@phdthesis{AhmadAbadi2007, author = {Ahmad Abadi, Mohammad}, title = {Development and application of novel genetic transformation technologies in maize (Zea mays L.)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-14572}, school = {Universit{\"a}t Potsdam}, year = {2007}, abstract = {Plant genetic engineering approaches are of pivotal importance to both basic and applied research. However, rapid commercialization of genetically engineered crops, especially maize, raises several ecological and environmental concerns largely related to transgene flow via pollination. In most crops, the plastid genome is inherited uniparentally in a maternal manner. Consequently, a trait introduced into the plastid genome would not be transferred to the sexually compatible relatives of the crops via pollination. Thus, beside its several other advantages, plastid transformation provides transgene containment, and therefore, is an environmentally friendly approach for genetic engineering of crop plants. Reliable in vitro regeneration systems allowing repeated rounds of regeneration are of utmost importance to development of plastid transformation technologies in higher plants. While being the world's major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. In one part of this work, a novel tissue culture and plant regeneration system was developed that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. Also, protocols were established for (i) the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants. Furthermore, several selection methods were tested for developing a plastid transformation system in maize. However, stable plastid transformed maize plants could not be yet recovered. Possible explanations as well as suggestions for future attempts towards developing plastid transformation in maize are discussed. Nevertheless, these results represent a first essential step towards developing chloroplast transformation technology for maize, a method that requires multiple rounds of plant regeneration and selection to obtain genetically stable transgenic plants. In order to apply the newly developed transformation system towards metabolic engineering of carotenoid biosynthesis, the daffodil phytoene synthase (PSY) gene was integrated into the maize genome. The results illustrate that expression of a recombinant PSY significantly increases carotenoid levels in leaves. The beta-carotene (pro-vitamin A) amounts in leaves of transgenic plants were increased by ~21\% in comparison to the wild-type. These results represent evidence for maize to have significant potential to accumulate higher amounts of carotenoids, especially beta-carotene, through transgenic expression of phytoene synthases. Finally, progresses were made towards developing transformation technologies in Peperomia (Piperaceae) by establishing an efficient leaf-based regeneration system. Also, factors determining plastid size and number in Peperomia, whose species display great interspecific variation in chloroplast size and number per cell, were investigated. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. Investigating plastid morphology in Peperomia species with giant chloroplasts, plasmatic connections between chloroplasts (stromules) were observed under the light microscope and in the absence of tissue fixation or GFP overexpression demonstrating the relevance of these structures in vivo. Furthermore, bacteria-like microorganisms were discovered within Peperomia cells, suggesting that this genus provides an interesting model not only for studying plastid biology but also for investigating plant-microbe interactions.}, language = {en} } @phdthesis{AranaCeballos2006, author = {Arana-Ceballos, Fernando Alberto}, title = {Biochemical and physiological studies of Arabidopsis thaliana Diacylglycerol Kinase 7 (AtDGK7)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-13729}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {A family of diacylglycerol kinases (DGK) phosphorylates the substrate diacylglycerol (DAG) to generate phosphatidic acid (PA) . Both molecules, DAG and PA, are involved in signal transduction pathways. In the model plant Arabidopsis thaliana, seven candidate genes (named AtDGK1 to AtDGK7) code for putative DGK isoforms. Here I report the molecular cloning and characterization of AtDGK7. Biochemical, molecular and physiological experiments of AtDGK7 and their corresponding enzyme are analyzed. Information from Genevestigator says that AtDGK7 gene is expressed in seedlings and adult Arabidopsis plants, especially in flowers. The AtDGK7 gene encodes the smallest functional DGK predicted in higher plants; but also, has an alternative coding sequence containing an extended AtDGK7 open reading frame, confirmed by PCR and submitted to the GenBank database (under the accession number DQ350135). The new cDNA has an extension of 439 nucleotides coding for 118 additional amino acids The former AtDGK7 enzyme has a predicted molecular mass of ~41 kDa and its activity is affected by pH and detergents. The DGK inhibitor R59022 also affects AtDGK7 activity, although at higher concentrations (i.e. IC50 ~380 µM). The AtDGK7 enzyme also shows a Michaelis-Menten type saturation curve for 1,2-DOG. Calculated Km and Vmax were 36 µM 1,2-DOG and 0.18 pmol PA min-1 mg of protein-1, respectively, under the assay conditions. Former protein AtDGK7 are able to phosphorylate different DAG analogs that are typically found in plants. The new deduced AtDGK7 protein harbors the catalytic DGKc and accessory domains DGKa, instead the truncated one as the former AtDGK7 protein (Gomez-Merino et al., 2005).}, language = {en} } @phdthesis{Tschoepe2007, author = {Tsch{\"o}pe, Okka}, title = {Managing open habitats for species conservation : the role of wild ungulate grazing, small-scale disturbances, and scale}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-13218}, school = {Universit{\"a}t Potsdam}, year = {2007}, abstract = {During the last decades, the global change of the environment has caused a dramatic loss of habitats and species. In Central Europe, open habitats are particularly affected. The main objective of this thesis was to experimentally test the suitability of wild megaherbivore grazing as a conservation tool to manage open habitats. We studied the effect of wild ungulates in a 160 ha game preserve in NE Germany in three successional stages (i) Corynephorus canescens-dominated grassland, (ii) ruderal tall forb vegetation dominated by Tanacetum vulgare and (iii) Pinus sylvestris-pioneer forest over three years. Our results demonstrate that wild megaherbivores considerably affected species composition and delayed successional pathways in open habitats. Grazing effects differed considerably between successional stages: species richness was higher in grazed ruderal and pioneer forest plots, but not in the Corynephorus sites. Species composition changed significantly in the Corynephorus and ruderal sites. Grazed ruderal sites had turned into sites with very short vegetation dominated by Agrostis spp. and the moss Brachythecium albicans, most species did not flower. Woody plant cover was significantly affected only in the pioneer forest sites. Young pine trees were severely damaged and tree height was considerably reduced, leading to a "Pinus-macchie"-appearance. Ecological patterns and processes are known to vary with spatial scale. Since grazing by megaherbivores has a strong spatial component, the scale of monitoring success of grazing may largely differ among and within different systems. Thus, the second aim of this thesis was to test whether grazing effects are consistent over different spatial scales, and to give recommendations for appropriate monitoring scales. For this purpose, we studied grazing effects on plant community structure using multi-scale plots that included three nested spatial scales (0.25 m2, 4 m2, and 40 m2). Over all vegetation types, the scale of observation directly affected grazing effects on woody plant cover and on floristic similarity, but not on the proportion of open soil and species richness. Grazing effects manifested at small scales regarding floristic similarity in pioneer forest and ruderal sites and regarding species richness in ruderal sites. The direction of scale-effects on similarity differed between vegetation types: Grazing effects on floristic similarity in the Corynephorus sites were significantly higher at the medium and large scale, while in the pioneer forest sites they were significantly higher at the smallest scale. Disturbances initiate vegetation changes by creating gaps and affecting colonization and extinction rates. The third intention of the thesis was to investigate the effect of small-scale disturbances on the species-level. In a sowing experiment, we studied early establishment probabilities of Corynephorus canescens, a key species of open sandy habitats. Applying two different regimes of mechanical ground disturbance (disturbed and undisturbed) in the three successional stages mentioned above, we focused on the interactive effects of small-scale disturbances, successional stage and year-to-year variation. Disturbance led to higher emergence in a humid and to lower emergence in a very dry year. Apparently, when soil moisture was sufficient, the main factor limiting C. canescens establishment was competition, while in the dry year water became the limiting factor. Survival rates were not affected by disturbance. In humid years, C. canescens emerged in higher numbers in open successional stages while in the dry year, emergence rates were higher in late stages, suggesting an important role of late successional stages for the persistence of C. canescens. We conclude that wild ungulate grazing is a useful tool to slow down succession and to preserve a species-rich, open landscape, because it does not only create disturbances, thereby supporting early successional stages, but at the same time efficiently controls woody plant cover. However, wild ungulate grazing considerably changed the overall appearance of the landscape. Additional measures like shifting exclosures might be necessary to allow vulnerable species to flower and reproduce. We further conclude that studying grazing impacts on a range of scales is crucial, since different parameters are affected at different spatial scales. Larger scales are suitable for assessing grazing impact on structural parameters like the proportion of open soil or woody plant cover, whereas species richness and floristic similarity are affected at smaller scales. Our results further indicate that the optimal strategy for promoting C. canescens is to apply disturbances just before seed dispersal and not during dry years. Further, at the landscape scale, facilitation by late successional species may be an important mechanism for the persistence of protected pioneer species.}, language = {en} } @phdthesis{Bieniawska2006, author = {Bieniawska, Zuzanna}, title = {Functional analysis of the sucrose synthase gene family in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-13132}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling.