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study, including all the participants who developed T2D (median follow-up 6.5 years) and a random subsample of the full cohort (subcohort: n=1248; T2D cases: n=820). The main analyses included two lipid profiles. The first was an assessment of seven TFA in plasma phospholipids, with a modified method for analysis of FA with very low abundances. The second lipid profile was derived from a high-throughout lipid profiling technology, which identified 940 distinct molecular species and allowed to quantify OCFAs and PUFAs composition across 15 lipid classes. Delta-5 desaturase (D5D) activity was estimated as 20:4/20:3-ratio. Using multivariable Cox regression models, we examined the associations of TFA subtypes with incident T2D and class-specific associations of OCFA and n-6 PUFAs with T2D risk.
Results: 16:1n-7t, 18:1n-7t, and c9t11-CLA were positively correlated with the intake of fat-rich dairy foods. iTFA 18:1 isomers were positively correlated with margarine. After adjustment for confounders and other TFAs, higher plasma phospholipid concentrations of two rTFAs were associated with a lower incidence of T2D: 18:1n-7t and t10c12-CLA. In contrast, the rTFA c9t11-CLA was associated with a higher incidence of T2D. rTFA 16:1n-7t and iTFAs (18:1n-6t, 18:1n-9t, 18:2n-6,9t) were not statistically significantly associated with T2D risk.
We observed heterogeneous integration of OCFA in different lipid classes, and the contribution of 15:0 versus 17:0 to the total OCFA abundance differed across lipid classes. Consumption of fat-rich dairy and fiber-rich foods were positively and red meat inversely correlated to OCFA abundance in plasma phospholipid classes. In women only, higher abundances of 15:0 in phosphatidylcholines (PC) and diacylglycerols (DG), and 17:0 in PC, lysophosphatidylcholines (LPC), and cholesterol esters (CE) were inversely associated with T2D risk. In men and women, a higher abundance of 15:0 in monoacylglycerols (MG) was also inversely associated with T2D. Conversely, a higher 15:0 concentration in LPC and triacylglycerols (TG) was associated with higher T2D risk in men. Women with a higher concentration of 17:0 as free fatty acids (FFA) also had higher T2D incidence.
The integration of n-6 PUFAs in lipid classes was also heterogeneous. 18:2 was highly abundant in phospholipids (particularly PC), CE, and TG; 20:3 represented a small fraction of FA in most lipid classes, and 20:4 accounted for a large proportion of circulating phosphatidylinositol (PI) and phosphatidylethanolamines (PE). Higher concentrations of 18:2 were inversely associated with T2D risk, especially within DG, TG, and LPC. However, 18:2 as part of MG was positively associated with T2D risk. Higher concentrations of 20:3 in phospholipids (PC, PE, PI), FFA, CE, and MG were linked to higher T2D incidence. 20:4 was unrelated to risk in most lipid classes, except positive associations were observed for 20:4 enriched in FFA and PE. The estimated D5D activities in PC, PE, PI, LPC, and CE were inversely associated with T2D and explained variance of estimated D5D activity by genomic variation in the FADS locus was only substantial in those lipid classes.
Conclusion: The TFAs' conformation is essential in their relationship to diabetes risk, as indicated by plasma rTFA subtypes concentrations having opposite directions of associations with diabetes risk. Plasma OCFA concentration is linked to T2D risk in a lipid class and sex-specific manner. Plasma n-6 PUFA concentrations are associated differently with T2D incidence depending on the specific FA and the lipid class. Overall, these results highlight the complexity of circulating FAs and their heterogeneous association with T2D risk depending on the specific FA structure, lipid class, and sex. My results extend the evidence of the relationship between diet, lipid metabolism, and subsequent T2D risk. In addition, my work generated several potential new biomarkers of dietary intake and prospective T2D risk.
Mitochondria and plastids are organelles with an endosymbiotic origin. During evolution, many genes are lost from the organellar genomes and get integrated in the nuclear genome, in what is known as intracellular/endosymbiotic gene transfer (IGT/EGT). IGT has been reproduced experimentally in Nicotiana tabacum at a gene transfer rate (GTR) of 1 event in 5 million cells, but, despite its centrality to eukaryotic evolution, there are no genetic factors known to influence the frequency of IGT in higher eukaryotes. The focus of this work was to determine the role of different DNA repair pathways of double strand break repair (DSBR) in the integration step of organellar DNA in the nuclear genome during IGT. Here, a CRISPR/Cas9 mutagenesis strategy was implemented in N. tabacum, with the aim of generating mutants in nuclear genes without expected visible phenotypes. This strategy led to the generation of a collection of independent mutants in the LIG4 (necessary for non-homologous end joining, NHEJ) and POLQ genes (necessary for microhomology mediated end joining, MMEJ). Targeting of other DSBR genes (KU70, KU80, RPA1C) generated mutants with unexpectedly strong developmental phenotypes.. These factors have telomeric roles, hinting towards a possible relationship between telomere length, and strength of developmental disruption upon loss of telomere structure in plants. The mutants were made in a genetic background encoding a plastid-encoded IGT reporter, that confers kanamycin resistance upon transfer to the nucleus. Through large scale independent experiments, increased IGT from the chloroplast to the nucleus was observed in lig4 mutants, as well as lines encoding a POLQ gene with a defective polymerase domain (polqΔPol). This shows that NHEJ or MMEJ have a double-sided relationship with IGT: while transferred genes may integrate using either pathway, the presence of both pathways suppresses IGT in wild-type somatic cells, thus demonstrating for the first time the extent on which nuclear genes control IGT frequency in plants. The IGT frequency increases in the mutants are likely mediated by increased availability of double strand breaks for integration. Additionally, kinetic analysis reveals that gene transfer (GT) events accumulate linearly as a function of time spent under antibiotic selection in the experiment, demonstrating that, contrary to what was previously thought, there is no such thing as a single GTR in somatic IGT experiments. Furthermore, IGT in tissue culture experiments appears to be the result of a "race against the clock" for integration in the nuclear genome, that starts when the organellar DNA arrives to the nucleus granting transient antibiotic resistance. GT events and escapes of kanamycin selection may be two possible outcomes from this race: those instances where the organellar DNA gets to integrate are recovered as GT events, and in those cases where timely integration fails, antibiotic resistance cannot be sustained, and end up considered as escapes. In the mutants, increased opportunities for integration in the nuclear genome change the overall ratio between IGT and escape events. The resources generated here are promising starting points for future research: (1) the mutant collection, for the further study of processes that depend on DNA repair in plants (2) the collection of GT lines obtained from these experiments, for the study of the effect of DSBR pathways over integration patterns and stability of transferred genes and (3) the developed CRISPR/Cas9 workflow for mutant generation, to make N. tabacum meet its potential as an attractive model for answering complex biological questions.
