@phdthesis{Ziege2022,
  author    = {Ziege, Ricardo},
  title     = {Growth dynamics and mechanical properties of E. coli biofilms},
  doi       = {10.25932/publishup-55986},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-559869},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xi, 123},
  year      = {2022},
  abstract  = {Biofilms are complex living materials that form as bacteria get embedded in a matrix of self-produced protein and polysaccharide fibres. The formation of a network of extracellular biopolymer fibres contributes to the cohesion of the biofilm by promoting cell-cell attachment and by mediating biofilm-substrate interactions. This sessile mode of bacteria growth has been well studied by microbiologists to prevent the detrimental effects of biofilms in medical and industrial settings. Indeed, biofilms are associated with increased antibiotic resistance in bacterial infections, and they can also cause clogging of pipelines or promote bio-corrosion. However, biofilms also gained interest from biophysics due to their ability to form complex morphological patterns during growth. Recently, the emerging field of engineered living materials investigates biofilm mechanical properties at multiple length scales and leverages the tools of synthetic biology to tune the functions of their constitutive biopolymers. This doctoral thesis aims at clarifying how the morphogenesis of Escherichia coli (E. coli) biofilms is influenced by their growth dynamics and mechanical properties. To address this question, I used methods from cell mechanics and materials science. I first studied how biological activity in biofilms gives rise to non-uniform growth patterns. In a second study, I investigated how E. coli biofilm morphogenesis and its mechanical properties adapt to an environmental stimulus, namely the water content of their substrate. Finally, I estimated how the mechanical properties of E. coli biofilms are altered when the bacteria express different extracellular biopolymers. On nutritive hydrogels, micron-sized E. coli cells can build centimetre-large biofilms. During this process, bacterial proliferation and matrix production introduce mechanical stresses in the biofilm, which release through the formation of macroscopic wrinkles and delaminated buckles. To relate these biological and mechanical phenomena, I used time-lapse fluorescence imaging to track cell and matrix surface densities through the early and late stages of E. coli biofilm growth. Colocalization of high cell and matrix densities at the periphery precede the onset of mechanical instabilities at this annular region. Early growth is detected at this outer annulus, which was analysed by adding fluorescent microspheres to the bacterial inoculum. But only when high rates of matrix production are present in the biofilm centre, does overall biofilm spreading initiate along the solid-air interface. By tracking larger fluorescent particles for a long time, I could distinguish several kinematic stages of E. coli biofilm expansion and observed a transition from non-linear to linear velocity profiles, which precedes the emergence of wrinkles at the biofilm periphery. Decomposing particle velocities to their radial and circumferential components revealed a last kinematic stage, where biofilm movement is mostly directed towards the radial delaminated buckles, which verticalize. The resulting compressive strains computed in these regions were observed to substantially deform the underlying agar substrates. The co-localization of higher cell and matrix densities towards an annular region and the succession of several kinematic stages are thus expected to promote the emergence of mechanical instabilities at the biofilm periphery. These experimental findings are predicted to advance future modelling approaches of biofilm morphogenesis. E. coli biofilm morphogenesis is further anticipated to depend on external stimuli from the environment. To clarify how the water could be used to tune biofilm material properties, we quantified E. coli biofilm growth, wrinkling dynamics and rigidity as a function of the water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that substrates with high water content promote biofilm spreading kinetics, while substrates with low water content promote biofilm wrinkling. The wrinkles observed on biofilm cross-sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, the micro-indentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to the water content of their substrate, which might be used for tuning their material properties in view of further applications. Biofilm material properties further depend on the composition and structure of the matrix of extracellular proteins and polysaccharides. In particular, E. coli biofilms were suggested to present tissue-like elasticity due to a dense fibre network consisting of amyloid curli and phosphoethanolamine-modified cellulose. To understand the contribution of these components to the emergent mechanical properties of E. coli biofilms, we performed micro-indentation on biofilms grown from bacteria of several strains. Besides showing higher dry masses, larger spreading diameters and slightly reduced water contents, biofilms expressing both main matrix