@phdthesis{Winck2011, author = {Winck, Flavia Vischi}, title = {Nuclear proteomics and transcription factor profiling in Chlamydomonas reinhardtii}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-53909}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5\% CO2 to 0.04\% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses.}, language = {en} } @phdthesis{Weiss2011, author = {Weiß, Julia}, title = {Computer assisted proteomics in a systems biology context}, address = {Potsdam}, pages = {VIII, 138, XVII S.}, year = {2011}, language = {en} } @phdthesis{Vosloh2011, author = {Vosloh, Daniel}, title = {Subcellular compartmentation of primary carbon metabolism in mesophyll cells of Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-55534}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Metabolismus in Pflanzenzellen ist stark kompartimentiert. Viele Stoffwechselwege haben Reaktionen in mehr als einem Kompartiment. Zum Beispiel wird w{\"a}hrend der Photosynthese in pflanzlichen Mesophyllzellen Kohlenstoff in Form von St{\"a}rke in den Chloroplasten synthetisiert, w{\"a}hrend es im Zytosol in Form von Sacharose gebildet und in der Vakuole gespeichert wird. Diese Reaktionen sind strikt reguliert um ein Gleichgewicht der Kohlenstoffpools der verschiedenen Kompartimente aufrecht zu erhalten und die Energieversorgung aller Teile der Zelle f{\"u}r anabolische Reaktionen sicher zu stellen. Ich wende eine Methode an, bei der die Zellen unter nicht-w{\"a}ssrigen Bedingungen fraktioniert werden und daher der metabolische Status der w{\"a}hrend der Ernte herrschte {\"u}ber den ganzen Zeitraum der Auftrennung beibehalten wird. Durch die Kombination von nichtw{\"a}ssriger Fraktionierung und verschiedener Massenspektrometrietechniken (Fl{\"u}ssigchromotagraphie- und Gaschromotagraphie basierende Massenspekrometrie) ist es m{\"o}glich die intrazellul{\"a}re Verteilung der meisten Intermediate des photosynthetischen Kohlenstoffstoffwechsels und der Produkte der nachgelagerten metabolischen Reaktionen zu bestimmen. Das Wissen {\"u}ber die in vivo Konzentrationen dieser Metabolite wurde genutzt um die {\"A}nderung der freien Gibbs Energie in vivo zu bestimmen. Mit Hilfe dessen kann bestimmt werden, welche Reaktion sich in einem Gleichgewichtszustand befinden und welche davon entfernt sind. Die Konzentration der Enzyme und der Km Werte wurden mit den Konzentrationen der Metabolite in vivo verglichen, um festzustellen, welche Enzyme substratlimitiert sind und somit sensitiv gegen{\"u}ber {\"A}nderungen der Substratkonzentration sind. Verschiedene Intermediate des Calvin-Benson Zyklus sind gleichzeitig Substrate f{\"u}r andere Stoffwechselwege, als da w{\"a}ren Dihyroxyaceton-phosphat (DHAP, Saccharosesynthese), Fructose 6-phosphat (Fru6P, St{\"a}rkesynthese), Erythrose 4-phosphat (E4P, Shikimat Stoffwechselweg) und Ribose 5-phosphat (R5P, Nukleotidbiosynthese). Die Enzyme, die diese Intermediate verstoffwechseln, liegen an den Abzweigungspunkten zu diesen Stoffwechselwegen. Diese sind Trisose phosphat isomerase (DHAP), Transketolase (E4P), Sedoheptulose-1,7 biphosphat aldolase (E4P) und Ribose-5-phosphat isomerase (R5P), welche nicht mit ihren Substraten ges{\"a}ttigt sind, da die jeweilige Substratkonzentration geringer als der zugeh{\"o}rige Km Wert ist. F{\"u}r metabolische Kontrolle bedeutet dies, dass diese Schritte am sensitivsten gegen{\"u}ber {\"A}nderungen der Substratkonzentrationen sind. Im Gegensatz dazu sind die regulierten irreversiblen Schritte von Fructose-1,6.biphosphatase und Sedoheptulose-1,7-biphosphatase relativ insensitiv gegen{\"u}ber {\"A}nderungen der Substratkonzentration. F{\"u}r den Stoffwechselweg der Saccharosesynthese konnte gezeigt werden, dass die zytosolische Aldolase eine geringer Bindeseitenkonzentration als Substratkonzentration (DHAP) aufweist, und dass die Konzentration von Saccharose-6-phosphat geringer als der Km Wert des synthetisierenden Enzyms Saccharose-phosphatase ist. Sowohl die Saccharose-phosphat-synthase, also auch die Saccharose-phosphatase sind in vivo weit von einem Gleichgewichtszustand entfernt. In Wildtyp Arabidopsis thaliana Columbia-0 Bl{\"a}ttern wurde der gesamte Pool von ADPGlc im Chloroplasten gefunden. Das Enzyme ADPGlc pyrophosphorylase ist im Chloroplasten lokalisiert und synthetisiert ADPGlc aus ATP und Glc1P. Dieses Verteilungsmuster spricht eindeutig gegen die Hypothese von Pozueta-Romero und Kollegen, dass ADPGlc im Zytosol durch ADP vermittelte Spaltung von Saccharose durch die Saccharose Synthase erzeugt wird. Basierend auf dieser Beobachtung und anderen ver{\"o}ffentlichten Ergebnissen wurde geschlußfolgert, dass der generell akzeptierte Stoffwechselweg der St{\"a}rkesynthese durch ADPGlc Produktion via ADPGlc pyrophosphorylase in den Chloroplasten korrekt ist, und die Hypothese des alternativen Stoffwechselweges unhaltbar ist. Innerhalb des Stoffwechselweges der Saccharosesynthsese wurde festgestellt, dass die Konzentration von ADPGlc geringer als der Km Wert des St{\"a}rkesynthase ist, was darauf hindeutet, dass das Enzym substratlimitiert ist. Eine generelle Beobachtung ist, dass viele Enzmye des Calvin-Benson Zyklus {\"a}hnliche Bindeseitenkonzentrationen wie Metabolitkonzentrationen aufweisen, wohingegen in den Synthesewegen von Saccharose und St{\"a}rke die Bindeseitenkonzentrationen der Enzyme viel geringer als die Metabolitkonzentrationen sind.}, language = {en} } @phdthesis{Tiller2011, author = {Tiller, Nadine}, title = {Plastid translation : functions of plastid-specific ribosomal proteins and identification of a factor mediating plastid-to-nucleus retrograde sifnalling}, address = {Potsdam}, pages = {122 S.}, year = {2011}, language = {en} } @phdthesis{Szecowka2011, author = {Szec{\´o}wka, Marek}, title = {Metabolic fluxes in photosynthetic and heterotrophic plant tissues}, address = {Potsdam}, pages = {XII, 145 S.}, year = {2011}, language = {en} } @phdthesis{Sun2011, author = {Sun, Xiaoliang}, title = {Towards understanding the dynamics of biological systems from -Omics data}, address = {Potsdam}, pages = {114 S.}, year = {2011}, language = {en} } @phdthesis{StoofLeichsenring2011, author = {Stoof-Leichsenring, Kathleen Rosemarie}, title = {Genetic analysis of diatoms and rotifers in tropical Kenyan lake sediments}, address = {Potsdam}, year = {2011}, language = {en} } @phdthesis{Sperfeld2011, author = {Sperfeld, Erik}, title = {Effects of temperature and co-limiting nutritional components on life history traits of Daphnia magna and its biochemical composition}, address = {Potsdam}, pages = {157 S.}, year = {2011}, language = {en} } @phdthesis{Siewert2011, author = {Siewert, Katharina}, title = {Autoaggressive human t cell receptorrs and their antigen specificities}, address = {Potsdam}, pages = {145 S.}, year = {2011}, language = {en} } @phdthesis{Sharma2011, author = {Sharma, Tripti}, title = {Regulation of potassium channels in plants : biophysical mechanisms and physiological implacations}, address = {Potsdam}, pages = {104 S.