The G protein-coupled estrogen receptor (GPER1) is acknowledged as an important mediator of estrogen signaling. Given the ubiquitous expression of GPER1, it is likely that the receptor plays a role in a variety of malignancies, not only in the classic hormonally regulated tissues (e.g., breast, ovary, and prostate), but also in the colon. As colorectal cancer (CRC) is the third most common cancer in both men and women worldwide and environmental factors and dietary habits are important risk factors, it is increasingly recognized that natural and synthetic hormones and their associated receptors might play a role in CRC. Through oral consumption, environmental contaminants with endocrine activity are in contact with the gastrointestinal mucosa, where they might exert their toxic effects. Although GPER1 has been shown to be engaged in physiological and pathophysiological processes, its role in CRC remains poorly understood. Thus, pro- as well as anti-tumorigenic effects are described in the literature. This thesis has uncovered novel roles of GPER1 in mediating major CRC-associated phenotypes in transformed and non-transformed colon cell lines. Exposure to the estrogens 17β-estradiol (E2), bisphenol-A (BPA) and diethylstilbestrol (DES) but also the androgen dihydrotestosterone (DHT) resulted in GPER1-dependent induction of supernumerary centrosomes, whole chromosomal instability (w-CIN) and aneuploidy. Indeed, both knockdown and inhibition of GPER1 attenuated the generation of (xeno)hormone-driven supernumerary centrosomes and karyotype instability. Mechanistically, (xeno)hormone-induced centrosome amplification was associated with transient multipolar mitosis and the generation of so called anaphase “lagging” chromosomes. The results of this thesis propose a GPER1/PKA/AKAP9-pathway in regulating centrosome numbers in colorectal cancer cells and the involvement of the centriolar protein centrin. Remarkably, exposure to (xeno)hormones resulted in atypical enlargement and unexpected phosphorylation of the centriole marker centrin in interphase. These findings provide a novel role for GPER1 in key CRC-prone lesions and shed light on underlying mechanisms that involve GPER1 function in the colon. Elucidating to what extent centrosomal proteins are involved in the GPER1-mediated aneugenic effect will be an important task for future studies. The present study was intended to lay a first foundation to understand the molecular basis and potential risk factors of CRC which might help to reduce the use of laboratory animals. Since numerous animal experiments are conducted in biomedical research, the development of alternative methods is indispensable. The Federal Institute for Risk Assessment (BfR) as the German Center for the Protection of Laboratory Animals (Bf3R) addresses this issue by uncovering underlying mechanisms leading to colorectal cancer as necessary prerequisite in order to develop alternative methods.
Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4).
We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin.
For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators.
In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation.
The light reactions of photosynthesis are carried out by a series of multiprotein complexes embedded in thylakoid membranes. Among them, photosystem I (PSI), acting as plastocyanin-ferderoxin oxidoreductase, catalyzes the final reaction. Together with light-harvesting antenna I, PSI forms a high-molecular-weight supercomplex of ~600 kDa, consisting of eighteen subunits and nearly two hundred co-factors. Assembly of the various components into a functional thylakoid membrane complex requires precise coordination, which is provided by the assembly machinery. Although this includes a small number of proteins (PSI assembly factors) that have been shown to play a role in the formation of PSI, the process as a whole, as well as the intricacy of its members, remains largely unexplored.
In the present work, two approaches were used to find candidate PSI assembly factors. First, EnsembleNet was used to select proteins thought to be functionally related to known PSI assembly factors in Arabidopsis thaliana (approach I), and second, co-immunoprecipitation (Co-IP) of tagged PSI assembly factors in Nicotiana tabacum was performed (approach II).
Here, the novel PSI assembly factors designated CO-EXPRESSED WITH PSI ASSEMBLY 1 (CEPA1) and Ycf4-INTERACTING PROTEIN 1 (Y4IP1) were identified. A. thaliana null mutants for CEPA1 and Y4IP1 showed a growth phenotype and pale leaves compared with the wild type. Biophysical experiments using pulse amplitude modulation (PAM) revealed insufficient electron transport on the PSII acceptor side. Biochemical analyses revealed that both CEPA1 and Y4IP1 are specifically involved in PSI accumulation in A. thaliana at the post-translational level but are not essential. Consistent with their roles as factors in the assembly of a thylakoid membrane protein complex, the two proteins localize to thylakoid membranes. Remarkably, cepa1 y4ip1 double mutants exhibited lethal phenotypes in early developmental stages under photoautotrophic growth. Finally, co-IP and native gel experiments supported a possible role for CEPA1 and Y4IP1 in mediating PSI assembly in conjunction with other PSI assembly factors (e.g., PPD1- and PSA3-CEPA1 and Ycf4-Y4IP1). The fact that CEPA1 and Y4IP1 are found exclusively in green algae and higher plants suggests eukaryote-specific functions. Although the specific mechanisms need further investigation, CEPA1 and Y4IP1 are two novel assembly factors that contribute to PSI formation.
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.
The musculoskeletal system provides support and enables movement to the body, and its deterioration is a crucial aspect of age-related functional decline. Mesenchymal stromal cells (MSCs) play an important role in musculoskeletal homeostasis due to their broad differentiation potentials and their ability to support osteogenic and myogenic tissue maintenance and regeneration. In the bone, MSCs differentiate either into osteochondrogenic progenitors to form osteocytes and chondrocytes, or increasingly with age into adipogenic progenitors which give rise to bone-resident adipocytes. In skeletal muscle, during healthy regeneration MSCs provide regulatory signals that activate local, tissue-specific stem cells, known as satellite cells, which regenerate contractile myofibres. This process involves a significant cross-talk to immune cells stemming from both lymphoid and myeloid lineages. During ageing, muscle-resident MSCs undergo increased adipogenic lineage commitment, causing niche changes that contribute to fatty infiltration in muscles. These shifts in cell populations in bone lead to the loss of osteogenic cells and subsequently osteoporosis, or in muscle to impaired regeneration and to the development of sarcopenia. However, the signals that drive transition of MSCs into their respective cellular fates remain elusive.