}, language = {en} } @phdthesis{Treplin2006, author = {Treplin, Simone}, title = {Inference of phylogenetic relationships in passerine birds (Aves: Passeriformes) using new molecular markers}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-11230}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {The aim of this study was to provide deeper insights in passerine phylogenetic relationships using new molecular markers. The monophyly of the largest avian order Passeriformes (~59\% of all living birds) and the division into its suborders suboscines and oscines are well established. Phylogenetic relationships within the group have been extremely puzzling, as most of the evolutionary lineages originated through rapid radiation. Numerous studies have hypothesised conflicting passerine phylogenies and have repeatedly stimulated further research with new markers. In the present study, I used three different approaches to contribute to the ongoing phylogenetic debate in Passeriformes. I investigated the recently introduced gene ZENK for its phylogenetic utility for passerine systematics in combination and comparison to three already established nuclear markers. My phylogenetic analyses of a comprehensive data set yielded highly resolved, consistent and strongly supported trees. I was able to show the high utility of ZENK for elucidating phylogenetic relationships within Passeriformes. For the second and third approach, I used chicken repeat 1 (CR1) retrotransposons as phylogenetic markers. I presented two specific CR1 insertions as apomorphic characters, whose presence/absence pattern significantly contributed to the resolution of a particular phylogenetic uncertainty, namely the position of the rockfowl species Picathartes spp. in the passerine tree. Based on my results, I suggest a closer relationship of these birds to crows, ravens, jays, and allies. For the third approach, I showed that CR1 sequences contain phylogenetic signal and investigated their applicability in more detail. In this context, I screened for CR1 elements in different passerine birds, used sequences of several loci to construct phylogenetic trees, and evaluated their reliability. I was able to corroborate existing hypotheses and provide strong evidence for some new hypotheses, e.g. I suggest a revision of the taxa Corvidae and Corvinae as vireos are closer related to crows, ravens, and allies. The subdivision of the Passerida into three superfamilies, Sylvioidea, Passeroidea, and Muscicapoidea was strongly supported. I found evidence for a split within Sylvioidea into two clades, one consisting of tits and the other comprising warblers, bulbuls, laughingthrushes, whitethroats, and allies. Whereas Passeridae appear to be paraphyletic, monophyly of weavers and estrild finches as a separate clade was strongly supported. The sister taxon relationships of dippers and the thrushes/flycatcher/chat assemblage was corroborated and I suggest a closer relationship of waxwings and kinglets to wrens, tree-creepers, and nuthatches.}, language = {en} } @phdthesis{Boelling2006, author = {B{\"o}lling, Christian}, title = {Comprehensive metabolite analysis in Chlamydomonas reinhardtii : method development and application to the study of environmental and genetic perturbations}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-11329}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled.}, language = {en} } @phdthesis{Mungur2006, author = {Mungur, Rajsree}, title = {Spatio-temporal analysis of florigenic signals in Arabidopsis thaliana, Sinapis alba and Brassica napus}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-9861}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {Daylength is one of several parameters controlling flowering time in many plant species. The day length is perceived in leaves, but how the floral signal is transduced to the shoot apex via the phloem to induce flowering remains to be elucidated. This study aimed at the identification of new candidates involved in the induction of flowering by employing three plant species, Arabidopsis thaliana, Sinapis alba and Brassica napus in combination with transcript profiling by Affymetrix chip hybridization, metabolite profiling by gas chromatography - mass spectrometry and targeted protein analysis using antibodies. All analyses were performed on tissue-specific samples and focused on phloem sap or phloem exudates. To find common transcript and metabolite candidates potentially associated with the floral transition, two independent induction systems in Arabidopsis were used: a photoextension system, whereby plants received fourteen additional hours of light, and a parallel dexamethasone-inducible system, which was centered on the induction of the known flowering gene CONSTANS (CO). Identification of signals preceding the CO cascade was possible using the light extension regime, while downstream events dependent on CO activation were compared in both systems. Altogether, a number of interesting transcript and metabolite candidates were identified in both systems with some degree of overlap. Sinapis alba was used to investigate the universality of the floral signals between species. Comparisons of metabolite data revealed a few common candidates that may prove interesting for further studies. In addition, a targeted approach was carried out to investigate the presence of the Flowering Locus T (FT) protein during different stages of flower development using an antibody. Interesting changes in the sizes of antigens from rape phloem were seen and appeared consistent in Arabidopsis and to a lesser extent in Sinapis. Overall, the broad surveying approaches for transcripts and metabolites used in this study revealed several new potential candidates involved in the induction and/or regulation of flowering. As far as the protein work, additional experiments will reveal the link between FT and floral induction as well as its role in maintaining the floral state using the abovementioned plant species.}, subject = {Florigen}, language = {en} } @phdthesis{Feulner2006, author = {Feulner, Philine}, title = {Adaptive radiation, speciation, and reproductive isolation in African weakly electric fish : (Genus Campylomormyrus, Mormyridae, Teleostei)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-9560}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {The ultimate aim of this study is to better understand the relevance of weak electricity in the adaptive radiation of the African mormyrid fish. The chosen model taxon, the genus Campylomormyrus, exhibits a wide diversity of electric organ discharge (EOD) waveform types. Their EOD is age, sex, and species specific and is an important character for discriminating among species that are otherwise cryptic. After having established a complementary set of molecular markers, I examined the radiation of Campylomormyrus by a combined approach of molecular data (sequence data from the mitochondrial cytochrome b and the nuclear S7 ribosomal protein gene, as well as 18 microsatellite loci, especially developed for the genus Campylomormyrus), observation of ontogeny and diversification of EOD waveform, and morphometric analysis of relevant morphological traits. I built up the first convincing phylogenetic hypothesis for the genus Campylomormyrus. Taking advantage of microsatellite data, the identified phylogenetic clades proved to be reproductively isolated biological species. This way I detected at least six species occurring in sympatry near Brazzaville/Kinshasa (Congo Basin). By combining molecular data and EOD analyses, I could show that there are three cryptic species, characterised by their own adult EOD types, hidden under a common juvenile EOD form. In addition, I confirmed that adult male EOD is species-specific and is more different among closely related species than among more distantly related ones. This result and the observation that the EOD changes with maturity suggest its function as a reproductive isolation mechanism. As a result of my morphometric shape analysis, I could assign species types to the identified reproductively isolated groups to produce a sound taxonomy of the group. Besides this, I could also identify morphological traits relevant for the divergences between the identified species. Among them, the variations I found in the shape of the trunk-like snout, suggest the presence of different trophic specializations; therefore, this trait might have been involved in the ecological radiation of the group. In conclusion, I provided a convincing scenario envisioning an adaptive radiation of weakly electric fish triggered by sexual selection via assortative mating due to differences in EOD characteristics, but caused by a divergent selection of morphological traits correlated with the feeding ecology.}, subject = {Phylogenie}, language = {en} } @phdthesis{GomezPorras2005, author = {G{\´o}mez-Porras, Judith Lucia}, title = {In silico identification of genes regulated by abscisic acid in Arabidopsis thaliana (L.) Heynh.}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-7401}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Abscisic acid (ABA) is a major plant hormone that plays an important role during plant growth and development. During vegetative growth ABA mediates (in part) responses to various environmental stresses such as cold, drought and high salinity. The response triggered by ABA includes changes in the transcript level of genes involved in stress tolerance. The aim of this project was the In silico identification of genes putatively regulated by ABA in A. thaliana. In silico predictions were combined with experimental data in order to evaluate the reliability of computational predictions. Taking advantage of the genome sequence of A. thaliana publicly available since 2000, 1 kb upstream sequences were screened for combinations of cis-elements known to be involved in the regulation of ABA-responsive genes. It was found that around 10 to 20 percent of the genes of A. thaliana might be regulated by ABA. Further analyses of the predictions revealed that certain combinations of cis-elements that confer ABA-responsiveness were significantly over-represented compared with results in random sequences and with random expectations. In addition, it was observed that other combinations that confer ABA-responsiveness in monocotyledonous species might not be functional in A. thaliana. It is proposed that ABA-responsive genes in A. thaliana show pairs of ABRE (abscisic acid responsive element) with MYB binding sites, DRE (dehydration responsive element) or with itself. The analysis of the distances between pairs of cis-elements suggested that pairs of ABREs are bound by homodimers of ABRE binding proteins. In contrast, pairs between MYB binding sites and ABRE, or DRE and ABRE showed a distance between cis-elements that suggested that the binding proteins interact through protein complexes and not directly. The comparison of computational predictions with experimental data confirmed that the regulatory mechanisms leading to the induction or repression of genes by ABA is very incompletely understood. It became evident that besides the cis-elements proposed in this study to be present in ABA-responsive genes, other known and unknown cis-elements might play an important role in the transcriptional regulation of ABA-responsive genes. For example, auxin-related cis elements, or the cis-elements recognized by the NAM-family of transcription factors (Non-Apical meristem). This work documents the use of computational and experimental approaches to analyse possible interactions between cis-elements involved in the regulation of ABA-responsive genes. The computational predictions allowed the distinction between putatively relevant combinations of cis-elements from irrelevant combinations of cis-elements in ABA-responsive genes. The comparison with experimental data allowed to identify certain cis-elements that have not been previously associated to the ABA-mediated transcriptional regulation, but that might be present in ABA-responsive genes (e.g. auxin responsive elements). Moreover, the efforts to unravel the gene regulatory network associated with the ABA-signalling pathway revealed that NAM-transcription factors and their corresponding binding sequences are important components of this network.