components also presented high rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, a lack of amyloid curli fibres provides much higher adhesive energies and more viscoelastic fluid-like material behaviour. Therefore, the combination of amyloid curli and phosphoethanolamine-modified cellulose fibres implies the formation of a composite material whereby the amyloid curli fibres provide rigidity to E. coli biofilms, whereas the phosphoethanolamine-modified cellulose rather acts as a glue. These findings motivate further studies involving purified versions of these protein and polysaccharide components to better understand how their interactions benefit biofilm functions. All three studies depict different aspects of biofilm morphogenesis, which are interrelated. The first work reveals the correlation between non-uniform biological activities and the emergence of mechanical instabilities in the biofilm. The second work acknowledges the adaptive nature of E. coli biofilm morphogenesis and its mechanical properties to an environmental stimulus, namely water. Finally, the last study reveals the complementary role of the individual matrix components in the formation of a stable biofilm material, which not only forms complex morphologies but also functions as a protective shield for the bacteria it contains. Our experimental findings on E. coli biofilm morphogenesis and their mechanical properties can have further implications for fundamental and applied biofilm research fields.},
  language  = {en}
}
@phdthesis{WijesinghaAhchige2022,
  author    = {Wijesingha Ahchige, Micha},
  title     = {Canalization of plant metabolism and yield},
  doi       = {10.25932/publishup-54884},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548844},
  school      = {Universit{\"a}t Potsdam},
  pages     = {VIII, 160},
  year      = {2022},
  abstract  = {Plant metabolism is the main process of converting assimilated carbon to different crucial compounds for plant growth and therefore crop yield, which makes it an important research topic. Although major advances in understanding genetic principles contributing to metabolism and yield have been made, little is known about the genetics responsible for trait variation or canalization although the concepts have been known for a long time. In light of a growing global population and progressing climate change, understanding canalization of metabolism and yield seems ever-more important to ensure food security. Our group has recently found canalization metabolite quantitative trait loci (cmQTL) for tomato fruit metabolism, showing that the concept of canalization applies on metabolism. In this work two approaches to investigate plant metabolic canalization and one approach to investigate yield canalization are presented. In the first project, primary and secondary metabolic data from Arabidopsis thaliana and Phaseolus vulgaris leaf material, obtained from plants grown under different conditions was used to calculate cross-environment coefficient of variations or fold-changes of metabolite levels per genotype and used as input for genome wide association studies. While primary metabolites have lower CV across conditions and show few and mostly weak associations to genomic regions, secondary metabolites have higher CV and show more, strong metabolite to genome associations. As candidate genes, both potential regulatory genes as well as metabolic genes, can be found, albeit most metabolic genes are rarely directly related to the target metabolites, suggesting a role for both potential regulatory mechanisms as well as metabolic network structure for canalization of metabolism. In the second project, candidate genes of the Solanum lycopersicum cmQTL mapping are selected and CRISPR/Cas9-mediated gene-edited tomato lines are created, to validate the genes role in canalization of metabolism. Obtained mutants appeared to either have strong aberrant developmental phenotypes or appear wild type-like. One phenotypically inconspicuous mutant of a pantothenate kinase, selected as candidate for malic acid canalization shows a significant increase of CV across different watering conditions. Another such mutant of a protein putatively involved in amino acid transport, selected as candidate for phenylalanine canalization shows a similar tendency to increased CV without statistical significance. This potential role of two genes involved in metabolism supports the hypothesis of structural relevance of metabolism for its own stability. In the third project, a mutant for a putative disulfide isomerase, important for thylakoid biogenesis, is characterized by a multi-omics approach. The mutant was characterized previously in a yield stability screening and showed a variegated leaf phenotype, ranging from green leaves with wild type levels of chlorophyll over differently patterned variegated to completely white leaves almost completely devoid of photosynthetic pigments. White mutant leaves show wild type transcript levels of photosystem assembly factors, with the exception of ELIP and DEG orthologs indicating a stagnation at an etioplast to chloroplast transition state. Green mutant leaves show an upregulation of these assembly factors, possibly acting as overcompensation for partially defective disulfide isomerase, which seems sufficient for proper chloroplast development as confirmed by a wild type-like proteome. Likely as a result of this phenotype, a general stress response, a shift to a sink-like tissue and abnormal thylakoid membranes, strongly alter the metabolic profile of white mutant leaves. As the severity and pattern of variegation varies from plant to plant and may be effected by external factors, the effect on yield instability, may be a cause of a decanalized ability to fully exploit the whole leaf surface area for photosynthetic activity.},