This thesis aims to elucidate the transcriptional shifts modulating cell states and cell types in musculoskeletal MSC fate determination. Single-cell RNA-sequencing (scRNA-seq) was used to characterise cell type-specific transcript regulation. State-of-the-art bioinformatics tools were combined with different analytical platforms that include both droplet-based scRNA-seq for large heterogeneous populations, and microfluidics-based scRNA-seq to assess small, rare subpopulations. For each platform, distinct computational pipelines were established including filtering steps to exclude low-quality cells, and data visualisation was performed by dimensionality reduction. Downstream analysis included clustering, cell type annotation, and differential gene expression to investigate transcriptional states in defined cell types during ageing and injury in the muscle and bone. Finally, a novel tool to assess publication activities in defined areas of research for the identified marker genes was developed.
The results in the bone indicate that ageing MSCs increasingly commit towards an adipogenic fate at the expense of osteogenic specialisation. The data also suggests that significant cell population shifts of MSC-type fibro-adipogenic progenitors during muscle ageing underlie the pathologies observed in homeostatic and post-injury regenerative conditions. High-throughput visualisation of publication activity for candidate genes enabled more effective biological evaluation of scRNA-seq data. These results expose critical age-related changes in the stem cell niches of skeletal muscle and bone, highlight their respective sensitivity to nutrition and pathology, and elucidate novel factors that modulate stem cell-based regeneration. Targeting these processes might improve musculoskeletal health in the context of ageing and prevent the negative effects of pathological lineage determination.
Selenium (Se) is an essential trace element that is ubiquitously present in the environment in small concentrations. Essential functions of Se in the human body are manifested through the wide range of proteins, containing selenocysteine as their active center. Such proteins are called selenoproteins which are found in multiple physiological processes like antioxidative defense and the regulation of thyroid hormone functions. Therefore, Se deficiency is known to cause a broad spectrum of physiological impairments, especially in endemic regions with low Se content. Nevertheless, being an essential trace element, Se could exhibit toxic effects, if its intake exceeds tolerable levels. Accordingly, this range between deficiency and overexposure represents optimal Se supply. However, this range was found to be narrower than for any other essential trace element. Together with significantly varying Se concentrations in soil and the presence of specific bioaccumulation factors, this represents a noticeable difficulty in the assessment of Se
epidemiological status. While Se is acting in the body through multiple selenoproteins, its intake occurs mainly in form of small organic or inorganic molecular mass species. Thus, Se exposure not only depends on daily intake but also on the respective chemical form, in which it is present.
The essential functions of selenium have been known for a long time and its primary forms in different food sources have been described. Nevertheless, analytical capabilities for a comprehensive investigation of Se species and their derivatives have been introduced only in the last decades. A new Se compound was identified in 2010 in the blood and tissues of bluefin tuna. It was called selenoneine (SeN) since it is an isologue of naturally occurring antioxidant ergothioneine (ET), where Se replaces sulfur. In the following years, SeN was identified in a number of edible fish species and attracted attention as a new dietary Se source and potentially strong antioxidant. Studies in populations whose diet largely relies on fish revealed that SeN
represents the main non-protein bound Se pool in their blood. First studies, conducted with enriched fish extracts, already demonstrated the high antioxidative potential of SeN and its possible function in the detoxification of methylmercury in fish. Cell culture studies demonstrated, that SeN can utilize the same transporter as ergothioneine, and SeN metabolite was found in human urine.
Until recently, studies on SeN properties were severely limited due to the lack of ways to obtain the pure compound. As a predisposition to this work was firstly a successful approach to SeN synthesis in the University of Graz, utilizing genetically modified yeasts. In the current study, by use of HepG2 liver carcinoma cells, it was demonstrated, that SeN does not cause toxic effectsup to 100 μM concentration in hepatocytes. Uptake experiments showed that SeN is not bioavailable to the used liver cells.
In the next part a blood-brain barrier (BBB) model, based on capillary endothelial cells from the porcine brain, was used to describe the possible transfer of SeN into the central nervous system (CNS). The assessment of toxicity markers in these endothelial cells and monitoring of barrier conditions during transfer experiments demonstrated the absence of toxic effects from SeN on the BBB endothelium up to 100 μM concentration. Transfer data for SeN showed slow but substantial transfer. A statistically significant increase was observed after 48 hours following SeN incubation from the blood-facing side of the barrier. However, an increase in Se content was clearly visible already after 6 hours of incubation with 1 μM of SeN. While the transfer rate of SeN after application of 0.1 μM dose was very close to that for 1 μM, incubation with 10 μM of SeN resulted in a significantly decreased transfer rate. Double-sided application of SeN caused no side-specific transfer of SeN, thus suggesting a passive diffusion mechanism of SeN across the BBB. This data is in accordance with animal studies, where ET accumulation was observed in the rat brain, even though rat BBB does not have the primary ET transporter – OCTN1. Investigation of capillary endothelial cell monolayers after incubation with SeN and reference selenium compounds showed no significant increase of intracellular selenium concentration. Speciesspecific Se measurements in medium samples from apical and basolateral compartments, as good as in cell lysates, showed no SeN metabolization. Therefore, it can be concluded that SeN may reach the brain without significant transformation.
As the third part of this work, the assessment of SeN antioxidant properties was performed in Caco-2 human colorectal adenocarcinoma cells. Previous studies demonstrated that the intestinal epithelium is able to actively transport SeN from the intestinal lumen to the blood side and accumulate SeN. Further investigation within current work showed a much higher antioxidant potential of SeN compared to ET. The radical scavenging activity after incubation with SeN was close to the one observed for selenite and selenomethionine. However, the SeN effect on the viability of intestinal cells under oxidative conditions was close to the one caused by ET. To answer the question if SeN is able to be used as a dietary Se source and induce the activity of selenoproteins, the activity of glutathione peroxidase (GPx) and the secretion of selenoprotein P (SelenoP) were measured in Caco-2 cells, additionally. As expected, reference selenium compounds selenite and selenomethionine caused efficient induction of GPx activity. In contrast to those SeN had no effect on GPx activity. To examine the possibility of SeN being embedded into the selenoproteome, SelenoP was measured in a culture medium. Even though Caco-2 cells effectively take up SeN in quantities much higher than selenite or selenomethionine, no secretion of SelenoP was observed after SeN incubation.
Summarizing, we can conclude that SeN can hardly serve as a Se source for selenoprotein synthesis. However, SeN exhibit strong antioxidative properties, which appear when sulfur in ET is exchanged by Se. Therefore, SeN is of particular interest for research not as part of Se metabolism, but important endemic dietary antioxidant.
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.