}, subject = {Bioinformatik}, language = {en} } @phdthesis{Schauer2006, author = {Schauer, Nicolas}, title = {Quantitative trait loci (QTL) for metabolite accumulation and metabolic regulation : metabolite profiling of interspecific crosses of tomato}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-7643}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {The advent of large-scale and high-throughput technologies has recently caused a shift in focus in contemporary biology from decades of reductionism towards a more systemic view. Alongside the availability of genome sequences the exploration of organisms utilizing such approach should give rise to a more comprehensive understanding of complex systems. Domestication and intensive breeding of crop plants has led to a parallel narrowing of their genetic basis. The potential to improve crops by conventional breeding using elite cultivars is therefore rather limited and molecular technologies, such as marker assisted selection (MAS) are currently being exploited to re-introduce allelic variance from wild species. Molecular breeding strategies have mostly focused on the introduction of yield or resistance related traits to date. However given that medical research has highlighted the importance of crop compositional quality in the human diet this research field is rapidly becoming more important. Chemical composition of biological tissues can be efficiently assessed by metabolite profiling techniques, which allow the multivariate detection of metabolites of a given biological sample. Here, a GC/MS metabolite profiling approach has been applied to investigate natural variation of tomatoes with respect to the chemical composition of their fruits. The establishment of a mass spectral and retention index (MSRI) library was a prerequisite for this work in order to establish a framework for the identification of metabolites from a complex mixture. As mass spectral and retention index information is highly important for the metabolomics community this library was made publicly available. Metabolite profiling of tomato wild species revealed large differences in the chemical composition, especially of amino and organic acids, as well as on the sugar composition and secondary metabolites. Intriguingly, the analysis of a set of S. pennellii introgression lines (IL) identified 889 quantitative trait loci of compositional quality and 326 yield-associated traits. These traits are characterized by increases/decreases not only of single metabolites but also of entire metabolic pathways, thus highlighting the potential of this approach in uncovering novel aspects of metabolic regulation. Finally the biosynthetic pathway of the phenylalanine-derived fruit volatiles phenylethanol and phenylacetaldehyde was elucidated via a combination of metabolic profiling of natural variation, stable isotope tracer experiments and reverse genetic experimentation.}, subject = {Tomate}, language = {en} } @phdthesis{Birkemeyer2005, author = {Birkemeyer, Claudia Sabine}, title = {Signal-metabolome interactions in plants}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-7144}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {From its first use in the field of biochemistry, instrumental analysis offered a variety of invaluable tools for the comprehensive description of biological systems. Multi-selective methods that aim to cover as many endogenous compounds as possible in biological samples use different analytical platforms and include methods like gene expression profile and metabolite profile analysis. The enormous amount of data generated in application of profiling methods needs to be evaluated in a manner appropriate to the question under investigation. The new field of system biology rises to the challenge to develop strategies for collecting, processing, interpreting, and archiving this vast amount of data; to make those data available in form of databases, tools, models, and networks to the scientific community. On the background of this development a multi-selective method for the determination of phytohormones was developed and optimised, complementing the profile analyses which are already in use (Chapter I). The general feasibility of a simultaneous analysis of plant metabolites and phytohormones in one sample set-up was tested by studies on the analytical robustness of the metabolite profiling protocol. The recovery of plant metabolites proved to be satisfactory robust against variations in the extraction protocol by using common extraction procedures for phytohormones; a joint extraction of metabolites and hormones from plant tissue seems practicable (Chapter II). Quantification of compounds within the context of profiling methods requires particular scrutiny (Chapter II). In Chapter III, the potential of stable-isotope in vivo labelling as normalisation strategy for profiling data acquired with mass spectrometry is discussed. First promising results were obtained for a reproducible quantification by stable-isotope in vivo labelling, which was applied in metabolomic studies. In-parallel application of metabolite and phytohormone analysis to seedlings of the model plant Arabidopsis thaliana exposed to sulfate limitation was used to investigate the relationship between the endogenous concentration of signal elements and the 'metabolic phenotype' of a plant. An automated evaluation strategy was developed to process data of compounds with diverse physiological nature, such as signal elements, genes and metabolites - all which act in vivo in a conditional, time-resolved manner (Chapter IV). Final data analysis focussed on conditionality of signal-metabolome interactions.}, subject = {Pflanzenhormon}, language = {en} } @phdthesis{Kolasa2005, author = {Kolasa, Anna}, title = {Identification and analysis of new phloem proteins from Brassicaceae and Cucurbitaceae}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-6939}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {The major aim of this work was the identification of new phloem sap proteins and a metabolic characterisation of this transport fluid. The experiments were performed on the three plant species C. sativus, C. maxima and B. napus. To characterise the phloem samples from B. napus, a new model plant for phloem analysis, western blot tests together with metabolite profiling were performed. GC-MS metabolite profiling and enzyme assays were used for measuring metabolites in the phloem of B. napus. Results from the phloem sap measurements showed, as expected, a typical sugar distribution for apoplasmic phloem loaders with sucrose being the predominant sugar. In stem extracts, the most abundant sugar was glucose with much lower fructose and sucrose levels. With the GC-MS approach it was possible to identify a number of metabolites which showed a differential distribution when phloem and stem tissue extracts were compared. For protein identification, two different approaches were employed (i) screening expression libraries with total phloem protein specific antisera and (ii) protein separation on 2 DE gels followed by ESI-MS/MS sequence analyses. For the first approach, three different phloem protein-specific antisera were produced and expression libraries were constructed. Phloem protein antisera were tested for specificity and some attempts to estimate specific epitopes were undertaken. Screening of the libraries resulted in the identification of 14 different proteins from all investigated species. Analyses of B. napus phloem sap proteins from 2 DE with ESI-MS/MS resulted in the identification of 5 different proteins. The phloem localisation of the identified proteins was additionally confirmed by western blot tests using specific antibodies. In order to functionally characterise some selected phloem proteins from B. napus, the group of potential calcium-binding polypeptides was analysed for functional Ca+2 binding properties and several Ca+2-binding proteins could be isolated. However, their sequences could as yet not be determined. Another approach used for functional protein characterisation was the analysis of Arabidopsis T-DNA insertion mutants. Four available mutants with insertions in phloem protein-specific genes were chosen from the SALK and GABI-Kat collections and selected homozygous lines were tested for the presence of the investigated proteins. In order to verify if the product of one of the mutated gene (GRP 7) is transported through the phloem, grafting experiments were performed followed by western blot analyses. Although the employed antiserum against GRP 7 protein did not allow distinguishing between the mutant and the wild type plants, successful Arabidopsis grafting could be established as a promising method for further studies on protein translocation through the phloem.}, subject = {Phloemproteine}, language = {en} } @phdthesis{Zhang2005, author = {Zhang, Baichen}, title = {Dissection of phloem transport in cucurbitaceae by metabolomic analysis}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-6644}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of Cucurbita maxima, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. The major results are summarized as below: 1) O-linked glycans in C.maxima were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock. 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites. 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology. 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed. 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network. 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines. 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem. 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF de novo sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem.}, subject = {phloem}, language = {en} } @phdthesis{Kolbe2005, author = {Kolbe, Anna}, title = {Redox-regulation of starch and lipid synthesis in leaves}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-6388}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels.}, subject = {Redoxreaktion}, language = {en} } @phdthesis{Nita2005, author = {Nita, Ana-Silvia}, title = {Genetic mapping and molecular characterization of tbr1 mutant in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5992}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Arabidopsis thaliana trichomes exhibit strong birefringence under polarized light, a characteristic of cell walls containing large amounts of highly ordered cellulose microfibrils. The tbr1 mutant of Arabidopsis lacks trichome birefringence and is deficient in secondary cell wall cellulose synthesis (Potikha and Delmer, 1995). The TBR gene was identified by recombinational mapping, candidate gene sequencing and molecular complementation using genomic cosmid clones, as well as a p35S:TBR genomic DNA construct, fully rescuing the mutant phenotype in both cases. The only mutant allele available (tbr-1) carries a substitution (G to E) in a conserved aminoacid domain of the protein. TBR gene structure was proved to have a longer size than the one found to be annotated at the time of identification in the data-base. A full cDNA clone containing the full transcript was available and also complementation experiments using different gene fragments (annotated and suggested) leaded to the result that TBR gene is indeed, longer. TBR encodes a novel plant-specific protein with predicted plasma membrane localization, therefore being consistent with idea that is required for-, or is a novel component of a functional cellulose synthase complex. TBR is part of an Arabidopsis gene/protein family, (TBL-trichome birefringence like) which, depending on homology, comprises up to 20 members, none of which has a biological or biochemical function attributed. T-DNA insertion lines in TBR gene and two close homologues have been screened by PCR, but no homozygous were found and no trichomes phenotype was identified. Promoter-GUS lines were produced for TBR, as well as for its two closest homologues (one being a segmentally duplicated gene on chromosome III), using 1.6-2 kb of promoter sequence upstream of the annotated start codons. The TBR promoter was the only one of the three that yielded trichome expression, this probably explaining the phenotype of the TBR mutant. Moreover, TBR is expressed in leaves, in growing lateral roots, and in vascular tissues of young Arabidopsis seedlings and plantlets. Later on, the expression appears in inflorescens, stems, flowers and green siliques. This expression pattern is largely overlapping with those of the two analyzed homologues and it corresponds with data of RT-PCR expression profiling performed for TBR and the two analyzed homologues in different tissues, at different developmental stages. Biochemical analysis of cell wall (leaves and trichomes), as GC and MALDI-TOF, were performed, but revealed no major differences between tbr1 and wild type plants. Scanning electron microscopy analysis and cell wall polysaccharides antibody labeling showed a clear difference in the trichomes cell wall structure between mutant plant and wild type.