  language  = {en}
}
@phdthesis{Welsch2022,
  author    = {Welsch, Maryna},
  title     = {Investigation of the stress tolerance regulatory network integration of the NAC transcription factor JUNGBRUNNEN1 (JUB1)},
  doi       = {10.25932/publishup-54731},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-547310},
  school      = {Universit{\"a}t Potsdam},
  pages     = {XIII, 116},
  year      = {2022},
  abstract  = {The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) is an important negative regulator of plant senescence, as well as of gibberellic acid (GA) and brassinosteroid (BR) biosynthesis in Arabidopsis thaliana. Overexpression of JUB1 promotes longevity and enhances tolerance to drought and other abiotic stresses. A similar role of JUB1 has been observed in other plant species, including tomato and banana. Our data show that JUB1 overexpressors (JUB1-OXs) accumulate higher levels of proline than WT plants under control conditions, during the onset of drought stress, and thereafter. We identified that overexpression of JUB1 induces key proline biosynthesis and suppresses key proline degradation genes. Furthermore, bZIP63, the transcription factor involved in proline metabolism, was identified as a novel downstream target of JUB1 by Yeast One-Hybrid (Y1H) analysis and Chromatin immunoprecipitation (ChIP). However, based on Electrophoretic Mobility Shift Assay (EMSA), direct binding of JUB1 to bZIP63 could not be confirmed. Our data indicate that JUB1-OX plants exhibit reduced stomatal conductance under control conditions. However, selective overexpression of JUB1 in guard cells did not improve drought stress tolerance in Arabidopsis. Moreover, the drought-tolerant phenotype of JUB1 overexpressors does not solely depend on the transcriptional control of the DREB2A gene. Thus, our data suggest that JUB1 confers tolerance to drought stress by regulating multiple components. Until today, none of the previous studies on JUB1´s regulatory network focused on identifying protein-protein interactions. We, therefore, performed a yeast two-hybrid screen (Y2H) which identified several protein interactors of JUB1, two of which are the calcium-binding proteins CaM1 and CaM4. Both proteins interact with JUB1 in the nucleus of Arabidopsis protoplasts. Moreover, JUB1 is expressed with CaM1 and CaM4 under the same conditions. Since CaM1.1 and CaM4.1 encode proteins with identical amino acid sequences, all further experiments were performed with constructs involving the CaM4 coding sequence. Our data show that JUB1 harbors multiple CaM-binding sites, which are localized in both the N-terminal and C-terminal regions of the protein. One of the CaM-binding sites, localized in the DNA-binding domain of JUB1, was identified as a functional CaM-binding site since its mutation strongly reduced the binding of CaM4 to JUB1. Furthermore, JUB1 transactivates expression of the stress-related gene DREB2A in mesophyll cells; this effect is significantly reduced when the calcium-binding protein CaM4 is expressed as well. Overexpression of both genes in Arabidopsis results in early senescence observed through lower chlorophyll content and an enhanced expression of senescence-associated genes (SAGs) when compared with single JUB1 overexpressors. Our data also show that JUB1 and CaM4 proteins interact in senescent leaves, which have increased Ca2+ levels when compared to young leaves. Collectively, our data indicate that JUB1 activity towards its downstream targets is fine-tuned by calcium-binding proteins during leaf senescence.},
  language  = {en}
}
@phdthesis{Tegtmeier2022,
  author    = {Tegtmeier, Laura},
  title     = {Functional analysis of ENTH domain proteins},
  doi       = {10.25932/publishup-57004},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-570049},
  school      = {Universit{\"a}t Potsdam},
  pages     = {106},
  year      = {2022},
  abstract  = {In plant cells, subcellular transport of cargo proteins relies to a large extent on post-Golgi transport pathways, many of which are mediated by clathrin-coated vesicles (CCVs). Vesicle formation is facilitated by different factors like accessory proteins and adaptor protein complexes (APs), the latter serving as a bridge between cargo proteins and the coat protein clathrin. One type of accessory proteins is defined by a conserved EPSIN N-TERMINAL HOMOLOGY (ENTH) domain and interacts with APs and clathrin via motifs in the C-terminal part. In Arabidopsis thaliana, there are three closely related ENTH domain proteins (EPSIN1, 2 and 3) and one highly conserved but phylogenetically distant outlier, termed MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1). In case of