Pichia pastoris (syn. Komagataella phaffi) is a distinguished expression system widely used in industrial production processes. Recent molecular research has focused on numerous approaches to increase recombinant protein yield in P. pastoris. For example, the design of expression vectors and synthetic genetic elements, gene copy number optimization, or co-expression of helper proteins
(transcription factors, chaperones, etc.). However, high clonal variability of transformants and low screening throughput have hampered significant success.
To enhance screening capacities, display-based methodologies inherit the potential for efficient isolation of producer clones via fluorescence-activated cell sorting (FACS). Therefore, this study focused on developing a novel clone selection method that is based on the non-covalent attachment of Fab fragments on the P. pastoris cell surface to be applicable for FACS.
Initially, a P. pastoris display system was developed, which is a prerequisite for the surface capture of secreted Fabs. A Design of Experiments approach was applied to analyze the influence of various genetic elements on antibody fragment display. The combined P. pastoris formaldehyde dehydrogenase promoter (PFLD1), Saccharomyces cerevisiae invertase 2 signal peptide (ScSUC2), - agglutinin (ScSAG1) anchor protein, and the ARS of Kluyveromyces lactis (panARS) conferred highest display levels.
Subsequently, eight single-chain variable fragments (scFv) specific for the constant part of the Fab heavy or light chain were individually displayed in P. pastoris. Among the tested scFvs, the anti-human CH1 IgG domain scFv allowed the most efficient Fab capture detected by flow cytometry.
Irrespective of the Fab sequence, exogenously added as well as simultaneously secreted Fabs were successfully captured on the cell surface. Furthermore, Fab secretion capacities were shown to correlate to the level of surface-bound Fabs as demonstrated for characterized producer clones.
Flow-sorted clones presenting high amounts of Fabs showed an increase in median Fab titers (factor of 21 to 49) compared to unsorted clones when screened in deep-well plates. For selected candidates, improved functional Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask production. Since the scFv capture matrix was encoded on an episomal plasmid with inherently unstable autonomously replicating sequences (ARS), efficient plasmid curing was observed after removing the selective pressure. Hence, sorted clones could be immediately used for production without the need to modify the expression host or vector. The resulting switchable display/secretion system provides a streamlined approach for the isolation of Fab producers and subsequent Fab production.
Development of electrochemical antibody-based and enzymatic assays for mycotoxin analysis in food
(2023)
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.
Aging is a complex process characterized by several factors, including loss of genetic and epigenetic information, accumulation of chronic oxidative stress, protein damage and aggregates and it is becoming an emergent drug target. Therefore, it is the utmost importance to study aging and agerelated diseases, to provide treatments to develop a healthy aging process. Skeletal muscle is one of the earliest tissues affected by age-related changes with progressive loss of muscle mass and function from 30 years old, effect known as sarcopenia. Several studies have shown the accumulation of protein aggregates in different animal models, as well as in humans, suggesting impaired proteostasis, a hallmark of aging, especially regarding degradation systems. Thus, different publications have explored the role of the main proteolytic systems in skeletal muscle from rodents and humans, like ubiquitin proteasomal system (UPS) and autophagy lysosomal system (ALS), however with contradictory results. Yet, most of the published studies are performed in muscles that comprise more than one fiber type, that means, muscles composed by slow and fast fibers. These fiber types, exhibit different metabolism and contraction speed; the slow fibers or type I display an oxidative metabolism, while fast fibers function towards a glycolytic metabolism ranging from fast oxidative to fast glycolytic fibers. To this extent, the aim of this thesis sought to understand on how aging impacts both fiber types not only regarding proteostasis but also at a metabolome and transcriptome network levels. Therefore, the first part of this thesis, presents the differences between slow oxidative (from Soleus muscle) and fast glycolytic fibers (Extensor digitorum longus, EDL) in terms of degradation systems and how they cope with oxidative stress during aging, while the second part explores the differences between young and old EDL muscle transcriptome and metabolome, unraveling molecular features. More specifically, the results from the present work show that slow oxidative muscle performs better at maintaining the function of UPS and ALS during aging than EDL muscle, which is clearly affected, accounting for the decline in the catalytic activity rates and accumulation of autophagy-related proteins. Strinkingly, transcriptome and metabolome analyses reveal that fast glycolytic muscle evidences significant downregulation of mitochondrial related processes and damaged mitochondria morphology during aging, despite of having a lower oxidative metabolism compared to oxidative fibers. Moreover, predictive analyses reveal a negative association between aged EDL gene signature and lifespan extending interventions such as caloric restriction (CR). Although, CR intervention does not alter the levels of mitochondrial markers in aged EDL muscle, it can reverse the higher mRNA levels of muscle damage markers. Together, the results from this thesis give new insights about how different metabolic muscle fibers cope with age-related changes and why fast glycolytic fibers are more susceptible to aging than slow oxidative fibers.
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.
Life on Earth is diverse and ranges from unicellular organisms to multicellular creatures like humans. Although there are theories about how these organisms might have evolved, we understand little about how ‘life’ started from molecules. Bottom-up synthetic biology aims to create minimal cells by combining different modules, such as compartmentalization, growth, division, and cellular communication.
All living cells have a membrane that separates them from the surrounding aqueous medium and helps to protect them. In addition, all eukaryotic cells have organelles that are enclosed by intracellular membranes. Each cellular membrane is primarily made of a lipid bilayer with membrane proteins. Lipids are amphiphilic molecules that assemble into molecular bilayers consisting of two leaflets. The hydrophobic chains of the lipids in the two leaflets face each other, and their hydrophilic headgroups face the aqueous surroundings. Giant unilamellar vesicles (GUVs) are model membrane systems that form large compartments with a size of many micrometers and enclosed by a single lipid bilayer. The size of GUVs is comparable to the size of cells, making them good membrane models which can be studied using an optical microscope. However, after the initial preparation, GUV membranes lack membrane proteins which have to be reconstituted into these membranes by subsequent preparation steps. Depending on the protein, it can be either attached via anchor lipids to one of the membrane leaflets or inserted into the lipid bilayer via its transmembrane domains.
The first step is to prepare the GUVs and then expose them to an exterior solution with proteins. Various protocols have been developed for the initial preparation of GUVs. For the second step, the GUVs can be exposed to a bulk solution of protein or can be trapped in a microfluidic device and then supplied with the protein solution. To minimize the amount of solution and for more precise measurements, I have designed a microfluidic device that has a main channel, and several dead-end side channels that are perpendicular to the main channel. The GUVs are trapped in the dead-end channels. This design exchanges the solution around the GUVs via diffusion from the main channel, thus shielding the GUVs from the flow within the main channel. This device has a small volume of just 2.5 μL, can be used without a pump and can be combined with a confocal microscope, enabling uninterrupted imaging of the GUVs during the experiments. I used this device for most of the experiments on GUVs that are discussed in this thesis.