}, year = {2011}, language = {en} } @phdthesis{Schuette2011, author = {Sch{\"u}tte, Moritz}, title = {Evolutionary fingerprints in genome-scale networks}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-57483}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Mathematical modeling of biological phenomena has experienced increasing interest since new high-throughput technologies give access to growing amounts of molecular data. These modeling approaches are especially able to test hypotheses which are not yet experimentally accessible or guide an experimental setup. One particular attempt investigates the evolutionary dynamics responsible for today's composition of organisms. Computer simulations either propose an evolutionary mechanism and thus reproduce a recent finding or rebuild an evolutionary process in order to learn about its mechanism. The quest for evolutionary fingerprints in metabolic and gene-coexpression networks is the central topic of this cumulative thesis based on four published articles. An understanding of the actual origin of life will probably remain an insoluble problem. However, one can argue that after a first simple metabolism has evolved, the further evolution of metabolism occurred in parallel with the evolution of the sequences of the catalyzing enzymes. Indications of such a coevolution can be found when correlating the change in sequence between two enzymes with their distance on the metabolic network which is obtained from the KEGG database. We observe that there exists a small but significant correlation primarily on nearest neighbors. This indicates that enzymes catalyzing subsequent reactions tend to be descended from the same precursor. Since this correlation is relatively small one can at least assume that, if new enzymes are no "genetic children" of the previous enzymes, they certainly be descended from any of the already existing ones. Following this hypothesis, we introduce a model of enzyme-pathway coevolution. By iteratively adding enzymes, this model explores the metabolic network in a manner similar to diffusion. With implementation of an Gillespie-like algorithm we are able to introduce a tunable parameter that controls the weight of sequence similarity when choosing a new enzyme. Furthermore, this method also defines a time difference between successive evolutionary innovations in terms of a new enzyme. Overall, these simulations generate putative time-courses of the evolutionary walk on the metabolic network. By a time-series analysis, we find that the acquisition of new enzymes appears in bursts which are pronounced when the influence of the sequence similarity is higher. This behavior strongly resembles punctuated equilibrium which denotes the observation that new species tend to appear in bursts as well rather than in a gradual manner. Thus, our model helps to establish a better understanding of punctuated equilibrium giving a potential description at molecular level. From the time-courses we also extract a tentative order of new enzymes, metabolites, and even organisms. The consistence of this order with previous findings provides evidence for the validity of our approach. While the sequence of a gene is actually subject to mutations, its expression profile might also indirectly change through the evolutionary events in the cellular interplay. Gene coexpression data is simply accessible by microarray experiments and commonly illustrated using coexpression networks where genes are nodes and get linked once they show a significant coexpression. Since the large number of genes makes an illustration of the entire coexpression network difficult, clustering helps to show the network on a metalevel. Various clustering techniques already exist. However, we introduce a novel one which maintains control of the cluster sizes and thus assures proper visual inspection. An application of the method on Arabidopsis thaliana reveals that genes causing a severe phenotype often show a functional uniqueness in their network vicinity. This leads to 20 genes of so far unknown phenotype which are however suggested to be essential for plant growth. Of these, six indeed provoke such a severe phenotype, shown by mutant analysis. By an inspection of the degree distribution of the A.thaliana coexpression network, we identified two characteristics. The distribution deviates from the frequently observed power-law by a sharp truncation which follows after an over-representation of highly connected nodes. For a better understanding, we developed an evolutionary model which mimics the growth of a coexpression network by gene duplication which underlies a strong selection criterion, and slight mutational changes in the expression profile. Despite the simplicity of our assumption, we can reproduce the observed properties in A.thaliana as well as in E.coli and S.cerevisiae. The over-representation of high-degree nodes could be identified with mutually well connected genes of similar functional families: zinc fingers (PF00096), flagella, and ribosomes respectively. In conclusion, these four manuscripts demonstrate the usefulness of mathematical models and statistical tools as a source of new biological insight. While the clustering approach of gene coexpression data leads to the phenotypic characterization of so far unknown genes and thus supports genome annotation, our model approaches offer explanations for observed properties of the coexpression network and furthermore substantiate punctuated equilibrium as an evolutionary process by a deeper understanding of an underlying molecular mechanism.}, language = {en} } @phdthesis{Schoenheit2011, author = {Sch{\"o}nheit, J{\"o}rg}, title = {A phagocyte-specific Irf8 gene enhancer establishes early conventional dendritic cell commitment}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-55482}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Haematopoietic development is a complex process that is strictly hierarchically organized. Here, the phagocyte lineages are a very heterogeneous cell compartment with specialized functions in innate immunity and induction of adaptive immune responses. Their generation from a common precursor must be tightly controlled. Interference within lineage formation programs for example by mutation or change in expression levels of transcription factors (TF) is causative to leukaemia. However, the molecular mechanisms driving specification into distinct phagocytes remain poorly understood. In the present study I identify the transcription factor Interferon Regulatory Factor 8 (IRF8) as the specification factor of dendritic cell (DC) commitment in early phagocyte precursors. Employing an IRF8 reporter mouse, I showed the distinct Irf8 expression in haematopoietic lineage diversification and isolated a novel bone marrow resident progenitor which selectively differentiates into CD8α+ conventional dendritic cells (cDCs) in vivo. This progenitor strictly depends on Irf8 expression to properly establish its transcriptional DC program while suppressing a lineage-inappropriate neutrophile program. Moreover, I demonstrated that Irf8 expression during this cDC commitment-step depends on a newly discovered myeloid-specific cis-enhancer which is controlled by the haematopoietic transcription factors PU.1 and RUNX1. Interference with their binding leads to abrogation of Irf8 expression, subsequently to disturbed cell fate decisions, demonstrating the importance of these factors for proper phagocyte cell development. Collectively, these data delineate a transcriptional program establishing cDC fate choice with IRF8 in its center.