}, subject = {tbr mutant}, language = {en} } @phdthesis{Schaefer2005, author = {Sch{\"a}fer, Arne}, title = {Characterization of ammoniumtransporters in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5937}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Nitrogen is often a limiting factor for plant growth due to its heterogenous distribution in the soil and to seasonal and diurnal changes in growth rates. In most soils, NH4+ and NO3 - are the predominant sources of inorganic nitrogen that are available for plant nutrition. In this context, plants have evolved mechanisms that enable them to optimize nitrogen acquisition, which include transporters specialized in the uptake of nitrogen and susceptible to a regulation that responds to nitrogen limiting or excess conditions. Although the average NH4+ concentrations of soils are generally 100 to 1000 times lower than those of NO3 - (Marschner, 1995), most plants preferentially take up NH4+ when both forms are present because unlike NO3- , NH4+ has not to be reduced prior to assimilation and thus requires less energy for assimilation (Bloom et al., 1992). Apart from high uptake rates in roots, high intracellular ammonium concentrations also result from quantitatively important internal breakdown of amino acids (Feng et al., 1998), and originates in high quantities during photorespiration (Mattson et al., 1997, Pearson et al., 1998). Thus, NH4+ is a key component of nitrogen metabolism for all plants and can accumulate to varying concentrations in all compartments of the cell, including the cytosol, the vacuole and in the apoplast (Wells and Miller, 2000; Nielsen and Schjoerring, 1998). Two related families of ammonium transporters (AMT1 and AMT2), containing six genes which encode transporter proteins that are specific for ammonium had been identified prior to this thesis and some genes had partially been characterised in Arabidopsis (Gazzarrini et al., 1999; Sohlenkamp et al. 2002; Kaiser et al., 2002). However, these studies were not sufficient to assign physiological functions to the individual transporters and AMT1.4 and AMT1.5 had not been studied prior to this thesis. Given this background, it was considered desirable to acquire a deeper knowledge of the physiological functions of the six Arabidopsis ammonium transporters. To this end, tissue specific expression profiles of the individual wildtype AtAMT genes were performed by quantitative real time PCR (qRT-PCR) and promoter-GUS expression. Modern approaches such as the use of T-DNA insertional mutants and RNAi hairpin constructs were employed to reduce the expression levels of AMT genes. Transcript levels were determined, and physiological, biochemical and developmental analysis such as growth tests on different media and 14C-MA and NH4+ uptake studies with the isolated insertional mutants and RNAi lines were performed to deepen the knowledge of the individual functions of the six AMTs in Arabidopsis. In addition, double mutants of the insertional mutants were created to investigate the extent in which homologous genes could compensate for lost transporter functions. The results described in this thesis show that the six AtAMT genes display a high degree of specifity in their tissue specific expression and are likely to play complementary roles in ammonium uptake into roots, in shoots, and in flowers. AtAMT1.1 is likely to be a 'work horse' for cellular ammonium transport and reassimilation. A major role is probably the recapture of photorespiratory NH3/NH4+ escaping from the cytosol. In roots, it is likely to transport NH4+ from the apoplast into cortical cells. AtAMT1.3 and AtAMT1.5 appear to be specialised in the acquisition of external NH4+ from the soil. Furthermore, AtAMT1.5 plays an additional role in the reassimilation of NH3/NH4+ released during the breakdown of storage proteins in the cotyledons of germinating seedlings. It was difficult to distinguish a specialisation between the transporters AtAMt1.2 and AtAMt1.1, however the root and flower specific expression patterns are different and indicate alternative functions of both. AtAMT1.4 has a very distinct expression which is restricted to the vascular bundels of leaves and to pollen only, where it is likely to be involved in the loading of NH4+ into the cells.The AtAMT2.1 expression pattern is confined to vascular bundels and meristematic active tissues in leaves where ammonium concentrations can reach very high levels. Additionally, the Vmax of AtAMT2 increases with increasing external pH, contrasting to AtAMT1.1. Thus, AtAMT2.1 it might be specialised in ammonium transport in ammonium rich environments, where the functions of other transporters are limited, enabling cells to take up NH4+ over a wide range of concentrations. The root hair expression ascribes an additional role in NH3/NH4+ acquisition where it possibly serves as a transporter that is able to acquire ammonium from basic soils where other transporters become less effective.RNAi lines showing a reduction in AtAMT gene mRNA levels and NH4+ transport kinetics, grew slower and flowering time was delayed. This indicates that NH4+ is a crucial and limiting factor for plant growth.}, subject = {Ammonium}, language = {en} } @phdthesis{Schwager2005, author = {Schwager, Monika}, title = {Climate change, variable colony sizes and temporal autocorrelation : consequences of living in changing environments}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5744}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Natural and human induced environmental changes affect populations at different time scales. If they occur in a spatial heterogeneous way, they cause spatial variation in abundance. In this thesis I addressed three topics, all related to the question, how environmental changes influence population dynamics. In the first part, I analysed the effect of positive temporal autocorrelation in environmental noise on the extinction risk of a population, using a simple population model. The effect of autocorrelation depended on the magnitude of the effect of single catastrophic events of bad environmental conditions on a population. If a population was threatened by extinction only, when bad conditions occurred repeatedly, positive autocorrelation increased extinction risk. If a population could become extinct, even if bad conditions occurred only once, positive autocorrelation decreased extinction risk. These opposing effects could be explained by two features of an autocorrelated time series. On the one hand, positive autocorrelation increased the probability of series of bad environmental conditions, implying a negative effect on populations. On the other hand, aggregation of bad years also implied longer periods with relatively good conditions. Therefore, for a given time period, the overall probability of occurrence of at least one extremely bad year was reduced in autocorrelated noise. This can imply a positive effect on populations. The results could solve a contradiction in the literature, where opposing effects of autocorrelated noise were found in very similar population models. In the second part, I compared two approaches, which are commonly used for predicting effects of climate change on future abundance and distribution of species: a "space for time approach", where predictions are based on the geographic pattern of current abundance in relation to climate, and a "population modelling approach" which is based on correlations between demographic parameters and the inter-annual variation of climate. In this case study, I compared the two approaches for predicting the effect of a shift in mean precipitation on a population of the sociable weaver Philetairus socius, a common colonially living passerine bird of semiarid savannahs of southern Africa. In the space for time approach, I compared abundance and population structure of the sociable weaver in two areas with highly different mean annual precipitation. The analysis showed no difference between the two populations. This result, as well as the wide distribution range of the species, would lead to the prediction of no sensitive response of the species to a slight shift in mean precipitation. In contrast, the population modelling approach, based on a correlation between reproductive success and rainfall, predicted a sensitive response in most model types. The inconsistency of predictions was confirmed in a cross-validation between the two approaches. I concluded that the inconsistency was caused, because the two approaches reflect different time scales. On a short time scale, the population may respond sensitively to rainfall. However, on a long time scale, or in a regional comparison, the response may be compensated or buffered by a variety of mechanisms. These may include behavioural or life history adaptations, shifts in the interactions with other species, or differences in the physical environment. The study implies that understanding, how such mechanisms work, and at what time scale they would follow climate change, is a crucial precondition for predicting ecological consequences of climate change. In the third part of the thesis, I tested why colony sizes of the sociable weaver are highly variable. The high variation of colony sizes is surprising, as in studies on coloniality it is often assumed that an optimal colony size exists, in which individual bird fitness is maximized. Following this assumption, the pattern of bird dispersal should keep colony sizes near an optimum. However, I showed by analysing data on reproductive success and survival that for the sociable weaver fitness in relation to colony size did not follow an optimum curve. Instead, positive and negative effects of living in large colonies overlaid each other in a way that fitness was generally close to one, and density dependence was low. I showed in a population model, which included an evolutionary optimisation process of dispersal that this specific shape of the fitness function could lead to a dispersal strategy, where the variation of colony sizes was maintained.}, subject = {Populationsbiologie}, language = {en} } @phdthesis{Czechowski2005, author = {Czechowski, Tomasz}, title = {Nitrogen signalling in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5445}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {Nitrogen is an essential macronutrient for plants and nitrogen fertilizers are indispensable for modern agriculture. Unfortunately, we know too little about how plants regulate their use of soil nitrogen, to maximize fertilizers-N use by crops and pastures. This project took a dual approach, involving forward and reverse genetics, to identify N-regulators in plants, which may prove useful in the future to improve nitrogen-use efficiency in agriculture. To identify nitrogen-regulated transcription factor genes in Arabidopsis that may control N-use efficiency we developed a unique resource for qRT-PCR measurements on all Arabidpsis transcription factor genes. Using closely spaced, gene-specific primer pairs and SYBR® Green to monitor amplification of double-stranded DNA, transcript levels of 83\% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4\% of reactions produced non-specific PCR products, and 13\% of TF transcripts were undetectable in these organs. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001-100 copies per cell. Real-time RT-PCR revealed 26 root-specific and 39 shoot-specific TF genes, most of which have not been identified as organ-specific previously. An enlarged and improved version of the TF qRT-PCR platform contains now primer pairs for 2256 Arabidopsis TF genes, representing 53 gene families and sub-families arrayed on six 384-well plates. Set-up of real-time PCR reactions is now fully robotized. One researcher is able to measure expression of all 2256 TF genes in a single biological sample in a just one working day. The Arabidopsis qRT-PCT platform was successfully used to identify 37 TF genes which transcriptionaly responded at the transcriptional level to N-deprivation or to nitrate per se. Most of these genes have not been characterized previously. Further selection of TF genes based on the responses of selected candidates to other macronutrients and abiotic stresses allowed to distinguish between TFs regulated (i) specifically by nitrogen (29 genes) (ii) regulated by general macronutrient or by salt and osmotic stress (6 genes), and (iii) responding to all major macronutrients and to abiotic stresses. Most of the N-regulated TF genes were also regulated by carbon. Further characterization of sixteen selected TF genes, revealed: (i) lack of transcriptional response to organic nitrogen, (ii) two major types of kinetics of induction by nitrate, (iii) specific responses for the majority of the genes to nitrate but not downstream products of nitrate assimilation. All sixteen TF genes were cloned into binary vectors for constitutive and ethanol inducible over expression, and the first generation of transgenic plants were obtained for almost all of them. Some of the plants constitutively over expressing TF genes under control of the 35S promoter revealed visible phenotypes in T1 generation. Homozygous T-DNA knock out lines were also obtained for many of the candidate TF genes. So far, one knock out line revealed a visible phenotype: retardation of flowering time. A forward genetic approach using an Arabidopsis ATNRT2.1 promoter : Luciferase reporter line, resulted in identification of eleven EMS mutant reporter lines affected in induction of ATNRT2.1 expression by nitrate. These lines could by divided in the following classes according to expression of other genes involved in primary nitrogen and carbon metabolism: (i) lines affected exclusively in nitrate transport, (ii) those affected in nitrate transport, acquisition, but also in glycolysis and oxidative pentose pathway, (iii) mutants affected moderately in nitrate transport, oxidative pentose pathway and glycolysis but not in primary nitrate assimilation. Thus, several different N-regulatory genes may have been mutated in this set of mutants. Map-based cloning has begun to identify the genes affected in these mutants.}, subject = {Stickstoff}, language = {en} } @phdthesis{Dolniak2005, author = {Dolniak, Blazej}, title = {Functional characterisation of NIC2, a member of the MATE family from Arabidopsis thaliana (L.) Heynh.}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5372}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {The multidrug and toxic compounds extrusion (MATE) family includes hundreds of functionally uncharacterised proteins from bacteria and all eukaryotic kingdoms except the animal kingdom, that function as drug/toxin::Na+ or H+ antiporters. In Arabidopsis thaliana the MATE family comprises 56 members, one of which is NIC2 (Novel Ion Carrier 2). Using heterologous expression systems including Escherichia coli and Saccharomyces cerevisiae, and the homologous expression system of Arabidopsis thaliana, the functional characterisation of NIC2 was performed. It has been demonstrated that NIC2 confers resistance of E. coli towards the chemically diverse compounds such as tetraethylammonium chloride (TEACl), tetramethylammonium chloride (TMACl) and a toxic analogue of indole-3-acetic acid, 5-fluoro-indole-acetic acid (F-IAA). Therefore, NIC2 may be able to transport a broad range of drug and toxic compounds. In wild-type yeast the expression of NIC2 increased the tolerance towards lithium and sodium, but not towards potassium and calcium. In A. thaliana, the overexpression of NIC2 led to strong phenotypic changes. Under normal growth condtions overexpression caused an extremely bushy phenotype with no apical dominance but an enhanced number of lateral flowering shoots. The amount of rossette leaves and flowers with accompanying siliques were also much higher than in wild-type plants and the senescence occurred earlier in the transgenic plants. In contrast, RNA interference (RNAi) used to silence NIC2 expression, induced early flower stalk development and flowering compared with wild-type plants. In additon, the main flower stalks were not able to grow vertically, but instead had a strong tendency to bend towards the ground. While NIC2 RNAi seedlings produced many lateral roots outgrowing from the primary root and the root-shoot junction, NIC2 overexpression seedlings displayed longer primary roots that were characterised by a 2 to 4 h delay in the gravitropic response. In addition, these lines exhibited an enhanced resistance to exogenously applied auxins, i.e. indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) when compared with the wild-type roots. Based on these results, it is suggested that the NIC2 overexpression and NIC2 RNAi phenotypes were due to decreased or increased levels of auxin, respectively. The ProNIC2:GUS fusion gene revealed that NIC2 is expressed in the stele of the elongation zone, in the lateral root cap, in new lateral root primordia, and in pericycle cells of the root system. In the vascular tissue of rosette leaves and inflorescence stems, the expression was observed in the xylem parenchyma cells, while in siliques it was also in vascular tissue, but as well in the dehiscence and abscission zones. The organ- and tissue-specific expression sites of NIC2 correlate with the sites of auxin action in mature Arabidopsis plants. Further experiments using ProNIC2:GUS indicated that NIC2 is an auxin-inducible gene. Additionally, during the gravitropic response when an endogenous auxin gradient across the root tip forms, the GUS activity pattern of the ProNIC2:GUS fusion gene markedly changed at the upper side of the root tip, while at the lower side stayed unchanged. Finally, at the subcellular level NIC2-GFP fusion protein localised in the peroxisomes of Nicotana tabacum BY2 protoplasts. Considering the experimental results, it is proposed that the hypothetical function of NIC2 is the efflux transport which takes part in the auxin homeostasis in plant tissues probably by removing auxin conjugates from the cytoplasm into peroxisomes.}, subject = {Ackerschmalwand}, language = {en} } @phdthesis{Rossmanith2005, author = {Rossmanith, Eva}, title = {Breeding biology, mating system and population dynamics of the Lesser Spotted Woodepcker (Picoides minor) : combining empirical and model investigations}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5328}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {The protection of species is one major focus in conservation biology. The basis for any management concept is the knowledge of the species autecology. In my thesis, I studied the life-history traits and population dynamics of the endangered Lesser Spotted Woodpecker (Picoides minor) in Central Europe. Here, I combine a range of approaches, from empirical investigations of a Lesser Spotted Woodpecker population in the Taunus low mountain range in Germany, the analysis of empirical data and the development of an individual-based stochastic model simulating the population dynamics. In the field studies I collected basic demographic data of reproductive success and mortality. Moreover, breeding biology and behaviour were investigated in detail. My results showed a significant decrease of the reproductive success with later timing of breeding, caused by deterioration in food supply. Moreover, mate fidelity was of benefit, since pairs composed of individuals that bred together the previous year started earlier with egg laying and obtained a higher reproductive success. Both sexes were involved in parental care, but the care was only shared equally during incubation and the early nestling stage. In the late nestling stage, parental care strategies differed between sexes: Females considerably decreased feeding rate with number of nestlings and even completely deserted small broods. Males fed their nestlings irrespective of brood size and compensated for the females absence. The organisation of parental care in the Lesser Spotted Woodpecker is discussed to provide the possibility for females to mate with two males with separate nests and indeed, polyandry was confirmed. To investigate the influence of the observed flexibility in the social mating system on the population persistence, a stochastic individual-based model simulating the population dynamics of the Lesser Spotted Woodpecker was developed, based on empirical results. However, pre-breeding survival rates could not be obtained empirically and I present in this thesis a pattern-oriented modelling approach to estimate pre-breeding survival rates by comparing simulation results with empirical pattern of population structure and reproductive success on population level. Here, I estimated the pre-breeding survival for two Lesser Spotted Woodpecker populations on different latitudes to test the reliability of the results. Finally, I used the same simulation model to investigate the effect of flexibility in the mating system on the persistence of the population. With increasing rate of polyandry in the population, the persistence increased and even low rates of polyandry had a strong influence. Even when presuming only a low polyandry rate and costs of polyandry in terms of higher mortality and lower reproductive success for the secondary male, the positive effect of polyandry on the persistence of the population was still strong. This thesis greatly helped to increase the knowledge of the autecology of an endangered woodpecker species. Beyond the relevance for the species, I could demonstrate here that in general flexibility in mating systems are buffer mechanisms and reduce the impact of environmental and demographic noise.}, subject = {Modellierung}, language = {en} } @phdthesis{Ott2005, author = {Ott, Thomas}, title = {Functional genomics of nodulins in the model legume Lotus japonicus}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-5298}, school = {Universit{\"a}t Potsdam}, year = {2005}, abstract = {During this PhD project three technical platforms were either improved or newly established in order to identify interesting genes involved in SNF, validate their expression and functionally characterise them. An existing 5.6K cDNA array (Colebatch et al., 2004) was extended to produce the 9.6K LjNEST array, while a second array, the 11.6K LjKDRI array, was also produced. Furthermore, the protocol for array hybridisation was substantially improved (Ott et al., in press). After functional classification of all clones according to the MIPS database and annotation of their corresponding tentative consensus sequence (TIGR) these cDNA arrays were used by several international collaborators and by our group (Krusell et al., 2005; in press). To confirm results obtained from the cDNA array analysis different sets of cDNA pools were generated that facilitate rapid qRT-PCR analysis of candidate gene expression. As stable transformation of Lotus japonicus takes several months, an Agrobacterium rhizogenes transformation system was established in the lab and growth conditions for screening transformants for symbiotic phenotypes were improved. These platforms enable us to identify genes, validate their expression and functionally characterise them in the minimum of time. The resources that I helped to establish, were used in collaboration with other people to characterise several genes like the potassium transporter LjKup and the sulphate transporter LjSst1, that were transcriptionally induced in nodules compared to uninfected roots, in more detail (Desbrosses et al., 2004; Krusell et al., 2005). Another gene that was studied in detail was LjAox1. This gene was identified during cDNA array experiments and detailed expression analysis revealed a strong and early induction of the gene during nodulation with high expression in young nodules which declines with the age of the nodule. Therefore, LjAox1 is an early nodulin. Promoter:gus fusions revealed an LjAox1 expression around the nodule endodermis. The physiological role of LjAox1 is currently being persued via RNAi. Using RNA interference, the synthesis of all symbiotic leghemoglobins was silenced simultaneously in Lotus japonicus. As a result, growth of LbRNAi lines was severely inhibited compared to wild-type plants when plants were grown under symbiotic conditions in the absence of mineral nitrogen. The nodules of these plants were arrested in growth 14 post inoculation and lacked the characteristic pinkish colour. Growing these transgenic plants in conditions where reduced nitrogen is available for the plant led to normal plant growth and development. This demonstrates that leghemoglobins are not required for plant development per se, and proves for the first time that leghemoglobins are indispensable for symbiotic nitrogen fixation. Absence of leghemoglobins in LbRNAi nodules led to significant increases in free-oxygen concentrations throughout the nodules, a decrease in energy status as reflected by the ATP/ADP ratio, and an absence of the bacterial nitrogenase protein. The bacterial population within nodules of LbRNAi plants was slightly reduced. Alterations of plant nitrogen and carbon metabolism in LbRNAi nodules was reflected in changes in amino acid composition and starch deposition (Ott et al., 2005). These data provide strong evidence that nodule leghemoglobins function as oxygen transporters that facilitate high flux rates of oxygen to the sites of respiration at low free oxygen concentrations within the infected cells.