In the first project of the thesis, a lipid mixture doped with an anchor lipid was used that can bind to a histidine chain (referred to as His-tag(ged) or 6H) via the metal cation Ni2+. This method is widely used for the biofunctionalization of GUVs by attaching proteins without a transmembrane domain. Fluorescently labeled His-tags which are bound to a membrane can be observed in a confocal microscope. Using the same lipid mixture, I prepared the GUVs with different protocols and investigated the membrane composition of the resulting GUVs by evaluating the amount of fluorescently labeled His-tagged molecules bound to their membranes. I used the microfluidic device described above to expose the outer leaflet of the vesicle to a constant concentration of the His-tagged molecules. Two fluorescent molecules with a His-tag were studied and compared: green fluorescent protein (6H-GFP) and fluorescein isothiocyanate (6H-FITC). Although the quantum yield in solution is similar for both molecules, the brightness of the membrane-bound 6H-GFP is higher than the brightness of the membrane-bound 6H-FITC. The observed difference in the brightness reveals that the fluorescence of the 6H-FITC is quenched by the anchor lipid via the Ni2+ ion. Furthermore, my measurements also showed that the fluorescence intensity of the membranebound His-tagged molecules depends on microenvironmental factors such as pH. For both 6H-GFP and 6H-FITC, the interaction with the membrane is quantified by evaluating the equilibrium dissociation constant. The membrane fluorescence is measured as a function of the fluorophores’ molar concentration. Theoretical analysis of these data leads to the equilibrium dissociation constants of (37.5 ± 7.5) nM for 6H-GFP and (18.5 ± 3.7) nM for 6H-FITC.
The anchor lipid mentioned previously used the metal cation Ni2+ to mediate the bond between the anchor lipid and the His-tag. The Ni2+ ion can be replaced by other transition metal ions. Studies have shown that Co3+ forms the strongest bonds with the His-tags attached to proteins. In these studies, strong oxidizing agents were used to oxidize the Co2+ mediated complex with the His-tagged protein to a Co3+ mediated complex. This procedure puts the proteins at risk of being oxidized as well. In this thesis, the vesicles were first prepared with anchor lipids without any metal cation. The Co3+ was added to these anchor lipids and finally the His-tagged protein was added to the GUVs to form the Co3+ mediated bond. This system was also established using the microfluidic device.
The different preparation procedures of GUVs usually lead to vesicles with a spherical morphology. On the other hand, many cell organelles have a more complex architecture with a non spherical topology. One fascinating example is provided by the endoplasmic reticulum (ER) which is made of a continuous membrane and extends throughout the cell in the form of tubes and sheets. The tubes are connected by three-way junctions and form a tubular network of irregular polygons. The formation and maintenance of these reticular networks requires membrane proteins that hydrolyize guanosine triphosphate (GTP). One of these membrane proteins is atlastin. In this thesis, I reconstituted the atlastin protein in GUV membranes using detergent-assisted reconstitution protocols to insert the proteins directly into lipid bilayers.
This thesis focuses on protein reconstitution by binding His-tagged proteins to anchor lipids and by detergent-assisted insertion of proteins with transmembrane domains. It also provides the design of a microfluidic device that can be used in various experiments, one example is the evaluation of the equilibrium dissociation constant for membrane-protein interactions. The results of this thesis will help other researchers to understand the protocols for preparing GUVs, to reconstitute proteins in GUVs, and to perform experiments using the microfluidic device. This knowledge should be beneficial for the long-term goal of combining the different modules of synthetic biology to make a minimal cell.
Gene expression data is analyzed to identify biomarkers, e.g. relevant genes, which serve for diagnostic, predictive, or prognostic use. Traditional approaches for biomarker detection select distinctive features from the data based exclusively on the signals therein, facing multiple shortcomings in regards to overfitting, biomarker robustness, and actual biological relevance. Prior knowledge approaches are expected to address these issues by incorporating prior biological knowledge, e.g. on gene-disease associations, into the actual analysis. However, prior knowledge approaches are currently not widely applied in practice because they are often use-case specific and seldom applicable in a different scope. This leads to a lack of comparability of prior knowledge approaches, which in turn makes it currently impossible to assess their effectiveness in a broader context.
Our work addresses the aforementioned issues with three contributions. Our first contribution provides formal definitions for both prior knowledge and the flexible integration thereof into the feature selection process. Central to these concepts is the automatic retrieval of prior knowledge from online knowledge bases, which allows for streamlining the retrieval process and agreeing on a uniform definition for prior knowledge. We subsequently describe novel and generalized prior knowledge approaches that are flexible regarding the used prior knowledge and applicable to varying use case domains. Our second contribution is the benchmarking platform Comprior. Comprior applies the aforementioned concepts in practice and allows for flexibly setting up comprehensive benchmarking studies for examining the performance of existing and novel prior knowledge approaches. It streamlines the retrieval of prior knowledge and allows for combining it with prior knowledge approaches. Comprior demonstrates the practical applicability of our concepts and further fosters the overall development and comparability of prior knowledge approaches. Our third contribution is a comprehensive case study on the effectiveness of prior knowledge approaches. For that, we used Comprior and tested a broad range of both traditional and prior knowledge approaches in combination with multiple knowledge bases on data sets from multiple disease domains. Ultimately, our case study constitutes a thorough assessment of a) the suitability of selected knowledge bases for integration, b) the impact of prior knowledge being applied at different integration levels, and c) the improvements in terms of classification performance, biological relevance, and overall robustness.
In summary, our contributions demonstrate that generalized concepts for prior knowledge and a streamlined retrieval process improve the applicability of prior knowledge approaches. Results from our case study show that the integration of prior knowledge positively affects biomarker results, particularly regarding their robustness. Our findings provide the first in-depth insights on the effectiveness of prior knowledge approaches and build a valuable foundation for future research.
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.
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.
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.