}, language = {en} } @phdthesis{Schudoma2011, author = {Schudoma, Christian}, title = {Bioinformatic approaches to sequence-structure relationships in RNA loops}, address = {Potsdam}, pages = {114}, year = {2011}, language = {en} } @phdthesis{Schroeder2011, author = {Schr{\"o}der, Florian}, title = {Funktionelle Charakterisierung der EXO/EXL-Proteinfamilie und NFXL2-Isoformen in Arabidopsis thaliana}, address = {Potsdam}, pages = {86 S.}, year = {2011}, language = {de} } @phdthesis{Samereier2011, author = {Samereier, Matthias}, title = {Functional analyses of microtubule and centrosome-associated proteins in Dictyostelium discoideum}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-52835}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium.}, language = {en} } @phdthesis{Ruprecht2011, author = {Ruprecht, Colin}, title = {Characterization of genetic modules involved in cellulose synthesis in Arabidopsis thaliana}, address = {Potsdam}, pages = {109 S.}, year = {2011}, language = {en} } @phdthesis{Rott2011, author = {Rott, Markus}, title = {Die Rolle der plastid{\"a}ren APT Synthase in der Regulation des photosynthetischen Elektronenflusses}, address = {Potsdam}, pages = {131 S.}, year = {2011}, language = {de} } @phdthesis{Rocha2011, author = {Rocha, Marcia Rosa}, title = {Time series analysis reveals links between functional traits, population dynamics and ecosystem functions in a diverse phytoplankton community}, address = {Potsdam}, pages = {126 S.}, year = {2011}, language = {en} } @phdthesis{Pyl2011, author = {Pyl, Eva-Theresa}, title = {Networks and growth in Arabidopsis: two strategies to pertub a complex system}, address = {Potsdam}, pages = {XIV, 145 S.}, year = {2011}, language = {en} } @phdthesis{Piepho2011, author = {Piepho, Maike}, title = {Phytoplankton lipids in a changing world : more than just water flea feed}, address = {Potsdam}, pages = {111 S.}, year = {2011}, language = {en} } @phdthesis{Nguyen2011, author = {Nguyen, Hung Minh}, title = {Regulation of leaf growth and development by transcription factors in Arabidopsis thaliana}, address = {Potsdam}, pages = {152 S.}, year = {2011}, language = {en} } @phdthesis{Nahavandi2011, author = {Nahavandi, Nahid}, title = {Genetic and morphological analysis on the evolution of the Ponto-Caspian amphipod Pontogammarus maeoticus}, address = {Potsdam}, pages = {54 S.}, year = {2011}, language = {en} } @phdthesis{Naaf2011, author = {Naaf, Tobias}, title = {Floristic homogenization and impoverishment : herb layer changes over two decades in deciduous forest patches of the Weser-Elbe region (NW Germany)}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-52446}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Human-induced alterations of the environment are causing biotic changes worldwide, including the extinction of species and a mixing of once disparate floras and faunas. One type of biological communities that is expected to be particularly affected by environmental alterations are herb layer plant communities of fragmented forests such as those in the west European lowlands. However, our knowledge about current changes in species diversity and composition in these communities is limited due to a lack of adequate long-term studies. In this thesis, I resurveyed the herb layer communities of ancient forest patches in the Weser-Elbe region (NW Germany) after two decades using 175 semi-permanent plots. The general objectives were (i) to quantify changes in plant species diversity considering also between-community (β) and functional diversity, (ii) to determine shifts in species composition in terms of species' niche breadth and functional traits and (iii) to find indications on the most likely environmental drivers for the observed changes. These objectives were pursued with four independent research papers (Chapters 1-4) whose results were brought together in a General Discussion. Alpha diversity (species richness) increased by almost four species on average, whereas β diversity tended to decrease (Chapter 1). The latter is interpreted as a beginning floristic homogenization. The observed changes were primarily the result of a spread of native habitat generalists that are able to tolerate broad pH and moisture ranges. The changes in α and β diversity were only significant when species abundances were neglected (Chapters 1 and 2), demonstrating that the diversity changes resulted mainly from gains and losses of low-abundance species. This study is one of the first studies in temperate Europe that demonstrates floristic homogenization of forest plant communities at a larger than local scale. The diversity changes found at the taxonomic level did not result in similar changes at the functional level (Chapter 2). The likely reason is that these communities are functionally "buffered". Single communities involve most of the functional diversity of the regional pool, i.e., they are already functionally rich, while they are functionally redundant among each other, i.e., they are already homogeneous. Independent of taxonomic homogenization, the abundance of 30 species decreased significantly (Chapter 4). These species included 12 ancient forest species (i.e., species closely tied to forest patches with a habitat continuity > 200 years) and seven species listed on the Red List of endangered plant species in NW Germany. If these decreases continue over the next decades, local extinctions may result. This biotic impoverishment would seriously conflict with regional conservation goals. Community assembly mechanisms changed at the local level particularly at sites that experienced disturbance by forest management activities between the sampling periods (Chapter 3). Disturbance altered community assembly mechanisms in two ways: (i) it relaxed environmental filters and allowed the coexistence of different reproduction strategies, as reflected by a higher diversity of reproductive traits at the time of the resurvey, and (ii) it enhanced light availability and tightened competitive filters. These limited the functional diversity with respect to canopy height and selected for taller species. Thirty-one winner and 30 loser species, which had significantly increased or decreased in abundance, respectively, were characterized by various functional traits and ecological performances to find indications on the most likely environmental drivers for the observed floristic changes (Chapter 4). Winner species had higher seed longevity, flowered later in the season and had more often an oceanic distribution compared to loser species. Loser species tended to have a higher specific leaf area, to be more susceptible to deer browsing and to have a performance optimum at higher soil pH values compared to winner species. Multiple logistic regression analyses indicated that disturbances due to forest management interventions were the primary cause of the species shifts. As one of the first European resurvey studies, this study provides indications that an enhanced browsing pressure due to increased deer densities and increasingly warmer winters are important drivers. The study failed to demonstrate that eutrophication and acidification due to atmospheric deposition substantially drive herb layer changes. The restriction of the sample to the most base-rich sites in the region is discussed as a likely reason. Furthermore, the decline of several ancient forest species is discussed as an indication that the forest patches are still paying off their "extinction debt", i.e., exhibit a delayed response to forest fragmentation.