the trans-Golgi network (TGN) located MTV1, clathrin association and a role in vacuolar transport have been shown previously (Sauer et al. 2013). In contrast, for EPSIN1 and EPSIN2 limited functional and localization data were available; and EPSIN3 remained completely uncharacterized prior to this study (Song et al. 2006; Lee et al. 2007). The molecular details of ENTH domain proteins in plants are still unknown. In order to systematically characterize all four ENTH proteins in planta, we first investigated expression and subcellular localization by analysis of stable reporter lines under their endogenous promotors. Although all four genes are ubiquitously expressed, their subcellular distribution differs markedly. EPSIN1 and MTV1 are located at the TGN, whereas EPSIN2 and EPSIN3 are associated with the plasma membrane (PM) and the cell plate. To examine potential functional redundancy, we isolated knockout T-DNA mutant lines and created all higher order mutant combinations. The clearest evidence for functional redundancy was observed in the epsin1 mtv1 double mutant, which is a dwarf displaying overall growth reduction. These findings are in line with the TGN localization of both MTV1 and EPS1. In contrast, loss of EPSIN2 and EPSIN3 does not result in a growth phenotype compared to wild type, however, a triple knockout of EPSIN1, EPSIN2 and EPSIN3 shows partially sterile plants. We focused mainly on the epsin1 mtv1 double mutant and addressed the functional role of these two genes in clathrin-mediated vesicle transport by comprehensive molecular, biochemical, and genetic analyses. Our results demonstrate that EPSIN1 and MTV1 promote vacuolar transport and secretion of a subset of cargo. However, they do not seem to be involved in endocytosis and recycling. Importantly, employing high-resolution imaging, genetic and biochemical experiments probing the relationship of the AP complexes, we found that EPSIN1/AP1 and MTV1/AP4 define two spatially and molecularly distinct subdomains of the TGN. The AP4 complex is essential for MTV1 recruitment to the TGN, whereas EPSIN1 is independent of AP4 but presumably acts in an AP1-dependent framework. Our findings suggest that this ENTH/AP pairing preference is conserved between animals and plants.},
  language  = {en}
}
@phdthesis{Sun2022,
  author    = {Sun, Xianlei},
  title     = {Elasticity of fiber meshes derived from multiblock copolymers influences cell behaviors},
  doi       = {10.25932/publishup-53528},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-535285},
  school      = {Universit{\"a}t Potsdam},
  pages     = {96},
  year      = {2022},
  abstract  = {Objective: The behaviors of endothelial cells or mesenchymal stem cells are remarkably influenced by the mechanical properties of their surrounding microenvironments. Here, electrospun fiber meshes containing various mechanical characteristics were developed from polyetheresterurethane (PEEU) copolymers. The goal of this study was to explore how fiber mesh stiffness affected endothelial cell shape, growth, migration, and angiogenic potential of endothelial cells. Furthermore, the effects of the E-modulus of fiber meshes on human adipose-derived stem cells (hADSCs) osteogenic potential was investigated. Methods: Polyesteretherurethane (PEEU) polymers with various poly(p-dioxanone) (PPDO) to poly (ε-caprolactone) (PCL) weight percentages (40 wt.\%, 50 wt.\%, 60 wt.\%, and 70 wt.\%) were synthesized, termed PEEU40, PEEU50, PEEU60, and PEEU70, accordingly. The electrospinning method was used for the preparation of PEEU fiber meshes. The effects of PEEU fiber meshes with varying elasticities on the human umbilical vein endothelial cells (HUVECs) shape, growth, migration and angiogenic potential were characterized. To determine how the E-modulus of fiber meshes affects the osteogenic potential of hADSCs, the cellular and nuclear morphologies and osteogenic differentiation abilities were evaluated. Results: With the increasing stiffness of PEEU fiber meshes, the aspect ratios of HUVECs cultivated on PEEU materials increased. HUVECs cultivated on high stiffness fiber meshes (4.5 ± 0.8 MPa) displayed a considerably greater proliferation rate and migratory velocity, in addition demonstrating increased tube formation capability, compared with those of the cells cultivated on lower stiffness fiber meshes (2.6 ± 0.8 MPa). Furthermore, in comparison to those cultivated on lower stiffness fiber meshes, hADSCs adhered to the highest stiffness fiber meshes PEEU70 had an elongated shape. The hADSCs grown on the softer PEEU40 fiber meshes showed a reduced nuclear aspect ratio (width to height) than those cultivated on the stiffer fiber meshes. Culturing hADSCs on stiffer fibers improved their osteogenic differentiation potential. Compared with cells cultured on PEEU40, osteocalcin expression and alkaline phosphatase (ALP) activity increased by 73 ± 10\% and 43 ± 16\%, respectively, in cells cultured on PEEU70. Conclusion: The mechanical characteristics of the substrate are crucial in the modulation of cell behaviors. These findings indicate that adjusting the elasticity of fiber meshes might be a useful method for controlling the blood vessels development and regeneration. Furthermore, the mechanical characteristics of PEEU fiber meshes might be modified to control the osteogenic potential of hADSCs.},