Housing in metabolic cages can induce a pronounced stress response. Metabolic cage systems imply housing mice on metal wire mesh for the collection of urine and feces in addition to monitoring food and water intake. Moreover, mice are single-housed, and no nesting, bedding, or enrichment material is provided, which is often argued to have a not negligible impact on animal welfare due to cold stress. We therefore attempted to reduce stress during metabolic cage housing for mice by comparing an innovative metabolic cage (IMC) with a commercially available metabolic cage from Tecniplast GmbH (TMC) and a control cage. Substantial refinement measures were incorporated into the IMC cage design. In the frame of a multifactorial approach for severity assessment, parameters such as body weight, body composition, food intake, cage and body surface temperature (thermal imaging), mRNA expression of uncoupling protein 1 (Ucp1) in brown adipose tissue (BAT), fur score, and fecal corticosterone metabolites (CMs) were included. Female and male C57BL/6J mice were single-housed for 24 h in either conventional Macrolon cages (control), IMC, or TMC for two sessions. Body weight decreased less in the IMC (females—1st restraint: 6.94%; 2nd restraint: 6.89%; males—1st restraint: 8.08%; 2nd restraint: 5.82%) compared to the TMC (females—1st restraint: 13.2%; 2nd restraint: 15.0%; males—1st restraint: 13.1%; 2nd restraint: 14.9%) and the IMC possessed a higher cage temperature (females—1st restraint: 23.7°C; 2nd restraint: 23.5 °C; males—1st restraint: 23.3 °C; 2nd restraint: 23.5 °C) compared with the TMC (females—1st restraint: 22.4 °C; 2nd restraint: 22.5 °C; males—1st restraint: 22.6 °C; 2nd restraint: 22.4 °C). The concentration of fecal corticosterone metabolites in the TMC (females—1st restraint: 1376 ng/g dry weight (DW); 2nd restraint: 2098 ng/g DW; males—1st restraint: 1030 ng/g DW; 2nd restraint: 1163 ng/g DW) was higher compared to control cage housing (females—1st restraint:
640 ng/g DW; 2nd restraint: 941 ng/g DW; males—1st restraint: 504 ng/g DW; 2nd restraint: 537 ng/g DW). Our results show the stress potential induced by metabolic cage restraint that is markedly influenced by the lower housing temperature. The IMC represents a first attempt to target cold stress reduction during metabolic cage application thereby producing more animal welfare friendly data.
The development of seeds in angiosperms starts with a complex process of double fertilization, involving the fusion of the maternal egg cell and central cell with two paternal sperm cells. This gives rise to the embryo and the nourishing endosperm, which are then enclosed by the seed coat, derived from the maternal integuments. The growth of the seed coat in Arabidopsis thaliana (Arabidopsis) is actively inhibited before fertilization by epigenetic regulators known as Polycomb Group (PcG) proteins. These proteins deposit a repressive histone mark called H3K27me3, which must be removed to enable seed coat formation. In this thesis, I explored the mechanism of removal of H3K27me3 marks from the integument cells following fertilization, which allows for seed coat formation. We hypothesized that this removal should be primarily facilitated by histone demethylases from the JMJ family and potentially influenced by the plant hormones Brassinosteroids (BRs). This hypothesis was supported by the expression patterns of the JMJ protein REF6 and of BR related genes, which are specifically expressed in the integuments and in the seed coat. Moreover, mutations in both these pathways lead to developmental defects, such as reduced ovule viability and delayed seed coat growth. Our research provides evidence suggesting that BR signalling is likely involved in recruiting JMJ-type histone demethylases to target loci responsible for seed coat growth. Moreover, we have discovered an additional pathway through which BRs regulate seed coat development, independent of their influence on H3K27me3 marks. This finding emphasizes the diverse roles of BRs in coordinating seed development, extending beyond their well-known involvement in plant growth and development. Furthermore, I explored the role of another epigenetic mark, DNA methylation, in fertilization-independent (or autonomous) seed formation in Arabidopsis. For this, we utilized epigenetic Recombinant Inbred Lines (epiRILs) and thus identified an epigenetic Quantitative Trait Locus (epiQTL) on chromosome II, potentially responsible for the larger autonomous seed size observed in DNA methylation mutants. Overall, this thesis significantly enhances our comprehension of the intricate relationship between epigenetic modifications, hormonal signaling, and plant reproductive processes. It offers valuable insights into the genetic mechanisms governing both sexual and asexual seed formation, while also presenting potential avenues for the engineer of advantageous traits in agricultural crops.
Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana
(2023)
Biostimulant SuperFifty based molecular priming to increase plant strength and stress tolerance
(2023)
In times of ongoing biodiversity loss, understanding how communities are structured and what mechanisms and local adaptations underlie the patterns we observe in nature is crucial for predicting how future ecological and anthropogenic changes might affect local and regional biodiversity. Aquatic zooplankton are a group of primary consumers that represent a critical link in the food chain, providing nutrients for the entire food web. Thus, understanding the adaptability and structure of zooplankton communities is essential. In this work, the genetic basis for the different temperature adaptations of two seasonally shifted (i.e., temperature-dependent) occurring freshwater rotifers of a formerly cryptic species complex (Brachionus calyciflorus) was investigated to understand the overall genetic diversity and evolutionary scenario for putative adaptations to different temperature regimes. Furthermore, this work aimed to clarify to what extent the different temperature adaptations may represent a niche partitioning process thus enabling co-existence. The findings were then embedded in a metacommunity context to understand how zooplankton communities assemble in a kettle hole metacommunity located in the northeastern German "Uckermark" and which underlying processes contribute to the biodiversity patterns we observe. Using a combined approach of newly generated mitochondrial resources (genomes/cds) and the analysis of a candidate gene (Heat Shock Protein 40kDa) for temperature adaptation, I showed that the global representatives of B. calyciflorus s.s.. are genetically more similar than B. fernandoi (average pairwise nucleotide diversity: 0.079 intraspecific vs. 0.257 interspecific) indicating that both species carry different standing genetic variation. In addition to differential expression in the thermotolerant B. calyciflorus s.s. and thermosensitive B. fernandoi, the HSP 40kDa also showed structural variation with eleven fixed and six positively selected sites, some of which are located in functional areas of the protein. The estimated divergence time of ~ 25-29 Myr combined with the fixed sites and a prevalence of ancestral amino acids in B. calyciflorus s.s. indicate that B. calyciflorus s.s. remained in the ancestral niche, while B. fernandoi partitioned into a new niche. The comparison of mitochondrial and nuclear markers (HPS 40kDa, ITS1, COI) revealed a hybridisation event between the two species. However, as hybridisation between the two species is rare, it can be concluded that the temporally isolated niches (i.e., seasonal-shifted occurrence) they inhabit based on their different temperature preferences most likely represent a pre-zygotic isolation mechanism that allows sympatric occurrence while maintaining species boundaries. To determine the processes underlying zooplankton community assembly, a zooplankton metacommunity comprising 24 kettle holes was sampled over a two-year period. Active (i.e., water samples) and dormant communities (i.e., dormant eggs hatched from sediment) were identified using a two-fragment DNA metabarcoding approach (COI and 18S). Species richness and diversity as well as community composition were analysed considering spatial, temporal and environmental parameters. The analysis revealed that environmental filtering based on parameters such as pH, size and location of the habitat patch (i.e., kettle hole) and surrounding field crops largely determined zooplankton community composition (explained variance: Bray-Curtis dissimilarities: 10.5%; Jaccard dissimilarities: 12.9%), indicating that adaptation to a particular habitat is a key feature of zooplankton species in this system. While the spatial configuration of the kettle holes played a minor role (explained variance: Bray-Curtis dissimilarities: 2.8% and Jaccard dissimilarities: 5.5%), the individual kettle hole sites had a significant influence on the community composition. This suggests monopolisation/priority effects (i.e., dormant communities) of certain species in individual kettle holes. As environmental filtering is the dominating process structuring zooplankton communities, this system could be significantly influenced by future land-use change, pollution and climate change.