}, subject = {Lotus japonicus}, language = {en} } @phdthesis{Usadel2004, author = {Usadel, Bj{\"o}rn}, title = {Untersuchungen zur Biosynthese der pflanzlichen Zellwand = [Identification and characterization of genes involved in plant cell wall synthesis]}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-2947}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the "hmmer" program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes.}, subject = {Zellwand}, language = {en} } @phdthesis{Steinhauser2004, author = {Steinhauser, Dirk}, title = {Inferring hypotheses from complex profile data - by means of CSB.DB, a comprehensive systems-biology database}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-2467}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {The past decades are characterized by various efforts to provide complete sequence information of genomes regarding various organisms. The availability of full genome data triggered the development of multiplex high-throughput assays allowing simultaneous measurement of transcripts, proteins and metabolites. With genome information and profiling technologies now in hand a highly parallel experimental biology is offering opportunities to explore and discover novel principles governing biological systems. Understanding biological complexity through modelling cellular systems represents the driving force which today allows shifting from a component-centric focus to integrative and systems level investigations. The emerging field of systems biology integrates discovery and hypothesis-driven science to provide comprehensive knowledge via computational models of biological systems. Within the context of evolving systems biology, investigations were made in large-scale computational analyses on transcript co-response data through selected prokaryotic and plant model organisms. CSB.DB - a comprehensive systems-biology database - (http://csbdb.mpimp-golm.mpg.de/) was initiated to provide public and open access to the results of biostatistical analyses in conjunction with additional biological knowledge. The database tool CSB.DB enables potential users to infer hypothesis about functional interrelation of genes of interest and may serve as future basis for more sophisticated means of elucidating gene function. The co-response concept and the CSB.DB database tool were successfully applied to predict operons in Escherichia coli by using the chromosomal distance and transcriptional co-responses. Moreover, examples were shown which indicate that transcriptional co-response analysis allows identification of differential promoter activities under different experimental conditions. The co-response concept was successfully transferred to complex organisms with the focus on the eukaryotic plant model organism Arabidopsis thaliana. The investigations made enabled the discovery of novel genes regarding particular physiological processes and beyond, allowed annotation of gene functions which cannot be accessed by sequence homology. GMD - the Golm Metabolome Database - was initiated and implemented in CSB.DB to integrated metabolite information and metabolite profiles. This novel module will allow addressing complex biological questions towards transcriptional interrelation and extent the recent systems level quest towards phenotyping.}, subject = {Datenbank}, language = {en} } @phdthesis{Helaly2004, author = {Helaly, Alaa El-din A.}, title = {Molecular studies on plants to enhance their stress tolerance}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-2427}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Environmental stresses such as drought, high salt and low temperature affect plant growth and decrease crop productivity extremely. It is important to improve stress tolerance of the crop plant to increase crop yield under stress conditions. The Arabidopsis thaliana salt tolerance 1 gene (AtSTO1) was originally identified by Lippuner et al., (1996). In this study around 27 members of STO-like proteins were identified in Arabidopsis thaliana, rice and other plant species. The STO proteins have two consensus motifs (CCADEAAL and FCV(L)EDRA). The STO family members can be regarded as a distinct class of C2C2 proteins considering their low sequence similarity to other GATA like proteins and poor conservation in the C-terminus. AtSTO1 was found to be induced by salt, cold and drought in leaves and roots of 4-week-old Arabidopsis thaliana wild-type plants. The expression of AtSTO1 under salt and cold stress was more pronounced in roots than in leaves. The data provided here revealed that the AtSTO1 protein is localized in the nucleus. The observation that AtSTO1 localizes in the nucleus is consistent with its proposed function as a transcription factor. AtSTO1-dependent phenotypes were observed when plant were grown at 50 mM NaCl on agar plates. Leaves of AtSTO1 overexpression lines were bigger with dark green coloration, whereas stunted growth and yellowish leaves were observed in wild-type and RNAi plants. Also, the AtSTO1 overexpression plants when exposed to long-term cold stress had a red leaf coloration which was much stronger than in wild-type and RNAi lines. Growth of AtSTO1 overexpression lines in long term under salt and cold stress was always associated with long roots which was more pronounced than in wild-type and RNAi lines. Proline accumulation increased more strongly in leaves and roots of AtSTO1 overexpression lines than in tissues of wild-type and RNAi lines when treated with 200 mM NaCl, exposed to cold stress or when watering was prevented for one day or two weeks. Also, soluble sugar content increased to higher levels under salt, cold and drought stress in AtSTO1 overexpression lines when compared to wild-type and RNAi lines. The increase in soluble sugar content was detected in AtSTO1 overexpression lines after long-term (2 weeks) growth of plants under these stresses. Anthocyanins accumulated in leaves of AtSTO1 overexpression lines when exposed to long term salt stress (200 mM NaCl for 2 weeks) or to 4°C for 6 and 8 weeks. Also, anthocyanin content was increased in flowers of AtSTO1 overexpression plants kept at 4°C for 8 weeks. Taken together these data indicate that overexpression of AtSTO1 enhances abiotic stress toleranc via a more pronounced accumulation of compatible solutes under stress.}, language = {en} } @phdthesis{Venevskaia2004, author = {Venevskaia, Irina}, title = {Modeling of vegetation diversity and a national conservation planning: example of Russia}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001863}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Die {\"u}bergreifende Zielsetzung meiner Studie ist eine Ausarbeitung quantitativer Methoden zur nationalen nationale Schutzplanung in {\"U}bereinstimmung mit dem internationalen Ansatz. Diese Zielsetzung erfordert eine L{\"o}sung der folgenden Probleme: 1) Wie l{\"a}sst sich Vegetationsvielfalt in grober Aufl{\"o}sung auf Basis abiotischen Faktoren einsch{\"a}tzen? 2) Wie ist der Ansatz 'globaler Hotspots' f{\"u}r die Eingrenzung nationaler Biodiversit{\"a}ts-Hotspots zu {\"u}bernehmen? 3) Wie erfolgt die Auswahl von quantitativen Schutzzielen unter Einbezug der Unterschiede nationaler Hotspots bei Umweltbedingungen und durch den Menschen Bedrohung? 4) Wie sieht der Entwurf eines großfl{\"a}chigen nationalen Naturschutzkonzepts aus, das die hierarchische Natur der Artenvielfalt reflektiert? Die Fallstudie f{\"u}r nationale Naturschutzplanung ist Russland. Die nachfolgenden theoretischen Schl{\"u}sse wurden gezogen: · Großr{\"a}umige Vegetationsdiversit{\"a}t ist weitgehend vorhersagbar durch klimabedingte latente W{\"a}rme f{\"u}r Verdunstung und topographische Landschaftsstruktur, beschrieben als H{\"o}hendifferenz. Das klimabasierte Modell reproduziert die beobachtete Artenanzahl von Gef{\"a}ßpflanzen f{\"u}r verschiedene Gebiete auf der Welt mit einem durchschnittlichen Fehler von 15\% · Nationale Biodiversit{\"a}ts-Hotspots k{\"o}nnen auf Grundlage biotischer oder abiotischer Daten kartographiert werden, indem als Korrektur f{\"u}r ein Land die quantitativen Kriterien f{\"u}r Planzenendemismus und Landnutzung des Ansatzes der 'globalen Hotspots' genutzt wird · Quantitative Naturschutzziele, die die Unterschiede zwischen nationalen Biodiversit{\"a}ts-Hotspots in Bezug auf Umweltbedingungen und der Bedrohung durch den Menschen miteinbeziehen, k{\"o}nnen mit nationalen Daten {\"u}ber Arten auf der Roten Liste gesetzt werden · Ein großr{\"a}umiger nationaler Naturschutzplan, der die hierarchische Natur der Artenvielfalt ber{\"u}cksichtigt, kann durch eine Kombination von abiotischer Methode im nationalen Bereich (Identifikation großr{\"a}umiger Hotspots) und biotischer Methode im regionalen Bereich (Datenanalyse der Arten auf der Roten Liste) entworfen werden}, language = {en} } @phdthesis{Junker2004, author = {Junker, Bj{\"o}rn H.}, title = {Sucrose breakdown in the potato tuber}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001673}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {In dieser Arbeit wurden verschiedene Ans{\"a}tze verfolgt, um das Verst{\"a}ndnis des Saccharose-zu-St{\"a}rke Stoffwechselweges in sich entwickelnden Kartoffelknollen zu untersuchen. Zun{\"a}chst wurde ein induzierbares Genexpressions-System aus dem Schimmelpilz Aspergillus nidulans f{\"u}r die Untersuchung des Metabolismus von Kartoffelknollen optimiert. Es wurde herausgefunden, dass dieses sogenannte alc system schneller auf Acetaldehyd reagiert als auf Ethanol, und dass Acetaldehyd weniger Seiteneffekte auf den Metabolismus hat. Die optimalen Induktionsbedingungen wurden dann benutzt um die Effekte einer zeitlich kontrollierten zytosolischen Expression einer Hefe-Invertase auf den Metabolismus der Kartoffelknolle zu untersuchen. Die beobachteten Unterschiede zwischen induzierter und konstitutiver Expression der Invertase f{\"u}hrten zu der Feststellung, dass die Glycolyse erst induziert wird nachdem ein ATP-Mangel durch erh{\"o}htes Saccharose-Cycling kreiert wurde. Weiterhin lassen die Ergebnisse darauf schließen, dass Maltose in der Kartoffelknolle eher ein Produkt der Kondensation zweier Glucose-Einheiten ist statt ein Produkt des St{\"a}rke-Abbaus zu sein. Im zweiten Teil dieser Arbeit wurde gezeigt, dass die Expression einer Hefe-Invertase in der Vakuole von Kartoffelknollen {\"a}hnliche Effekte auf deren Metabolismus hat wie die Expression des gleichen Enzymes im Apoplasten. Diese Beobachtung ist ein weiterer Beleg f{\"u}r die Pr{\"a}senz eines Mechanismus, bei dem Saccharose mittels Endozytose in die Vakuole aufgenommen wird anstatt {\"u}ber Transporter direkt ins Zytosol aufgenommen zu werden. Zum Schluß wird ein kinetisches Modell des Saccharose-Abbaus vorgestellt, das in der Lage ist diesen Teil des Stoffwechsels der Kartoffelknolle quantitativ zu simulieren. Weiterhin kann dieses Modell die metabolischen Effekte der Einf{\"u}hrung einer Hefe-Invertase in das Zytosol von Kartoffelknollen mit erstaunlicher Pr{\"a}zision vorhersagen. Zusammengefasst zeigen die Ergebnisse dieser Arbeit, dass induzierbare Genexpression sowie Computermodelle von Stoffwechselwegen n{\"u}tzliche Hilfsmittel f{\"u}r eine Verbesserung des Verst{\"a}ndnisses des Pflanzenmetabolismus sind.