  language  = {en}
}
@phdthesis{Shevtsova2022,
  author    = {Shevtsova, Iuliia},
  title     = {Recent and future vegetation change in the treeline region of Chukotka (NE Russia) inferred from field data, satellite data and modelling},
  doi       = {10.25932/publishup-54845},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548452},
  school      = {Universit{\"a}t Potsdam},
  pages     = {149},
  year      = {2022},
  abstract  = {Vegetation change at high latitudes is one of the central issues nowadays with respect to ongoing climate changes and triggered potential feedback. At high latitude ecosystems, the expected changes include boreal treeline advance, compositional, phenological, physiological (plants), biomass (phytomass) and productivity changes. However, the rate and the extent of the changes under climate change are yet poorly understood and projections are necessary for effective adaptive strategies and forehanded minimisation of the possible negative feedbacks. The vegetation itself and environmental conditions, which are playing a great role in its development and distribution are diverse throughout the Subarctic to the Arctic. Among the least investigated areas is central Chukotka in North-Eastern Siberia, Russia. Chukotka has mountainous terrain and a wide variety of vegetation types on the gradient from treeless tundra to northern taiga forests. The treeline there in contrast to subarctic North America and north-western and central Siberia is represented by a deciduous conifer, Larix cajanderi Mayr. The vegetation varies from prostrate lichen Dryas octopetala L. tundra to open graminoid (hummock and non-hummock) tundra to tall Pinus pumila (Pall.) Regel shrublands to sparse and dense larch forests. Hence, this thesis presents investigations on recent compositional and above-ground biomass (AGB) changes, as well as potential future changes in AGB in central Chukotka. The aim is to assess how tundra-taiga vegetation develops under changing climate conditions particularly in Fareast Russia, central Chukotka. Therefore, three main research questions were considered: 1) What changes in vegetation composition have recently occurred in central Chukotka? 2) How have the above-ground biomass AGB rates and distribution changed in central Chukotka? 3) What are the spatial dynamics and rates of tree AGB change in the upcoming millennia in the northern tundra-taiga of central Chukotka? Remote sensing provides information on the spatial and temporal variability of vegetation. I used Landsat satellite data together with field data (foliage projective cover and AGB) from two expeditions in 2016 and 2018 to Chukotka to upscale vegetation types and AGB for the study area. More specifically, I used Landsat spectral indices (Normalised Difference Vegetation Index (NDVI), Normalised Difference Water Index (NDWI) and Normalised Difference Snow Index (NDSI)) and constrained ordination (Redundancy analysis, RDA) for further k-means-based land-cover classification and general additive model (GAM)-based AGB maps for 2000/2001/2002 and 2016/2017. I also used Tandem-X DEM data for a topographical correction of the Landsat satellite data and to derive slope, aspect, and Topographical Wetness Index (TWI) data for forecasting AGB. Firstly, in 2016, taxa-specific projective cover data were collected during a Russian-German expedition. I processed the field data and coupled them with Landsat spectral Indices in the RDA model that was used for k-means classification. I could establish four meaningful land-cover classes: (1) larch closed-canopy forest, (2) forest tundra and shrub tundra, (3) graminoid tundra and (4) prostrate herb tundra and barren areas, and accordingly, I produced the land cover maps for 2000/2001/2002 and 2016/20017. Changes in land-cover classes between the beginning of the century (2000/2001/2002) and the present time (2016/2017) were estimated and interpreted as recent compositional changes in central Chukotka. The transition from graminoid tundra to forest tundra and shrub tundra was interpreted as shrubification and amounts to a 20\% area increase in the tundra-taiga zone and 40\% area increase in the northern taiga. Major contributors of shrubification are alder, dwarf birch and some species of the heather family. Land-cover change from the forest tundra and shrub tundra class to the larch closed-canopy forest class is interpreted as tree infilling and is notable in the northern taiga. We find almost no land-cover changes in the present treeless tundra. Secondly, total AGB state and change were investigated for the same areas. In addition to the total vegetation AGB, I provided estimations for the different taxa present at the field sites. As an outcome, AGB in the study region of central Chukotka ranged from 0 kg m-2 at barren areas to 16 kg m-2 in closed-canopy forests with the larch trees contributing the highest. A comparison of changes in AGB within the investigated period from 2000 to 2016 shows that the greatest changes (up to 1.25 kg m 2 yr 1) occurred in the northern taiga and in areas where land cover changed to larch closed-canopy forest. Our estimations indicate