}, language = {en} } @phdthesis{Mutwil2011, author = {Mutwil, Marek}, title = {Integrative transcriptomic approaches to analyzing plant co-expression networks}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-50752}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {It is well documented that transcriptionally coordinated genes tend to be functionally related, and that such relationships may be conserved across different species, and even kingdoms. (Ihmels et al., 2004). Such relationships was initially utilized to reveal functional gene modules in yeast and mammals (Ihmels et al., 2004), and to explore orthologous gene functions between different species and kingdoms (Stuart et al., 2003; Bergmann et al., 2004). Model organisms, such as Arabidopsis, are readily used in basic research due to resource availability and relative speed of data acquisition. A major goal is to transfer the acquired knowledge from these model organisms to species that are of greater importance to our society. However, due to large gene families in plants, the identification of functional equivalents of well characterized Arabidopsis genes in other plants is a non-trivial task, which often returns erroneous or inconclusive results. In this thesis, concepts of utilizing co-expression networks to help infer (i) gene function, (ii) organization of biological processes and (iii) knowledge transfer between species are introduced. An often overlooked fact by bioinformaticians is that a bioinformatic method is as useful as its accessibility. Therefore, majority of the work presented in this thesis was directed on developing freely available, user-friendly web-tools accessible for any biologist.}, language = {en} } @phdthesis{Mittag2011, author = {Mittag, Sonnhild}, title = {Vom Rosetta-Stone-Protein NitFhit zur Tumorsuppressorwirkung der humanen Nitrilase1}, address = {Potsdam}, pages = {113 S.}, year = {2011}, language = {de} } @phdthesis{Massie2011, author = {Massie, Thomas Michael}, title = {Dynamic behavior of phytoplankton populations far from steady state : chemostat experiments and mathematical modeling}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-58102}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Nature changes continuously and is only seemingly at equilibrium. Environmental parameters like temperature, humidity or insolation may strongly fluctuate on scales ranging from seconds to millions of years. Being part of an ecosystem, species have to cope with these environmental changes. For ecologists, it is of special interest how individual responses to environmental changes affect the dynamics of an entire population - and, if this behavior is predictable. In this context, the demographic structure of a population plays a decisive role since it originates from processes of growth and mortality. These processes are fundamentally influenced by the environment. But, how exactly does the environment influence the behavior of populations? And what does the transient behavior look like? As a result from environmental influences on demography, so called cohorts form. They are age or size classes that are disproportionally represented in the demographic distribution of a population. For instance, if most old and young individuals die due to a cold spell, the population finally consists of mainly middle-aged individuals. Hence, the population got synchronized. Such a population tends to show regular fluctuations in numbers (denoted as oscillations) since the alternating phases of individual growth and population growth (due to reproduction) are now performed synchronously by the majority of the population.That is, one time the population growths, and the other time it declines due to mortality. Synchronous behavior is one of the most pervasive phenomena in nature. Gravitational synchrony in the solar system; fireflies flashing in unison; coordinate firing of pacemaker cells in the heart; electrons in a superconductor marching in lockstep. Whatever scale one looks at, in animate as well as inanimate systems, one is likely to encounter synchrony. In experiments with phytoplankton populations, I could show that this principle of synchrony (as used by physicists) could well-explain the oscillations observed in the experiments, too. The size of the fluctuations depended on the strength by which environmental parameters changed as well as on the demographic state of a population prior to this change. That is, two population living in different habitats can be equally influenced by an environmental change, however, the resulting population dynamics may be significantly different when both populations differed in their demographic state before. Moreover, specific mechanisms relevant for the dynamic behavior of populations, appear only when the environmental conditions change. In my experiments, the population density declined by 50\% after ressource supply was doubled. This counter-intuitive behavior can be explained by increasing ressource consumption. The phytoplankton cells grew larger and enhanced their individual constitution. But at the same time, reproduction was delayed and the population density declined due to the losses by mortality. Environmental influences can also synchronize two or more populations over large distances, which is denoted as Moran effect. Assume two populations living on two distant islands. Although there is no exchange of individuals between them, both populations show a high similarity when comparing their time series. This is because the globally acting climate synchronizes the regionally acting weather on both island. Since the weather fluctuations influence the population dynamics, the Moran effect states that the synchrony between the environment equals the one between the populations. My experiments support this theory and also explain deviations arising when accounting for differences in the populations and the habitats they are living in. Moreover, model simulations and experiments astonishingly show that the synchrony between the populations can be higher than between the environment, when accounting for differences in the environmental fluctuations ("noise color").}, language = {de} } @phdthesis{Li2011, author = {Li, Xiaoqing}, title = {Structural and dynamic analysis of circadian oscillators and modelling seasonal response in Soay sheep}, address = {Potsdam}, pages = {163 S.}, year = {2011}, language = {en} } @phdthesis{Lehmann2011, author = {Lehmann, Martin}, title = {Back to the roots : regulation of arabidopsis root metabolism during oxidative stress}, address = {Potsdam}, pages = {154 S.}, year = {2011}, language = {en} } @phdthesis{Lachmuth2011, author = {Lachmuth, Susanne}, title = {Towards a mechanistic understanding of how demography, genetic differentiation and environmental factors interact to generate the invasion dynamics of senecio inaequidens}, address = {Potsdam}, pages = {172 S.