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 Förster 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.
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.
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.
Die Fluoreszenz-Calcium-Imaging-Methode wird auch heute noch als gängige Methode verwendet, vor allem wegen der geringeren Kosten für das Wirkstoffscreening in der pharmazeutischen Forschung, wobei Ionenkanäle 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 Überexpression dieser ionenpermeablen Proteine in Zellen auftreten können, wie z. B. Zelltoxizität, geringere Proteinexpression und die Beseitigung der exprimierten Proteine aufgrund veränderter Domänen sowie die zeitaufwändige Pflege von Zelllinien. Die Synthese von Ionenkanälen in zellfreien Proteinsyntheseplattformen für das künftige 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 für die schnelle pharmakologische Analyse von Wirkstoffen. Das erste Ziel dieser wissenschaftlichen Arbeit bestand darin, die grundlegenden Prinzipien der Calcium-Imaging-Methode zur Untersuchung von Ionenkanälen in zellbasierten Systemen zu untersuchen. Hierfür 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 überexprimierten transient-receptor-potential-Ionenkanälen (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 überexprimiert. In dieser Studie wurden insbesondere die Aktivierung klassischer GPCR-Signalwege wie Phospholipase C und Proteinkinase C durch Angiotensin-(1-7) über Mas und die Beteiligung von TRP-Kanälen nachgewiesen. Die zellbasierte-Calcium-Imaging-Methode für 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 für die Integration synthetisierter Ionenkanäle 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 Ionenkanäle zu untersuchen. Die Enzymaktivität 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-Ionenkanäle wurden mit dem Fura-5N-AM- Fluoreszenzfarbstoff in mit Carboxylesterase vorsynthetisierten Mikrosomen nachgewiesen.
Zusammenfassend lässt sich sagen, dass das Prinzip der zellbasierten Calcium-Imaging -Methode vielversprechend an das eukaryotische zellfreie Sf21-System angepasst werden konnte, um Ionenkanäle zu analysieren. Nach entsprechender Forschung könnte die etablierte Methode in Zukunft auch auf andere Membranproteine ausgeweitet werden. Dies umfasst die Untersuchung anderer zell-frei exprimierte GPCRs oder anderer Ionenkanäle wie Kalium-, Natrium- und Chlorid-Ionenkanäle.
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.
Over the last decades, interest in the impact of the intestinal microbiota on host health has steadily increased. Diet is a major factor that influences the gut microbiota and thereby indirectly affects human health. For example, a high fat diet rich in saturated fatty acids led to an intestinal proliferation of the colitogenic bacterium Bilophila (B.) wadsworthia by stimulating the release of the bile acid taurocholate (TC). TC contains the sulfonated head group taurine, which undergoes conversion to sulfide (H2S) by B. wadsworthia. In a colitis prone murine animal model (IL10 / mice), the bloom of B. wadsworthia was accompanied by an exacerbation of intestinal inflammation. B. wadsworthia is able to convert taurine and also other sulfonates to H2S, indicating the potential association of sulfonate utilization and the stimulation of colitogenic bacteria.
This potential link raised the question, whether dietary sulfonates or their sulfonated metabolites stimulate the growth of colitogenic bacteria such as B. wadsworthia and whether these bacteria convert sulfonates to H2S. Besides taurine, which is present in meat, fish and life-style beverages, other dietary sulfonates are part of daily human nutrition. Sulfolipids such as sulfoquinovosyldiacylglycerols (SQDG) are highly abundant in salad, parsley and the cyanobacterium Arthrospira platensis (Spirulina). Based on previous findings, Escherichia (E.) coli releases the polar headgroup sulfoquinovose (SQ) from SQDG. Moreover, E. coli is able to convert SQ to 2,3 dihydroxypropane 1 sulfonate (DHPS) under anoxic conditions. DHPS is also converted to H2S by B. wadsworthia or by other potentially harmful gut bacteria such as members of the genus Desulfovibrio. However, only few studies report the conversion of sulfonates to H2S by bacteria directly isolated from the human intestinal tract. Most sulfonate utilizing bacteria were obtained from environmental sources such as soil or lake sediment or from potentially intestinal sources such as sewage.
In the present study, fecal slurries from healthy human subjects were incubated with sulfonates under strictly anoxic conditions, using formate and lactate as electron donors. Fecal slurries that converted sulfonates to H2S, were used as a source for the isolation of H2S forming bacteria. Isolates were identified based on their 16S ribosomal RNA (16S rRNA) gene sequence. In addition, conventional C57BL/6 mice were fed a semisynthetic diet supplemented with the SQDG rich Spirulina (SD) or a Spirulina free control diet (CD). During the intervention, body weight, water and food intake were monitored and fecal samples were collected. After three weeks, mice were killed and organ weight and size were measured, intestinal sulfonate concentrations were quantified, gut microbiota composition was determined and parameters of intestinal and hepatic fat metabolism were analyzed.