}, language = {en} } @phdthesis{Scheich2004, author = {Scheich, Christoph}, title = {High-throughput evaluation of protein folding conditions and expression constructs for structural genomics}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001552}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Das E. coli Expressionssystem ist das am h{\"a}ufigsten angewandte hinsichtlich der rekombinante Proteinexpression f{\"u}r strukturelle und funktionelle Analysen aufgrund der hohen erzielten Ausbeuten und der einfachen Handhabbarkeit. Allerdings ist insbesondere die Expression eukaryotischer Proteine in E. coli problematisch, z.B. wenn das Protein nicht korrekt gefaltet ist und in unl{\"o}slichen Inclusion Bodies anf{\"a}llt. In manchen F{\"a}llen ist die Analyse von Deletionskonstrukten oder einzelnen Proteindom{\"a}nen der Untersuchung des Voll{\"a}ngeproteins vorzuziehen. Dies umfasst die Herstellung eines Satzes von Expressionskonstrukten, welche charakterisiert werden m{\"u}ssen. In dieser Arbeit werden Methoden optimiert und evaluiert f{\"u}r die in vitro-Faltung von Inclusion Body-Proteinen sowie die Entwicklung einer Hochdurchsatz-Charakterisierung von Expressionskonstrukten. Die {\"U}berf{\"u}hrung von Inclusion Body-Proteinen in den nativen Zustand beinhaltet zwei Schritte: (a) Aufl{\"o}sen mit einen chaotropen Reagenz oder starkem ionischen Detergenz und (b) Faltung des Proteins durch Beseitigung des Chaotrops begleitet von dem Transfer in einen geeigneten Puffer. Die Ausbeute an nativ gefaltetem Protein ist oft stark eingeschr{\"a}nkt aufgrund von Aggregation und Fehlfaltung; sie kann allerdings durch die Zugabe bestimmter Additive zum Faltungspuffer erh{\"o}ht werden. Solche Additive m{\"u}ssen empirisch identifiziert werden. In dieser Arbeit wurde eine Testprozedur f{\"u}r Faltungsbedingungen entwickelt. Zur Reduzierung der m{\"o}glichen Kombinationen der getesteten Additive wurden sowohl empirische Beobachtungen aus der Literatur als auch bekannte Eigenschaften der Additive ber{\"u}cksichtigt. Zur Verminderung der eingesetzten Proteinmenge und des Arbeitsaufwandes wurde der Test automatisiert und miniaturisiert mittels eines Pipettierroboters. 20 Bedingungen zum schnellen Verd{\"u}nnen von denaturierten Proteinen werden hierbei getestet und zwei Bedingungen zur Faltung von Proteinen mit dem Detergenz/Cyclodextrin Protein-Faltungssystem von Rozema et al. (1996). 100 \&\#181;g Protein werden pro Bedingung eingesetzt. Zus{\"a}tzlich werden acht Bedingungen f{\"u}r die Faltung von His-Tag-Fusionsproteinen (ca. 200 \&\#181;g), welche an eine Metallchelat-Matrix immobilisiert sind, getestet. Die Testprozedur wurde erfolgreich angewendet zur Faltung eines humanen Proteins, der p22 Untereinheit von Dynactin, welche in E. coli in Inclusion Bodies exprimiert wird. So wie es sich bei vielen Proteinen darstellt, war auch f{\"u}r p22 Dynactin kein biologischer Nachweistest vorhanden, um den Erfolg des Faltungsexperimentes zu messen. Die L{\"o}slichkeit des Proteins kann nicht als eindeutiges Kriterium dienen, da neben nativ gefaltetem Protein, l{\"o}sliche fehlgefaltete Spezies und Mikroaggregate auftreten k{\"o}nnen. Diese Arbeit evaluiert Methoden zur Detektion kleiner Mengen nativen Proteins nach dem automatisierten Faltungstest. Bevor p22 Dynactin gefaltet wurde, wurden zwei Modellenzyme zur Evaluierung eingesetzt, bovine Carboanhydrase II (CAB) und Malat Dehydrogenase aus Schweineherz-Mitochondrien. Die wiedererlangte Aktivit{\"a}t nach der R{\"u}ckfaltung wurde korreliert mit verschiedenen biophysikalischen Methoden. Bindungsstudien mit 8-Anilino-1-Naphtalenesulfons{\"a}ure ergaben keine brauchbaren Informationen bei der R{\"u}ckfaltung von CAB aufgrund der zu geringen Sensitivit{\"a}t und da fehlgefaltete Proteine nicht eindeutig von nativem Protein unterschieden werden konnten. Tryptophan Fluoreszenzspektren der r{\"u}ckgefalteten CAB wurden zur Einsch{\"a}tzung des Erfolges der R{\"u}ckfaltung angewandt. Die Verschiebung des Intensit{\"a}tsmaximum zu einer niedrigeren Wellenl{\"a}nge im Vergleich zum denaturiert entfalteten Protein sowie die Fluoreszenzintensit{\"a}t korrelierten mit der wiedererlangten enzymatischen Aktivit{\"a}t. F{\"u}r beide Modellenzyme war analytische hydrophobe Interaktionschromatographie (HIC) brauchbar zur Identifizierung r{\"u}ckgefalteter Proben mit aktivem Enzym. Kompakt gefaltetes, aktives Enzym eluierte in einem distinkten Peak im abnehmenden Ammoniumsulfat-Gradienten. Das Detektionslimit f{\"u}r analytische HIC lag bei 5 \&\#181;g. Im Falle von CAB konnte gezeigt werden, dass Tryptophan-Fluoreszenz-Spektroskopie und analytische HIC in Kombination geeignet sind um Falsch-Positive oder Falsch-Negative, welche mit einem der Monitore erhalten wurden, auszuschließen. Diese beiden Methoden waren ebenfalls geeignet zur Identifizierung der Faltungsbedingungen von p22 Dynactin. Tryptophan-Fluoreszenz-Spektroskopie kann jedoch zu Falsch-Positiven f{\"u}hren, da in machen F{\"a}llen Spektren von l{\"o}slichen Mikroaggregaten kaum unterscheidbar sind von Spektren des nativ gefalteten Proteins. Dies zusammenfassend wurde eine schnelle und zuverl{\"a}ssige Testprozedur entwickelt, um Inclusion Body-Proteine einer strukturellen und funktionellen Analyse zug{\"a}nglich zu machen. In einem separaten Projekt wurden 88 verschiedene E. coli-Expressionskonstrukte f{\"u}r 17 humane Proteindom{\"a}nen, welche durch Sequenzanalyse identifiziert wurden, mit einer Hochdurchsatzreinigung und \–faltungsanalytik untersucht, um f{\"u}r die Strukturanalyse geeignete Kandidaten zu erhalten. Nach Expression in einem Milliliter im 96er Mikrotiterplattenformat und automatisierter Proteinreinigung wurden l{\"o}slich exprimierte Proteindom{\"a}nen direkt analysiert mittels 1D \&\#185;H-NMR Spektroskopie. Hierbei zeigte sich, dass insbesondere isolierte Methylgruppen-Signale unter 0.5 ppm sensitive und zuverl{\"a}ssige Sonden sind f{\"u}r gefaltetes Protein. Zus{\"a}tzlich zeigte sich, dass \– {\"a}hnlich zur Evaluierung des Faltungstests \– analytische HIC effizient eingesetzt werden kann zur Identifizierung von Konstrukten, welche kompakt gefaltetes Protein ergeben. Sechs Konstrukte, welche zwei Dom{\"a}nen repr{\"a}sentieren, konnten schnell als tauglich f{\"u}r die Strukturanalyse gefunden werden. Die Struktur einer dieser Dom{\"a}nen wurde k{\"u}rzlich von Mitarbeitern gel{\"o}st, die andere Struktur wurde im Laufe dieses Projektes von einer anderen Gruppe ver{\"o}ffentlicht.}, language = {en} } @phdthesis{Daub2004, author = {Daub, Carsten Oliver}, title = {Analysis of integrated transcriptomics and metabolomics data : a systems biology approach}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001251}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Moderne Hochdurchsatzmethoden erlauben die Messung einer Vielzahl von komplement{\"a}ren Daten und implizieren die Existenz von regulativen Netzwerken auf einem systembiologischen Niveau. Ein {\"u}blicher Ansatz zur Rekonstruktion solcher Netzwerke stellt die Clusteranalyse dar, die auf einem {\"A}hnlichkeitsmaß beruht. Wir verwenden das informationstheoretische Konzept der wechselseitigen Information, das urspr{\"u}nglich f{\"u}r diskrete Daten definiert ist, als {\"A}hnlichkeitsmaß und schlagen eine Erweiterung eines f{\"u}r gew{\"o}hnlich f{\"u}r die Anwendung auf kontinuierliche biologische Daten verwendeten Algorithmus vor. Wir vergleichen unseren Ansatz mit bereits existierenden Algorithmen. Wir entwickeln ein geschwindigkeitsoptimiertes Computerprogramm f{\"u}r die Anwendung der wechselseitigen Information auf große Datens{\"a}tze. Weiterhin konstruieren und implementieren wir einen web-basierten Dienst fuer die Analyse von integrierten Daten, die durch unterschiedliche Messmethoden gemessen wurden. Die Anwendung auf biologische Daten zeigt biologisch relevante Gruppierungen, und rekonstruierte Signalnetzwerke zeigen {\"U}bereinstimmungen mit physiologischen Erkenntnissen.}, language = {en} } @phdthesis{Rose2003, author = {Rose, Andreas}, title = {Analysis of phenolic compounds by dint of GDH-biosensors and immunoassays}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001048}, school = {Universit{\"a}t Potsdam}, year = {2003}, abstract = {In den letzten Jahren gerieten phenolische Substanzen, wie z.B. Chlor-, Nitrophenol oder Alkylphenolethoxylate aufgrund ihrer Toxizit{\"a}t sowie ihres kanzerogenen und endokrinen Potentials in das Interesse der {\"O}ffentlichkeit. Diese Substanzen gelangen in großen Mengen, z.B. aus industriellen Prozessen (Papier-, Kunststoff-, oder Lederindustrie) oder als Abbauprodukte von Pflanzenschutzmitteln in die Umwelt. Ziel dieser Arbeit war es, einfache biochemische Bestimmungsmethoden f{\"u}r verschiedene phenolische Umweltschadstoffe auf Basis biochemischer Erkennungselemente zu entwickeln. Diese sollten als Screeningmethoden in der Vor-Ort-Analytik einsetzbar sein. Die Anwendung sollte kosteng{\"u}nstig und einfach durchzuf{\"u}hren sein, so dass die Messung kein hochwissenschaftliches Personal erfordert. Daher stand im Hintergrund der Arbeit die Integration der Analysenmethode in ein kompaktes Handger{\"a}t. Zu diesem Zweck wurde ein Biosensor entwickelt der zur direkten Messung und in Kombination mit einem Immunoassay einsetzbar ist: 1.) Elektrochemischer Biosensor Ein elektrochemischer Biosensor stellt die Verbindung zwischen einer Elektrode und der biologischen Komponente dar. Als Messprinzip wurde die Amperometrie gew{\"a}hlt. Hierbei wird die Pr{\"a}senz des nachzuweisenden Stoffes durch die angelegte Spannung am Sensor visualisiert, da beim Vorhandensein ein Stromfluss gemessen wird. Um die Signalintensit{\"a}t zu erh{\"o}hen k{\"o}nnen Enzyme als Katalysatoren genutzt werden, die in der Lage sind die R{\"u}ckreaktion der Elektrodenreaktion zu realisieren. In diesem Fall wurde Glucose-Dehydrogenase (GDH) verwendet, die oxidierte phenolische Verbindungen reduzieren kann. Zusammen mit der Oxidation an der Sensoroberfl{\"a}che bildet sich ein Verst{\"a}rkungszyklus aus, der das urspr{\"u}ngliche Signal vielfach erh{\"o}ht. Wir waren in der Lage, GDH durch Einbetten in ein Polymerennetzwerk auf der Oberfl{\"a}che einer gedruckten Platin-Dickschicht-Elektrode zu immobilisieren. Als Resultat erhielten wir einen sehr empfindlichen und {\"a}ußerst stabilen Biosensor. Seine schnelle Ansprechzeit erm{\"o}glicht den Einsatz in automatisierten Fließsystemen zur Messung großer Probenzahlen. Der Einsatz in einem manuell betriebenen Handger{\"a}t konnte ebenfalls realisiert werden und brachte nur geringe Beeintr{\"a}chtigungen in bezug auf die Empfindlichkeit der Messung. Die erfolgreiche Implementierung des Biosensors in das Handger{\"a}t wurde in Rahmen eines internationalen Workshops in Barcelona, anhand der {\"U}berpr{\"u}fung der Reinigungsleistung von Kl{\"a}rwerken, gezeigt. 