}, year = {2011}, language = {en} } @phdthesis{Krueger2011, author = {Kr{\"u}ger, Anne}, title = {Molekulare Charakterisierung von NE81 und CP75, zwei kernh{\"u}llen- und centrosomassoziierten Proteinen in Dictyostelium discoideum}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-53915}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Lamine bilden zusammen mit laminassoziierten Proteinen die nukle{\"a}re Lamina. Diese ist notwendig f{\"u}r die mechanische Stabilit{\"a}t von Zellen, die Organisation des Chromatins, der Genexpression, dem Fortgang des Zellzyklus und der Zellmigration. Die vielf{\"a}ltigen Funktionen der Lamine werden durch die Pathogenese von Laminopathien belegt. Zu diesen Erkrankungen, welche ihre Ursache in Mutationen innerhalb der laminkodierenden Gene, oder der Gene laminassoziierter bzw. laminprozessierender Proteine haben, z{\"a}hlen unter anderem das „Hutchinson-Gilford Progerie Syndrom", die „Emery-Dreifuss" Muskeldystrophie und die dilatierte Kardiomyopathie. Trotz der fundamentalen Bedeutung der Lamine, wurden diese bisher nur in Metazoen und nicht in einzelligen Organismen detektiert. Der am{\"o}bide Organismus Dictyostelium discoideum ist ein haploider Eukaryot, der h{\"a}ufig als Modellorganismus in den verschiedensten Bereichen der Zellbiologie eingesetzt wird. Mit der Entdeckung von NE81, einem Protein das mit der inneren Kernh{\"u}lle von Dictyostelium discoideum assoziiert ist, wurde erstmals ein Protein identifiziert, dass man aufgrund seiner Eigenschaften als lamin{\"a}hnliches Protein in einem niederen Eukaryoten bezeichnen kann. Diese Merkmale umfassen die Existenz lamintypischer Sequenzen, wie die CDK1-Phosphorylierungsstelle, direkt gefolgt von einer zentralen „Rod"-Dom{\"a}ne, sowie eine typische NLS und die hoch konservierte CaaX-Box. F{\"u}r die Etablierung des NE81 als „primitives" Lamin, wurden im Rahmen dieser Arbeit verschiedene Experimente durchgef{\"u}hrt, die strukturelle und funktionelle Gemeinsamkeiten zu den Laminen in anderen Organismen aufzeigen konnten. Die Herstellung eines polyklonalen Antik{\"o}rpers erm{\"o}glichte die Verifizierung der subzellul{\"a}ren Lokalisation des NE81 durch Elektronenmikroskopie und gab Einblicke in das Verhalten des endogenen Proteins innerhalb des Zellzyklus. Mit der Generierung von NE81-Nullmutanten konnte demonstriert werden, dass NE81 eine wichtige Rolle bei der nukle{\"a}ren Integrit{\"a}t und der Chromatinorganisation von Zellen spielt. Des Weiteren f{\"u}hrte die Expression von zwei CaaX-Box deletierten NE81 - Varianten dazu, den Einfluss des Proteins auf die mechanische Stabilit{\"a}t der Zellen nachweisen zu k{\"o}nnen. Auch die Bedeutung der hochkonservierten CaaX-Box f{\"u}r die Lokalisation des Proteins wurde durch die erhaltenen Ergebnisse deutlich. Mit der Durchf{\"u}hrung von FRAP-Experimente konnte außerdem die strukturgebende Funktion von NE81 innerhalb des Zellkerns bekr{\"a}ftigt werden. Zus{\"a}tzlich wurde im Rahmen dieser Arbeit damit begonnen, den Einfluss der Isoprenylcysteincarboxylmethyltransferase auf die Lokalisation des Proteins aufzukl{\"a}ren. Die Entdeckung eines lamin{\"a}hnlichen Proteins in einem einzelligen Organismus, der an der Schwelle zu den Metazoen steht, ist f{\"u}r die evolution{\"a}re Betrachtung der Entwicklung der sozialen Am{\"o}be und f{\"u}r die Erforschung der molekularen Basis von Laminopathien in einem einfachen Modellorganismus sehr interessant. Die Arbeit mit Dictyostelium discoideum k{\"o}nnte daher Wege aufzeigen, dass Studium der Laminopathien am Tiermodell drastisch zu reduzieren. In den letzten Jahren hat die Erforschung unbekannter Bestandteile des Centrosoms in Dictyostelium discoideum große Fortschritte gemacht. Eine zu diesem Zwecke von unserer Arbeitsgruppe durchgef{\"u}hrte Proteomstudie, f{\"u}hrte zur Identifizierung weiterer, potentiell centrosomaler Kandidatenproteine. Der zweite Teil dieser Arbeit besch{\"a}ftigt sich mit der Charakterisierung eines solchen Kandidatenproteins, dem CP75. Es konnte gezeigt werden, dass CP75 einen echten, centrosomalen Bestandteil darstellt, der mikrotubuli-unabh{\"a}ngig mit der Core Struktur des Zellorganells assoziiert ist. Weiterhin wurde deutlich, dass die Lokalisation am Centrosom in Abh{\"a}ngigkeit vom Zellzyklus erfolgt und CP75 vermutlich mit CP39, einem weiteren centrosomalen Core Protein, interagiert.}, language = {de} } @phdthesis{Krehl2011, author = {Krehl, Susanne}, title = {Das Selenoprotein Glutathionperoxidase-2 : physiologische Funktion und Einfluss auf die entz{\"u}ndungsassoziierte Colonkarzinogenese}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-50220}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Bei der Entdeckung der Glutathionperoxidase-2 (GPx2) wurde zun{\"a}chst davon ausgegangen, dass die Funktion dieses Enzyms im Kryptengrund des Colons einzig in der Reduktion von H2O2 besteht. Im Laufe der weiteren Erforschung zeigte sich, dass GPx2 auch in verschiedenen Tumorgeweben vermehrt exprimiert wird. Dabei wird diskutiert, ob die Wirkung von GPx2 im Tumor eher als pro- oder als antikarzinogen einzustufen ist. Mehrere Experimente in vitro und in vivo zeigten antiinflammatorische Eigenschaften der GPx2. Aufgrund dieser Befunde wird derzeit {\"u}ber weitere Funktionen der GPx2 spekuliert. In dieser Arbeit wurde die physiologische Funktion von GPx2 n{\"a}her erforscht, dazu wurden Wildtyp- und GPx2-Knockout-M{\"a}use in Hinblick auf Ver{\"a}nderungen der Enzymexpression und der Colonmorphologie untersucht. Es wurden drei verschiedene Selendi{\"a}ten verf{\"u}ttert: selenarmes, selenad{\"a}quates und selensupplementiertes Futter. Unter physiologischen Bedingungen ist am Kryptengrund des Colons, innerhalb der proliferierenden Zone, die Mitoserate am h{\"o}chsten. Der Großteil der apoptotischen Zellen ist hingegen an der Kryptenspitze vorzufinden. Durch den Knockout von GPx2 kam es zu einer signifikanten Erh{\"o}hung der Apoptoserate am Kryptengrund. Dabei war der gr{\"o}ßte Effekt auf selenarmem Futter zu verzeichnen. Hierbei wurde sogar eine Ver{\"a}nderung der Colonmorphologie dokumentiert, da die Verschiebung der Proliferationszone in Richtung Kryptenspitze eine Verl{\"a}ngerung der Krypten nach sich zog. Im Wildtyp wurden keine Apoptosen im Kryptengrund detektiert. GPx1 wird unter physiologischen Bedingungen im Gegensatz zur GPx2 in der Kryptenspitze exprimiert und ist im Selenmangel nicht mehr detektierbar. Der Knockout von GPx2 erh{\"o}hte die GPx1-Expression im Kryptengrund auf allen drei Selendi{\"a}ten. Diese {\"U}berexpression von GPx1 am Kryptengrund soll vermutlich den Verlust von GPx2 an dieser Stelle kompensieren. Da jedoch dort die massive Apoptoserate detektiert wurde, kann die GPx1 nicht die komplette Funktion von GPx2 kompensieren. Diese Ergebnisse deuten darauf hin, dass die Funktion von GPx2 nicht nur in der Reduktion von H2O2 liegt. Vielmehr kann eine Rolle bei der Aufrechterhaltung der Hom{\"o}ostase von Zellen postuliert werden. Ein weiterer Bestandteil dieser Arbeit war die Kl{\"a}rung der Frage, welchen Einfluss GPx2 auf die entz{\"u}ndungsassoziierte Colonkarzinogenese aus{\"u}bt. In dem hierf{\"u}r verwendeten AOM/DSS-Model wird der karzinogene Prozess durch Entz{\"u}ndung vorangetrieben. Es erfolgte sowohl im Wildtyp als auch im GPx2-Knockout zum einen die Bewertung des Entz{\"u}ndungsstatus des Colons und zum anderen wurde die Anzahl von ACF und Tumoren verglichen. Das Colon im GPx2-Knockout war wesentlich st{\"a}rker entz{\"u}ndet als im Wildtyp. Diese Ergebnisse best{\"a}tigen die f{\"u}r die GPx2 postulierte antiinflammatorische Funktion. Normalerweise f{\"u}hrt eine Erh{\"o}hung der Mitoseanzahl zur Regeneration des entz{\"u}ndeten Gewebes. Jedoch beeinflusst der Verlust von GPx2 vermutlich den Ablauf der Entz{\"u}ndung, indem beispielsweise die Regeneration des Gewebes durch die enorm hohe Apoptoserate am Kryptengrund verlangsamt wird. Des Weiteren hatten sich im GPx2-Knockout tendenziell mehr Tumore entwickelt. Somit korrelierte die Entz{\"u}ndung des Colons mit der Entwicklung von Tumoren. Der Verlust von GPx2 beg{\"u}nstigte vermutlich sowohl die Tumorinitiation als auch die Tumorprogression. Allerdings stimulierte die Expression von GPx2 ebenfalls das Tumorwachstum. Es kann geschlussfolgert werden, dass eine ad{\"a}quate GPx2-Expression vor Entz{\"u}ndung sch{\"u}tzt und somit das Risiko f{\"u}r Colonkrebs senkt. Ob GPx2 aber insgesamt pro- oder antikarzinogen wirkt, h{\"a}ngt vermutlich vom Stadium des Colonkarzinogenese ab.