Human fecal slurries converted taurine, isethionate, cysteate, 3 sulfolacate, SQ and DHPS to H2S. However, inter individual differences in the degradation of these sulfonates were observed. Taurine, isethionate, and 3 sulfolactate were utilized by fecal microbiota of all donors, while SQ, DHPS and cysteate were converted to H2S only by microbiota from certain individuals. Bacterial isolates from human feces able to convert sulfonates to H2S were identified as taurine-utilizing Desulfovibrio strains, taurine- and isethionate-utilizing B. wadsworthia, or as SQ- and 3-sulfolactate- utilizing E. coli. In addition, a co culture of E. coli and B. wadsworthia led to complete degradation of SQ to H2S, with DHPS as an intermediate. Of the human fecal isolates, B. wadsworthia and Desulfovibrio are potentially harmful. E. coli strains might be also pathogenic, but isolated E. coli strains from human feces were identified as commensal gut bacteria.
Feeding SD to mice increased the cecal and fecal SQ concentration and altered the microbiota composition, but the relative abundance of SQDG or SQ converting bacteria and colitogenic bacteria was not enriched in mice fed SD for 21 days. SD did not affect the relative abundance of Enterobacteriaceae, to which the SQDG- and SQ-utilizing E. coli strain belong to. Furthermore, the abundance of B. wadsworthia decreased from day 2 to day 9 in feces, but recovered afterwards in the same mice. In cecum, the family Desulfovibrionaceae, to which B. wadsworthia and Desulfovibrio belong to, were reduced. No changes in the number of B. wadsworthia in cecal contents or of Desulfovibrionaceae in feces were observed. SD led to a mild activation of the immune system, which was not observed in control mice fed CD. Mice fed SD had an increased body weight, a higher adipose tissue weight, and a decreased liver weight compared to the control mice, suggesting an impact of Spirulina supplementation on fat metabolism. However, expression levels of genes involved in intestinal and hepatic intracellular lipid uptake and availability were reduced. Further investigations on the lipid metabolism at protein level could help to clarify these discrepancies.
In summary, humans differ in the ability of their fecal microbiota to utilize dietary sulfonates. While sulfonates stimulated the proliferation of potentially colitogenic isolates from human fecal slurries, the increased availability of SQ in Spirulina fed conventional mice did not lead to an enrichment of such bacteria. Presence or absence of these bacteria may explain the inter individual differences in sulfonate conversion observed for fecal slurries. This work provides new insights in the ability of intestinal bacteria to utilize sulfonates and thus, contributes to a better understanding of microbiota-mediated effects on dietary sulfonate utilization. Interestingly, feeding of the Spirulina-supplemented diet led to body-weight gain in mice in the first two days of intervention, the reasons for which are unknown.
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.
Heat stress (HS) is one of the major abiotic stresses which adversely affects the survival and growth of plants due to their sessile nature. To combat the detrimental effects of HS and develop thermotolerance, plants have evolved several defense mechanisms. Thermomemory is one such molecular mechanism whereby plants that have been acclimated (or primed/P) by a moderate HS can respond more efficiently and continue their growth after exposure to a severe or lethal HS (called triggering/T), while unprimed plants cannot survive. Thermomemory is known to be regulated by several transcription factors (TFs), epigenetic changes, chromatin remodellers, post-transcriptional changes and it also involves protein stability control and primary metabolism adjustment. Recent research has suggested that the shoot apical meristem (SAM) in Arabidopsis thaliana has a distinct transcriptional thermomemory which is possibly regulated by eight TFs called HEAT SHOCK FACTORS (HSFs). The main objective of this PhD thesis is to investigate the role of HSFA7b (one of the eight HSFs), in regulating thermomemory at the SAM by identifying the molecular networks it regulates. HSFA7a, a close homolog of HSFA7b, is also one of the eight HSFs that are involved in regulating thermomemory at the SAM. Thermomemory was found to be defective in the hsfa7b and hsfa7a hsfa7b mutants; the percentage survival of these seedlings was significantly lower than in wild-type (WT) seedlings after the priming and triggering (PT) treatment. Transcriptome and ChIP analyses were performed to identify the molecular networks controlled by HSFA7b and its close homolog HSFA7a, in regulating thermomemory at the SAM. The chromatin regulator SPLAYED (SYD) was found to be regulated by both HSFA7a and HSFA7b at the SAM during thermomemory. SYD is directly involved in SAM maintenance by directly regulating WUSCHEL (WUS), a master regulator of stem cell maintenance. WUS expression was down-regulated at the SAM of PT treated hsfa7a/b mutants compared to WT-Col-0 seedlings. HSFA7a and HSFA7b also jointly regulate the expression of orphan gene QUA QUINE STARCH (QQS) during thermomemory. Starch accumulation negatively correlates with QQS expression and this trend was observed in WT plants in response to thermopriming. The remobilization of starch was affected in the hsfa7a/b mutants compared to WT plants during the recovery period after T treatment. These findings indicate that defects in SAM maintenance and starch remobilization could possibly contribute to the reduced thermomemory in the hsfa7a/b mutants. Moreover, transcriptome and ChIP analysis indicate that ethylene signaling genes are directly regulated by HSFA7b during thermomemory. Transcriptome analysis of the HSFA7b-IOE line indicates that HSFA7b positively regulates the expression of HEAT STRESS ASSOCIATED 32 (HSA32), an important thermomemory gene, and HSFA7b strongly suppresses the expression of the reactive oxygen species (ROS) responsive REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) gene, which is also a repressed target of SYD. In Arabidopsis, the HSFA7b transcript undergoes alternative splicing at high temperatures to form two splice variants: one correctly/constitutively spliced variant which is functional and codes for the HSFA7b protein and one intron retained splice variant. Higher accumulation of the functional HSFA7b splice variant was found at the SAM compared to other tissues. Moreover, accumulation of the functional splice variant was higher in P and PT plants compared to control plants, whereas higher levels of the intron retained splice variant is found in plants subjected directly to the T treatment. The intron retained HSFA7b splice variant is degraded by the non-sense mediated decay (NMD) pathway as a means of regulating transcript level essential for protein synthesis at high temperatures. Importantly, HSFA7b protein accumulation was observed in plants subjected to PT treatment that survive and continue growth, but not in plants subjected directly to T treatment that do not survive, indicating that constitutive/ correct splicing of the HSFA7b transcript is a component of thermomemory. Taken together, these findings suggest that HSFA7a and HSFA7b jointly regulate SAM maintenance via the chromatin remodeller SYD and starch remobilization via QQS. In addition to them, HSFA7b also regulates the expression of ethylene signaling genes, heat responsive genes and the ROS responsive RRTF1. Furthermore, constitutive/correct splicing in the HSFA7b transcript is also an essential component of thermomemory.
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.
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.