a general increase in total AGB throughout the investigated tundra-taiga and northern taiga, whereas the tundra showed no evidence of change in AGB within the 15 years from 2002 to 2017. In the third manuscript, potential future AGB changes were estimated based on the results of simulations of the individual-based spatially explicit vegetation model LAVESI using different climate scenarios, depending on Representative Concentration Pathways (RCPs) RCP 2.6, RCP 4.5 and RCP 8.5 with or without cooling after 2300 CE. LAVESI-based AGB was simulated for the current state until 3000 CE for the northern tundra-taiga study area for larch species because we expect the most notable changes to occur will be associated with forest expansion in the treeline ecotone. The spatial distribution and current state of tree AGB was validated against AGB field data, AGB extracted from Landsat satellite data and a high spatial resolution image with distinctive trees visible. The simulation results are indicating differences in tree AGB dynamics plot wise, depending on the distance to the current treeline. The simulated tree AGB dynamics are in concordance with fundamental ecological (emigrational and successional) processes: tree stand formation in simulated results starts with seed dispersion, tree stand establishment, tree stand densification and episodic thinning. Our results suggest mostly densification of existing tree stands in the study region within the current century in the study region and a lagged forest expansion (up to 39\% of total area in the RCP 8.5) under all considered climate scenarios without cooling in different local areas depending on the closeness to the current treeline. In scenarios with cooling air temperature after 2300 CE, forests stopped expanding at 2300 CE (up to 10\%, RCP 8.5) and then gradually retreated to their pre-21st century position. The average tree AGB rates of increase are the strongest in the first 300 years of the 21st century. The rates depend on the RCP scenario, where the highest are as expected under RCP 8.5. Overall, this interdisciplinary thesis shows a successful integration of field data, satellite data and modelling for tracking recent and predicting future vegetation changes in mountainous subarctic regions. The obtained results are unique for the focus area in central Chukotka and overall, for mountainous high latitude ecosystems.},
  language  = {en}
}
@phdthesis{Schulte2022,
  author    = {Schulte, Luise},
  title     = {Dynamics of Larix (Mill.) species in Siberia during the last 50,000 years inferred from sedimentary ancient DNA},
  doi       = {10.25932/publishup-55878},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-558782},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xi, 121},
  year      = {2022},
  abstract  = {The deciduous needle tree larch (Larix Mill.) covers more than 80\% of the Asian boreal forests. Only a few Larix species constitute the vast forests and these species differ markedly in their ecological traits, most importantly in their ability to grow on and stabilize underlying permafrost. The pronounced dominance of the summergreen larches makes the Asian boreal forests unique, as the rest of the northern hemisphere boreal forests is almost exclusively dominated by evergreen needle-leaf forests. Global warming is impacting the whole world but is especially pronounced in the arctic and boreal regions. Although adapted to extreme climatic conditions, larch forests are sensitive to varying climatic conditions. By their sheer size, changes in Asian larch forests as range shifts or changes in species composition and the resulting vegetation-climate feedbacks are of global relevance. It is however still uncertain if larch forests will persist under the ongoing warming climate or if they will be replaced by evergreen forests. It is therefore of great importance to understand how these ecosystems will react to future climate warmings and if they will maintain their dominance. One step in the better understanding of larch dynamics is to study how the vast dominant forests developed and why they only established in northern Asia. A second step is to study how the species reacted to past changes in the climate. The first objective of this thesis was to review and identify factors promoting Asian larch dominance. I achieved this by synthesizing and comparing reported larch occurrences and influencing components on the northern hemisphere continents in the present and in the past. The second objective was to find a possibility to directly study past Larix populations in Siberia and specifically their genetic variation, enabling the study of geographic movements. For this, I established chloroplast enrichment by hybridization capture from sedimentary ancient DNA (sedaDNA) isolated from lake sediment records. The third objective was to use the established method to track past larch populations, their glacial refugia during the Last Glacial Maximum (LGM) around 21,000 years before present (ka BP), and their post-glacial migration patterns. To study larch promoting factors, I compared the present state of larch species ranges, areas of dominance, their bioclimatic niches, and the distribution on different extents and thaw depths of