}, language = {de} } @phdthesis{Klie2011, author = {Klie, Sebastian}, title = {Integrative analysis of hight-throughput "omics"-data and structured biological knowledge}, address = {Potsdam}, pages = {102 S.}, year = {2011}, language = {en} } @phdthesis{Kittner2011, author = {Kittner, Madeleine}, title = {Folding and aggregation of amyloid peptides}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-53570}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Aggregation of the Amyloid β (Aβ) peptide to amyloid fibrils is associated with the outbreak of Alzheimer's disease. Early aggregation intermediates in form of soluble oligomers are of special interest as they are believed to be the major toxic components in the process. These oligomers are of disordered and transient nature. Therefore, their detailed molecular structure is difficult to access experimentally and often remains unknown. In the present work extensive, fully atomistic replica exchange molecular dynamics simulations were performed to study the preaggregated, monomer states and early aggregation intermediates (dimers, trimers) of Aβ(25-35) and Aβ(10-35)-NH2 in aqueous solution. The folding and aggregation of Aβ(25-35) were studied at neutral pH and 293 K. Aβ(25-35) monomers mainly adopt β-hairpin conformations characterized by a β-turn formed by residues G29 and A30, and a β-sheet between residues N27-K28 and I31-I32 in equilibrium with coiled conformations. The β-hairpin conformations served as initial configurations to model spontaneous aggregation of Aβ(25-35). As expected, within the Aβ(25-35) dimer and trimer ensembles many different poorly populated conformations appear. Nevertheless, we were able to distinguish between disordered and fibril-like oligomers. Whereas disordered oligomers are rather compact with few intermolecular hydrogen bonds (HBs), fibril-like oligomers are characterized by the formation of large intermolecular β-sheets. In most of the fibril-like dimers and trimers individual peptides are fully extended forming in- or out-of-register antiparallel β-sheets. A small amount of fibril-like trimers contained V-shaped peptides forming parallel β-sheets. The dimensions of extended and V-shaped oligomers correspond well to the diameters of two distinct morphologies found for Aβ(25-35) fibrils. The transition from disordered to fibril-like Aβ(25-35) dimers is unfavorable but driven by energy. The lower energy of fibril-like dimers arises from favorable intermolecular HBs and other electrostatic interactions which compete with a loss in entropy. Approximately 25 \% of the entropic cost correspond to configurational entropy. The rest relates to solvent entropy, presumably caused by hydrophobic and electrostatic effects. In contrast to the transition towards fibril-like dimers the first step of aggregation is driven by entropy. Here, we compared structural and thermodynamic properties of the individual monomer, dimer and trimer ensembles to gain qualitative information about the aggregation process. The β-hairpin conformation observed for monomers is successively dissolved in dimer and trimer ensembles while instead intermolecular β-sheets are formed. As expected upon aggregation the configurational entropy decreases. Additionally, the solvent accessible surface area (SASA), especially the hydrophobic SASA, decreases yielding a favorable solvation free energy which overcompensates the loss in configurational entropy. In summary, the hydrophobic effect, possibly combined with electrostatic effects, yields an increase in solvent entropy which is believed to be one major driving force towards aggregation. Spontaneous folding of the Aβ(10-35)-NH2 monomer was modeled using two force fields, GROMOS96 43a1 and OPLS/AA, and compared to primary NMR data collected at pH 5.6 and 283 K taken from the literature. Unexpectedly, the two force fields yielded significantly different main conformations. Comparison between experimental and calculated nuclear Overhauser effect (NOE) distances is not sufficient to distinguish between the different force fields. Additionally, the comparison with scalar coupling constants suggest that the chosen protonation in both simulations corresponds to a pH lower than in the experiment. Based on this analysis we were unable to determine which force field yields a better description of this system. Dimerization of Aβ(10-35)-NH2 was studied at neutral pH and 300 K. Dimer conformations arrange in many distinct, poorly populated and rather complex alignments or interlocking patterns which are rather stabilized by side chain interactions than by specific intermolecular hydrogen bonds. Similar to Aβ(25-35) dimers, transition towards β-sheet-rich, fibril-like Aβ(10-35) dimers is driven by energy competing with a loss in entropy. Here, transition is mediated by favorable peptide-solvent and solvent-solvent interactions mainly arising from electrostatic interactions.}, language = {en} } @phdthesis{Kartal2011, author = {Kartal, {\"O}nder}, title = {The role of interfacial and 'entropic' enzymes in transitory starch degradation : a mathematical modeling approach}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-53947}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Plants and some unicellular algae store carbon in the form of transitory starch on a diurnal basis. The turnover of this glucose polymer is tightly regulated and timely synthesis as well as mobilization is essential to provide energy for heterotrophic growth. Especially for starch degradation, novel enzymes and mechanisms have been proposed recently. However, the catalytic properties of these enzymes and their coordination with metabolic regulation are still to be discovered. This thesis develops theoretical methods in order to interpret and analyze enzymes and their role in starch degradation. In the first part, a novel description of interfacial enzyme catalysis is proposed. Since the initial steps of starch degradation involve reactions at the starch-stroma interface it is necessary to have a framework which allows the derivation of interfacial enzyme rate laws. A cornerstone of the method is the introduction of the available area function - a concept from surface physics - to describe the adsorption step in the catalytic cycle. The method is applied to derive rate laws for