2.) Kombination mit Immunoassays Der Einsatzbereich der GDH-Biosensoren l{\"a}sst sich durch die Kombination mit anderen Techniken erweitern, wobei der Sensor zur Visualisierung der Nachweisreaktion dient. In diesem Fall kann der Sensor zur Bestimmung der Enzymaktivit{\"a}t von ß?Galactosidase (ßGal) verwendet werden. Der Nachweis geringster Enzymmengen wurde realisiert. Die ßGal wird zur Markierung eines Analytanalogen in Immunoassays verwendet, um die Bindung von Antik{\"o}rper und Analytmolek{\"u}l sichtbar zu machen. Im Immunoassay bildet sich ein Gleichgewicht zwischen Antik{\"o}rper, unmarkiertem Analyt und markiertem Analytanalog (Tracer) aus. {\"U}ber die Bestimmung der Enzymaktivit{\"a}t kann man die Analytkonzentration in der Probe errechnen. Wir haben unseren GDH-Biosensor erfolgreich mit zwei Techniken kombiniert. Zum Einen mit einem Assay zur Bestimmung von Nitrophenol, der in einem automatisiertem Fließsystem realisiert wurde. Hier wird die Mischung aus Antik{\"o}rpern, Analyt und Tracer {\"u}ber eine S{\"a}ule gegeben und gesp{\"u}lt. Die gebundenen Bestandteile werden durch den GDH-Biosensor quantifiziert. Zum Anderen wurde ein Kapillarimmunoassay entwickelt, der in das Handger{\"a}t integriert werden kann. Dabei wird der Antik{\"o}rper direkt an der Kapillare fixiert. Die Probe wird mit Tracer vermischt und in die Kapillare gegeben. Dort bildet sich das Gleichgewicht aus und weitere Probenbestandteile werden im Sp{\"u}lschritt eliminiert. Die Analytkonzentration wird durch die Bestimmung des gebunden Tracers (Aktivit{\"a}t der ßGal) mit Hilfe des GDH-Biosensors realisiert.}, language = {en} } @phdthesis{Lemke2004, author = {Lemke, Britt}, title = {Identification of Epo-independent red cell progenitors : the E-cad+ progenitors}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0001432}, school = {Universit{\"a}t Potsdam}, year = {2004}, abstract = {Erythrozyten z{\"a}hlen zu den am h{\"a}ufigsten vorkommenden terminal differenzierten Zelltypen des menschlichen K{\"o}rpers. Durchschnittlich werden t{\"a}glich ca. 2 x 1011 von ihnen im K{\"o}rper eines erwachsenen Menschen produziert. Die reifen Erythrozyten entstehen aus multipotenten h{\"a}matopoetischen Stammzellen, die {\"u}ber Stadien von erythroiden Vorl{\"a}uferzellen, erst den sogenannten burst forming units-erythroid (BFU-E) und sp{\"a}ter den colony forming units-erythroid (CFU-E), zu kernlosen h{\"a}moglobinisierten Zellen differenzieren. F{\"u}r die Untersuchung der molekularen Mechanismen der humanen Erythropoese ist die effektive in vitro Amplifizierung einer weitgehend homogenen Population der Vorl{\"a}uferzellen der einzelnen Entwicklungsstadien notwendig. Den Wachstumsfaktoren stem cell factor (SCF) und Erythropoietin (Epo) f{\"a}llt dabei eine entscheidende Rolle zu. Unter ihrem synergistischen Einfluß lassen sich Epo-abh{\"a}ngige Zellpopulationen, die sich aus BFU-E und CFU-E Typ Zellen zusammensetzen, ausreichend amplifizieren (Panzenb{\"o}ck et al., 1998). Freyssinier et al., 1999 beschrieb erstmals die Isolierung einer Epo-unabh{\"a}ngigen Population von Vorl{\"a}uferzellen (CD36+ Vorl{\"a}uferzellen), die ebenfalls erythroide Eigenschaften aufweisen. Ziel dieser Arbeit war die Isolierung und Charakterisierung von Epo-unabh{\"a}ngigen Vorl{\"a}uferzellen, die eine fr{\"u}he erythroide und m{\"o}glichst homogene Vorl{\"a}uferzellpopulation darstellen und m{\"o}glicherweise ein h{\"o}heres Proliferationspotential aufweisen. F{\"u}r die Identifizierung der Epo-unabh{\"a}ngigen Vorl{\"a}uferzellen, wurden CD34+ Zellen aus Nabelschnurblut aufgereinigt und unter serumfreien Kulturbedingungen und unter Zusatz der Wachstumsfaktoren SCF, Interleukin 3 (IL-3) und eines Fusionsproteins aus IL-6 und l{\"o}slichem IL-6 Rezeptor (hyper-IL-6) {\"u}ber einen Zeitraum von 8 Tagen kultiviert. Anschließend wurde eine Population von E-cadherin positiven (E-cad+) Zellen {\"u}ber immunomagnetische Selektion isoliert. Diese neu gewonnenen Epo-unabh{\"a}ngigen E-cad+ Vorl{\"a}uferzellen wurden hinsichtlich ihres proliferativen Potentials und ihrer Differenzierungseigenschaften mit SCF/Epo-Vorl{\"a}uferzellen und CD36+ Vorl{\"a}uferzellen verglichen. Von allen drei Zelltypen wurden des weiteren detailierte molekulargenetische Analysen mittels DNA microarray Technologie durchgef{\"u}hrt und die resultierenden Genexpressionsmuster miteinander verglichen. Die Ergebnisse zeigen, dass die E-cad+ Vorl{\"a}uferzellen eine fr{\"u}he, weitgehend homogene Epo-unabh{\"a}ngige Population vom BFU-E Typ darstellen und durch entsprechende {\"A}nderungen der Kulturbedingungen zu einer in vitro Differenzierung angeregt werden k{\"o}nnen. Die E-cad+ Vorl{\"a}uferzellen sind hinsichtlich ihres proliferativen Potentials, ihrer Reaktion auf verschiedene Wachstumsfaktoren, der Expression spezifischer Oberfl{\"a}chenmolek{\"u}le und ihrer Genexpressionsmuster mit SCF/Epo-Vorl{\"a}uferzellen und CD36+ Vorl{\"a}uferzellen vergleichbar. Aufgrund der Identifizierung unterschiedlich exprimierter Gene zwischen den Epo-unabh{\"a}ngigen E-cad+ und den Epo-abh{\"a}ngigen SCF/Epo Vorl{\"a}uferzellen konnten Kanditatengene wie Galectin-3, Cyclin D1, der Anti-M{\"u}llerian Hormonrezeptor, Prostata-Differenzierungsfaktor und insulin-like growth factor binding protein 4 identifiziert werden, die als potentielle Regulatoren der Erythropoese in Betracht kommen k{\"o}nnten. Es konnte weiterhin gezeigt werden, dass CD36+ Vorl{\"a}uferzellen, die aus der selben Zellpopulation wie die E-cad+ Vorl{\"a}uferzellen immunomagnetisch selektioniert wurden, eine heterogene Population darstellen, die sowohl E-cadherin positive als auch negative Zellen enth{\"a}lt. Die Analyse der Genexpressionsmuster zeigte, dass in den CD36+ Vorl{\"a}uferzellen zwar auch die Expression erythroid-spezifischen Gene nachgewiesen werden kann, hier aber im Gegensatz zu den E-cad+ Vorl{\"a}uferzellen auch f{\"u}r Megakaryozyten spezifische Gene stark exprimiert sind. Die Ergebnisse dieser Arbeit tragen zu einem neuen Modell der in vivo Abl{\"a}ufe der Entwicklung roter Blutzellen bei und werden der weiteren Untersuchung der molekularen Mechanismen der Erythropoese dienen.}, language = {en} } @phdthesis{Bissinger2003, author = {Bissinger, Vera}, title = {Factors determining growth and vertical distribution of planktonic algae in extremely acidic mining lakes (pH 2.7)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0000695}, school = {Universit{\"a}t Potsdam}, year = {2003}, abstract = {Die vorliegende Dissertation besch{\"a}ftigt sich mit den Faktoren, die das Wachstum und die Vertikalverteilung von Planktonalgen in extrem sauren Tagebaurestseen (TBS; pH 2-3) beeinflussen. Im exemplarisch untersuchten TBS 111 (pH 2.7; Lausitzer Revier) dominiert die Goldalge Ochromonas sp. in oberen und die Gr{\"u}nalge Chlamydomonas sp. in tieferen Wasserschichten, wobei letztere ein ausgepr{\"a}gtes Tiefenchlorophyll-Maximum (DCM) ausbildet. Es wurde ein deutlicher Einfluss von Limitation durch anorganischen Kohlenstoff (IC) auf das phototrophe Wachstum von Chlamydomonas sp. in oberen Wasserschichten nachgewiesen, die mit zunehmender Tiefe von Lichtlimitation abgel{\"o}st wird. Im Vergleich mit Arbeiten aus neutralen Seen zeigte Chlamydomonas sp. erniedrigte maximale Wachstumsraten, einen gesteigerten Kompensationspunkt und erh{\"o}hte Dunkelrespirationsraten, was auf gesteigerte metabolische Kosten unter den extremen physikalisch-chemischen Bedingungen hinweist. Die Photosyntheseleistungen von Chlamydomonas sp. waren in Starklicht-adaptierten Zellen durch IC-Limitation deutlich verringert. Außerdem ergaben die ermittelten minimalen Zellquoten f{\"u}r Phosphor (P) einen erh{\"o}hten P-Bedarf unter IC-Limitation. Anschließend konnte gezeigt werden, dass Chlamydomonas sp. ein mixotropher Organismus ist, der seine Wachstumsraten {\"u}ber die osmotrophe Aufnahme gel{\"o}sten organischen Kohlenstoffs (DOC) erh{\"o}hen kann. Dadurch ist dieser Organismus f{\"a}hig, in tieferen, Licht-limitierten Wasserschichten zu {\"u}berleben, die einen h{\"o}heren DOC-Gehalt aufweisen. Da die Vertikalverteilung der Algen im TBS 111 jedoch weder durch IC-Limitation, P-Verf{\"u}gbarkeit noch die in situ DOC-Konzentrationen abschließend erkl{\"a}rt werden konnte (bottom-up Kontrolle), wurde eine neue Theorie zur Entstehung der Vertikalverteilung gepr{\"u}ft. Grazing der phagotrophen und phototrophen Alge Ochromonas sp. auf der phototrophen Alge Chlamydomonas sp. erwies sich als herausragender Faktor, der {\"u}ber top-down Kontrolle die Abundanz der Beute in h{\"o}heren Wasserschichten beeinflussen kann. Gemeinsam mit der Tatsache, dass Chlamydomonas sp. DOC zur Wachstumssteigerung verwendet, f{\"u}hrt dies zu einer Akkumulation von Chlamydomonas sp. in der Tiefe, ausgepr{\"a}gt als DCM. Daher erscheint grazing als der Hauptfaktor, der die beobachtete Vertikalschichtung der Algen im TBS 111 hervorruft. Die erzielten Ergebnisse liefern grundlegende Informationen, um die Auswirkungen von Strategien zur Neutralisierung der TBS auf das Nahrungsnetz absch{\"a}tzen zu k{\"o}nnen.}, language = {en} } @phdthesis{Streffer2002, author = {Streffer, Katrin}, title = {Highly sensitive measurements of substrates and inhibitors on the basis of tyrosinase sensors and recycling systems}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-0000632}, school = {Universit{\"a}t Potsdam}, year = {2002}, abstract = {Analytische Chemie heute meint nicht l{\"a}nger nur die große Messtechnik, die zeit- und kostenintensiv ist, die außerdem nur von qualifiziertem Personal zu bedienen ist und deren Resultate nur durch dieses Personal auswertbar sind. Meist erfordert diese sagen wir 'klassische analytische Messtechnik' auch noch spezielle R{\"a}umlichkeiten und oft eine relative große Menge an speziell vorbereiteten Proben. Neben dieser klassischen analytischen Messtechnik hat sich besonders in den letzten Jahren eine auf bestimmte Stoffgruppen und Anforderungen zugeschnittene Messtechnik durchgesetzt, die oft auch durch einen Laien bedient werden kann. Meist sind es sehr kleine Ger{\"a}te. Auch die ben{\"o}tigten Probenvolumina sind klein und eine spezielle Probenvorbereitung ist nicht erforderlich. Ausserdem sind die Ger{\"a}te einfach zu handhaben, billig sowohl in ihrer Herstellung als auch im Gebrauch und meist erlauben sie sogar eine kontinuierliche Messwerterfassung. Zahlreiche dieser in den letzten Jahren entwickelten Ger{\"a}te greifen zur{\"u}ck auf 40 Jahre Forschung auf dem Gebiet der Biosensorik. Seit Clark und Lyons im Jahr 1962 in der Lage waren, mit einer einfachen Sauerstoffelektrode, erg{\"a}nzt durch ein Enzym, Glucose zu messen, war die Entwicklung neuer Messtechnik nicht mehr aufzuhalten. Biosensoren, spezielle Messf{\"u}hler, die aus einer Kombination aus biologischer Komponente (erlaubt eine spezifische Erkennung des Analyten auch ohne vorherige Reinigung der Probe) und einem physikalischen Messf{\"u}hler (wandelt den prim{\"a}ren physikochemischen Effekt in ein elektronisch messbares Signal um) bestehen, eroberten den Markt. Im Rahmen dieser Doktorarbeit wurden verschiedene Tyrosinasesensoren entwickelt, die je nach Herkunft und Eigenschaften der verwendeten Tyrosinase unterschiedliche Anforderungen erf{\"u}llen. Beispielsweise wurde einer dieser Tyrosinasesensoren f{\"u}r die Bestimmung phenolischer Verbindungen in Fluss- und Seewasserproben eingesetzt, und die mit diesem Sensor gemessenen Ergebnisse konnten sehr gut mit dem entsprechenden DIN-Test zur Bestimmung phenolischer Verbindungen korreliert werden. Ein anderer entwickelter Sensor zeigte eine sehr hohe Empfindlichkeit f{\"u}r Catecholamine, Substanzen die speziell in der medizinischen Diagnostik von Wichtigkeit sind. Ausserdem zeigten die ebenfalls im Rahmen dieser Doktorarbeit durchgef{\"u}hrten Untersuchungen zweier verschiedener Tyrosinasen, dass, will man in Zukunft noch empfindlichere Tyrosinasesensoren entwickeln, eine spezielle Tyrosinase (Tyrosinase aus Streptomyces antibioticus) die bessere Wahl sein wird, als die bisher im Bereich der Biosensorforschung verwendete Tyrosinase aus Agaricus bisporus. Desweiteren wurden erste Erfolge auf molekularbiologischem Gebiet erreicht, das heisst, dass Tyrosinasemutanten mit speziellen, vorher {\"u}berlegten Eigenschaften, hergestellt werden sollen. Diese Erfolge k{\"o}nnen dazu genutzt werden, eine neue Generation an Tyrosinasesensoren zu entwickeln, Tyrosinasesensoren in denen Tyrosinase gerichtet gebunden werden kann, sowohl an den entsprechenden physikalischen Messf{\"u}hler oder auch an ein anderes Enzym. Davon verspricht man sich deutlich minimierte Wege, die die zu bestimmende Substanz (oder deren Produkt) sonst zur{\"u}cklegen m{\"u}sste, was am Ende zu einer deutlich erh{\"o}hten Empfindlichkeit des resultierenden Biosensors f{\"u}hren sollte.}, subject = {Enzymelektrode ; Monophenolmonooxygenase}, language = {en} }