permafrost. The species comparison showed that the bioclimatic niches greatly overlap between the American and Asian species and that it is only in the extremely continental climates in which only the Asian larch species can persist. I revealed that the area of dominance is strongly connected to permafrost extent but less linked to permafrost seasonal thaw depths. Comparisons of the paleorecord of larch between the continents suggest differences in the recolonization history. Outside of northern Asia and Alaska, glacial refugial populations of larch were confined to the southern regions and thus recolonization could only occur as migration from south to north. Alaskan larch populations could not establish wide-range dominant forest which could be related to their own genetically depletion as separated refugial population. In Asia, it is still unclear whether or not the northern refugial populations contributed and enhanced the postglacial colonization or whether they were replaced by populations invading from the south in the course of climate warming. Asian larch dominance is thus promoted partly by adaptions to extremely continental climates and by adaptations to grow on continuous permafrost but could be also connected to differences in glacial survival and recolonization history of Larix species. Except for extremely rare macrofossil findings of fossilized cones, traditional methods to study past vegetation are not able to distinguish between larch species or populations. Within the scope of this thesis, I therefore established a method to retrieve genetic information of past larch populations to distinguish between species. Using the Larix chloroplast genome as target, I successfully applied the method of DNA target enrichment by hybridization capture on sedaDNA samples from lake records and showed that it is able to distinguish between larch species. I then used the method on samples from lake records from across Siberia dating back up to 50 ka BP. The results allowed me to address the question of glacial survival and post-glacial recolonization mode in Siberian larch species. The analyzed pattern showed that LGM refugia were almost exclusively constituted by L. gmelinii, even in sites of current L. sibirica distribution. For included study sites, L. sibirica migrated into its extant northern distribution area only in the Holocene. Consequently, the post-glacial recolonization of L. sibirica was not enhanced by northern glacial refugia. In case of sites in extant distribution area of L. gmelinii, the absence of a genetic turn-over point to a continuous population rather than an invasion of southern refugia. The results suggest that climate has a strong influence on the distribution of Larix species and that species may also respond differently to future climate warming. Because species differ in their ecological characteristics, species distribution is also relevant with respect to further feedbacks between vegetation and climate. With this thesis, I give an overview of present and past larch occurrences and evaluate which factors promote their dominance. Furthermore, I provide the tools to study past Larix species and give first important insights into the glacial history of Larix populations.},
  language  = {en}
}
@phdthesis{Pellegrino2022,
  author    = {Pellegrino, Antonio},
  title     = {miRNA profiling for diagnosis of chronic pain in polyneuropathy},
  doi       = {10.25932/publishup-58385},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-583858},
  school      = {Universit{\"a}t Potsdam},
  pages     = {viii, 97, xi},
  year      = {2022},
  abstract  = {This dissertation aimed to determine differential expressed miRNAs in the context of chronic pain in polyneuropathy. For this purpose, patients with chronic painful polyneuropathy were compared with age matched healthy patients. Taken together, all miRNA pre library preparation quality controls were successful and none of the samples was identified as an outlier or excluded for library preparation. Pre sequencing quality control showed that library preparation worked for all samples as well as that all samples were free of adapter dimers after BluePippin size selection and reached the minimum molarity for further processing. Thus, all samples were subjected to sequencing. The sequencing control parameters were in their optimal range and resulted in valid sequencing results with strong sample to sample correlation for all samples. The resulting FASTQ file of each miRNA library was analyzed and used to perform a differential expression analysis. The differentially expressed and filtered miRNAs were subjected to miRDB to perform a target prediction. Three of those four miRNAs were downregulated: hsa-miR-3135b, hsa-miR-584-5p and hsa-miR-12136, while one was upregulated: hsa-miR-550a-3p. miRNA target prediction showed that chronic pain in polyneuropathy might be the result of a combination of miRNA mediated high blood flow/pressure and neural activity dysregulations/disbalances. Thus, leading to the promising conclusion that these four miRNAs could serve as potential biomarkers for the diagnosis of chronic pain in polyneuropathy. Since TRPV1 seems to be one of the major contributors of nociception and is associated with