two hydrolases, the Beta-amylase (BAM3) and the Isoamylase (DBE/ISA3), as well as to the Glucan, water dikinase (GWD) and a Phosphoglucan phosphatase (DSP/SEX4). The second part uses the interfacial rate laws to formulate a kinetic model of starch degradation. It aims at reproducing the stimulatory effect of reversible phosphorylation by GWD and DSP on the breakdown of the granule. The model can describe the dynamics of interfacial properties during degradation and suggests that interfacial amylopectin side-chains undergo spontaneous helix-coil transitions. Reversible phosphorylation has a synergistic effect on glucan release especially in the early phase dropping off during degradation. Based on the model, the hypothesis is formulated that interfacial phosphorylation is important for the rapid switch from starch synthesis to starch degradation. The third part takes a broader perspective on carbohydrate-active enzymes (CAZymes) but is motivated by the organization of the downstream pathway of starch breakdown. This comprises Alpha-1,4-glucanotransferases (DPE1 and DPE2) and Alpha-glucan-phosphorylases (Pho or PHS) both in the stroma and in the cytosol. CAZymes accept many different substrates and catalyze numerous reactions and therefore cannot be characterized in classical enzymological terms. A concise characterization is provided by conceptually linking statistical thermodynamics and polymer biochemistry. Each reactant is interpreted as an energy level, transitions between which are constrained by the enzymatic mechanisms. Combinations of in vitro assays of polymer-active CAZymes essential for carbon metabolism in plants confirmed the dominance of entropic gradients. The principle of entropy maximization provides a generalization of the equilibrium constant. Stochastic simulations confirm the results and suggest that randomization of metabolites in the cytosolic pool of soluble heteroglycans (SHG) may contribute to a robust integration of fluctuating carbon fluxes coming from chloroplasts.}, language = {en} } @phdthesis{Ivakov2011, author = {Ivakov, Alexander}, title = {Metabolic interactions in leaf development in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-59730}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Das Wachstum und {\"U}berleben von Pflanzen basiert auf der Photosynthese in den Bl{\"a}ttern. Diese beinhaltet die Aufnahme von Kohlenstoffdioxid aus der Atmosph{\"a}re und das simultane Einfangen von Lichtenergie zur Bildung organischer Molek{\"u}le. Diese werden nach dem Eintritt in den Metabolismus in viele andere Komponenten umgewandelt, welche die Grundlage f{\"u}r die Zunahme der Biomasse bilden. Bl{\"a}tter sind Organe, die auf die Fixierung von Kohlenstoffdioxid spezialisiert sind. Die Funktionen der Bl{\"a}tter beinhalten vor allem die Optimierung und Feinregulierung vieler Prozesse, um eine effektive Nutzung von Ressourcen und eine maximale Photosynthese zu gew{\"a}hrleisten. Es ist bekannt, dass sich die Morphologie der Bl{\"a}tter den Wachstumsbedingungen der Pflanze anpasst und eine wichtige Rolle bei der Optimierung der Photosynthese spielt. Trotzdem ist die Regulation dieser Art der Anpassung bisher nicht verstanden. Die allgemeine Zielsetzung dieser vorliegenden Arbeit ist das Verst{\"a}ndnis wie das Wachstum und die Morphologie der Bl{\"a}tter im Modellorganismus Arabidopsis thaliana reguliert werden. Besondere Aufmerksamkeit wurde hierbei der M{\"o}glichkeit geschenkt, dass es interne metabolische Signale in der Pflanze geben k{\"o}nnte, die das Wachstum und die Entwicklung von Bl{\"a}ttern beeinflussen. Um diese Fragestellung zu untersuchen, muss das Wachstum und die Entwicklung von Bl{\"a}ttern oberhalb des Levels des einzelnen Organs und im Kontext der gesamten Pflanze betrachtet werden, weil Bl{\"a}tter nicht eigenst{\"a}ndig wachsen, sondern von Ressourcen und regulatorischen Einfl{\"u}ssen der ganzen Pflanze abh{\"a}ngig sind. Aufgrund der Komplexit{\"a}t dieser Fragestellung wurden drei komplement{\"a}re Ans{\"a}tze durchgef{\"u}hrt. Im ersten und spezifischsten Ansatz wurde untersucht ob eine flussabw{\"a}rts liegende Komponente des Zucker-Signalwegs, Trehalose-6-Phosphat (Tre-6-P), das Blattwachstum und die Blattentwicklung beinflussen kann. Um diese Frage zu beantworten wurden transgene Arabidopsis-Linien mit einem gest{\"o}rten Gehalt von Tre-6-P durch die Expression von bakteriellen Proteinen die in dem metabolismus von trehalose beteiligt sind. Die Pflanzen-Linien wurden unter Standard-Bendingungen in Erde angebaut und ihr Metabolismus und ihre Blattmorphologie untersucht. Diese Experimente f{\"u}hrten auch zu einem unerwarteten Projekt hinsichtlich einer m{\"o}glichen Rolle von Tre-6-P in der Regulation der Stomata. In einem zweiten, allgemeineren Ansatz wurde untersucht, ob {\"A}nderungen im Zucker-Gehalt der Pflanzen die Morphogenese der Bl{\"a}tter als Antwort auf Licht beeinflussen. Dazu wurden eine Reihe von Mutanten, die im Zentralmetabolismus beeintr{\"a}chtigt sind, in derselben Lichtbedingung angezogen und bez{\"u}glich ihrer Blattmorphologie analysiert. In einem dritten noch allgemeineren Ansatz wurde die nat{\"u}rliche Variation von morphologischen Auspr{\"a}gungen der Bl{\"a}tter und Rosette anhand von wilden Arabidopsis {\"O}kotypen untersucht, um zu verstehen wie sich die Blattmorphologie auf die Blattfunktion und das gesamte Pflanzenwachstum auswirkt und wie unterschiedliche Eigenschaften miteinander verkn{\"u}pft sind. Das Verh{\"a}ltnis der Blattanzahl zum Gesamtwachstum der Pflanze und Blattgr{\"o}ße wurde gesondert weiter untersucht durch eine Normalisierung der Blattanzahl auf das Frischgewicht der Rosette, um den Parameter „leafing Intensity" abzusch{\"a}tzen. Leafing Intensity integrierte Blattanzahl, Blattgr{\"o}ße und gesamtes Rosettenwachstum in einer Reihe von Kompromiss-Interaktionen, die in einem Wachstumsvorteil resultieren, wenn Pflanzen weniger, aber gr{\"o}ßere Bl{\"a}tter pro Einheit Biomasse ausbilden. Dies f{\"u}hrte zu einem theoretischen Ansatz in dem ein einfaches allometrisch mathematisches Modell konstruiert wurde, um Blattanzahl, Blattgr{\"o}ße und Pflanzenwachstum im Kontext der gesamten Pflanze Arabidopsis zu verkn{\"u}pfen.}, language = {en} } @phdthesis{Hojka2011, author = {Hojka, Marta}, title = {Lifetimes and biogenesis of photosynthetic complexes in higher plants (Nicotiana tabacum)}, address = {Potsdam}, pages = {138 S.}, year = {2011}, language = {en} } @phdthesis{Hartmann2011, author = {Hartmann, Stefanie}, title = {Phylogenomics: comparative genome analysis ursing large-scale gene family data}, address = {Potsdam}, year = {2011}, language = {en} } @phdthesis{Guenl2011, author = {G{\"u}nl, Markus}, title = {Impact of apoplastic glycoside hydrolases on xyloglucan structure and function in arabidopsis}, address = {Potsdam}, pages = {145 S.}, year = {2011}, language = {en} } @phdthesis{Griessner2011, author = {Grießner, Matthias}, title = {Grenzfl{\"a}chenmodifizierung von Mikrosystemen f{\"u}r biochemische Assays}, address = {Potsdam}, pages = {95 S.