neuropathic pain, the influence of PKA phosphorylated ARMS on the sensitivity of TRPV1 as well as the part of AKAP79 during PKA phosphorylation of ARMS was characterized. Therefore, possible PKA-sites in the sequence of ARMS were identified. This revealed five canonical PKA-sites: S882, T903, S1251/52, S1439/40 and S1526/27. The single PKA-site mutants of ARMS revealed that PKA-mediated ARMS phosphorylation seems not to influence the interaction rate of TRPV1/ARMS. While phosphorylation of ARMST903 does not increase the interaction rate with TRPV1, ARMSS1526/27 is probably not phosphorylated and leads to an increased interaction rate. The calcium flux measurements indicated that the higher the interaction rate of TRPV1/ARMS, the lower the EC50 for capsaicin of TRPV1, independent of the PKA phosphorylation status of ARMS. In addition, the western blot analysis confirmed the previously observed TRPV1/ARMS interaction. More importantly, AKAP79 seems to be involved in the TRPV1/ARMS/PKA signaling complex. To overcome the problem of ARMS-mediated TRPV1 sensitization by interaction, ARMS was silenced by shRNA. ARMS silencing resulted in a restored TRPV1 desensitization without affecting the TRPV1 expression and therefore could be used as new topical therapeutic analgesic alternative to stop ARMS mediated TRPV1 sensitization.},
  language  = {en}
}
@phdthesis{Oberkofler2022,
  author    = {Oberkofler, Vicky},
  title     = {Molecular basis of HS memory in Arabidopsis thaliana},
  doi       = {10.25932/publishup-56954},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569544},
  school      = {Universit{\"a}t Potsdam},
  pages     = {181},
  year      = {2022},
  abstract  = {Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years. On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first. This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains. In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.},
  language  = {en}
}
@phdthesis{Nwosu2022,
  author    = {Nwosu, Ebuka Canisius},
  title     = {Sedimentary DNA-based reconstruction of cyanobacterial communities from Lake Tiefer See, NE Germany, for the last 11,000 years},
  doi       = {10.25932/publishup-56359},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-563590},
  school      = {Universit{\"a}t Potsdam},
  pages     = {xxvi, 214},
  year      = {2022},
  abstract  = {Climate change and human-driven eutrophication promote the spread of harmful cyanobacteria blooms in lakes worldwide, which affects water quality and impairs the aquatic food chain. In recent times, sedimentary ancient DNA-based (sedaDNA) studies were used to probe how centuries of climate and environmental changes have affected cyanobacterial assemblages in temperate lakes. However, there is a lack of information on the consistency between sediment-deposited cyanobacteria communities versus those of the water column, and on the individual role of natural climatic changes versus human pressure on cyanobacteria community dynamics over multi-millennia time scales. Therefore, this thesis uses sedimentary ancient DNA of Lake Tiefer See in northeastern Germany to trace the deposition of cyanobacteria along the water column into the sediment, and to reconstruct cyanobacteria communities spanning the last 11,000 years using a set of molecular techniques including quantitative PCR, biomarkers, metabarcoding, and metagenome sequence analyses. The results of this thesis proved that cyanobacterial composition and species richness did not significantly differ among different water depths, sediment traps, and surface sediments. This means that the cyanobacterial community composition from the sediments reflects the water column communities. However, there is a skewed sediment deposition of different cyanobacteria groups because of DNA alteration and/or deterioration during transport along the water column to the sediment. Specifically, single filament taxa, such as Planktothrix, are poorly represented in sediments despite being abundant in the water column as shown by an additional study of the thesis on cyanobacteria seasonality. In contrast, aggregate-forming taxa, like Aphanizomenon, are relatively overrepresented in sediment although they are not abundant in the water column. These different deposition patterns of cyanobacteria taxa should be considered in future DNA-based paleolimnological investigations. The thesis also reveals a substantial increase in total cyanobacteria abundance during the Bronze Age which is not apparent in prior phases of the early to middle Holocene and is suggested to be caused by human farming, deforestation, and excessive nutrient addition to the lake. Not only cyanobacterial abundance was influenced by human activity but also cyanobacteria community composition differed significantly between phases of no, moderate, and intense human impact. The data presented in this thesis are the first on sedimentary cyanobacteria DNA since the early Holocene in a temperate lake. The results bring together archaeological, historical climatic, and limnological data with deep DNA-sequencing and paleoecology to reveal a legacy impact of human pressure on lake cyanobacteria populations dating back to approximately 4000 years.},
  language  = {en}
}