}, year = {2011}, language = {de} } @phdthesis{Giorgi2011, author = {Giorgi, Federico Manuel}, title = {Expression-based reverse engineering of plant transcriptional networks}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-56760}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Regulation of gene transcription plays a major role in mediating cellular responses and physiological behavior in all known organisms. The finding that similar genes are often regulated in a similar manner (co-regulated or "co-expressed") has directed several "guilt-by-association" approaches in order to reverse-engineer the cellular transcriptional networks using gene expression data as a compass. This kind of studies has been considerably assisted in the recent years by the development of high-throughput transcript measurement platforms, specifically gene microarrays and next-generation sequencing. In this thesis, I describe several approaches for improving the extraction and interpretation of the information contained in microarray based gene expression data, through four steps: (1) microarray platform design, (2) microarray data normalization, (3) gene network reverse engineering based on expression data and (4) experimental validation of expression-based guilt-by-association inferences. In the first part test case is shown aimed at the generation of a microarray for Thellungiella salsuginea, a salt and drought resistant close relative to the model plant Arabidopsis thaliana; the transcripts of this organism are generated on the combination of publicly available ESTs and newly generated ad-hoc next-generation sequencing data. Since the design of a microarray platform requires the availability of highly reliable and non-redundant transcript models, these issues are addressed consecutively, proposing several different technical solutions. In the second part I describe how inter-array correlation artifacts are generated by the common microarray normalization methods RMA and GCRMA, together with the technical and mathematical characteristics underlying the problem. A solution is proposed in the form of a novel normalization method, called tRMA. The third part of the thesis deals with the field of expression-based gene network reverse engineering. It is shown how different centrality measures in reverse engineered gene networks can be used to distinguish specific classes of genes, in particular essential genes in Arabidopsis thaliana, and how the use of conditional correlation can add a layer of understanding over the information flow processes underlying transcript regulation. Furthermore, several network reverse engineering approaches are compared, with a particular focus on the LASSO, a linear regression derivative rarely applied before in global gene network reconstruction, despite its theoretical advantages in robustness and interpretability over more standard methods. The performance of LASSO is assessed through several in silico analyses dealing with the reliability of the inferred gene networks. In the final part, LASSO and other reverse engineering methods are used to experimentally identify novel genes involved in two independent scenarios: the seed coat mucilage pathway in Arabidopsis thaliana and the hypoxic tuber development in Solanum tuberosum. In both cases an interesting method complementarity is shown, which strongly suggests a general use of hybrid approaches for transcript expression-based inferences. In conclusion, this work has helped to improve our understanding of gene transcription regulation through a better interpretation of high-throughput expression data. Part of the network reverse engineering methods described in this thesis have been included in a tool (CorTo) for gene network reverse engineering and annotated visualization from custom transcription datasets.}, language = {en} } @phdthesis{Fettke2011, author = {Fettke, J{\"o}rg}, title = {Analysen St{\"a}rke-bezogener Kohlenstofffl{\"u}sse}, address = {Potsdam}, year = {2011}, language = {de} } @phdthesis{Dimova2011, author = {Dimova, Rumiana}, title = {Probing the membrane nanoregime with optical microscopy}, address = {Potsdam}, pages = {50 S.}, year = {2011}, language = {en} } @phdthesis{Devers2011, author = {Devers, Emanuel}, title = {Phosphate homeostasis and novel microRNAs are involved in the regulation of the arbuscular mycorrhizal symbiosis in Medicago truncatula}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-55572}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Die arbuskul{\"a}re Mykorrhiza ist die wahrscheinlich {\"a}lteste Form der Wurzelsymbiosen zwischen Pflanzen und Pilzen und hat sich vor 420 Millionen Jahren entwickelt. In dieser Symbiose, die zwischen nahezu allen Landpflanzen und Pilzen des Reiches Glomeromycota ausgebildet wird, versorgt der Pilz die Pflanze mit N{\"a}hrstoffen, wobei die verbesserte Versorgung mit Phosphat f{\"u}r die Pflanze sicher den gr{\"o}ßten Vorteil darstellt. Im Gegenzug erh{\"a}lt der Pilz Zucker, welche die Pflanze aus der Photosynthese bereitstellt. Zu hohe Phosphatkonzentrationen im Boden oder D{\"u}nger f{\"u}hren allerdings zu einer Verringerung in der Auspr{\"a}gung der arbuskul{\"a}ren Mykorrhiza. Diese Unterdr{\"u}ckung der Symbiose wird nicht durch eine lokale Reaktion der Wurzeln ausgel{\"o}st, sondern in erster Linie durch einen hohen Phosphatgehalt im Pflanzenspross. Somit handelt es sich also um eine systemische, also dem Gesamtsystem „Pflanze" betreffende Antwort. Die molekularen Mechanismen dieser Anpassung sind noch wenig bekannt und sind vor allem f{\"u}r die Agrarwirtschaft von besonderem Interesse. Eine Mikro-RNA (miRNA) des bereits bekannten Phosphathom{\"o}ostasesignalwegs (PHR1-miRNA399-PHO2 Signalweg) akkumuliert verst{\"a}rkt in mykorrhizierten Wurzeln. Das deutet daraufhin, dass dieser Signalweg und diese miRNA eine wichtige Rolle in der Regulation der arbuskul{\"a}ren Mykorrhiza spielen. Ziel dieser Studie war es neue Einblicke in die molekularen Mechanismen, die zur Unterdr{\"u}ckung der arbuskul{\"a}ren Mykorrhiza bei hohen Phosphatkonzentrationen f{\"u}hren, zu gewinnen. Dabei sollte der Einfluss von PHO2, sowie von miRNAs in dieser Symbiose genauer untersucht werden. Ein funktionelles Ortholog von PHO2, MtPho2, wurde in der Pflanze Medicago truncatula identifiziert. MtPho2-Mutanten, welche nicht mehr in der Lage waren ein funktionales PHO2 Protein zu exprimieren, zeigten schnellere Kolonisierung durch den AM-Pilz. Jedoch wurde auch in den mtpho2-Mutanten die Symbiose durch hohe Phosphatkonzentrationen unterdr{\"u}ckt. Dies bedeutet, dass PHO2 und somit der PHR1-miRNA399-PHO2 Signalweg eine wichtige Funktion w{\"a}hrend der fortschreitenden Kolonisierung der Wurzel durch den Pilz hat, aber und weitere Mechanismen in der Unterd{\"u}ckung der Symbiose bei hohen Phosphatkonzentrationen beteiligt sein m{\"u}ssen. Die Analyse von Transkriptionsprofilen von Spross- und Wurzeln mittels Microarrays zeigte, dass die Unterdr{\"u}ckung der AM Symbiose durch hohe Phosphatkonzentrationen m{\"o}glicherweise auf eine Unterdr{\"u}ckung der Expression einer Reihe symbiosespezifischer Gene im Spross der Pflanze beruht. Um die Rolle weiterer miRNA in der AM Symbiose zu untersuchen, wurden mittels einer Hochdurchsatz-Sequenzierung 243 neue und 181 aus anderen Pflanzen bekannte miRNAs in M. truncatula entdeckt. Zwei dieser miRNAs, miR5229 und miR160f*, sind ausschließlich w{\"a}hrend der arbuskul{\"a}ren Mykorrhiza zu finden und weitere miRNAs werden w{\"a}hrend dieser Symbiose verst{\"a}rkt gebildet. Interessanterweise f{\"u}hren einige dieser miRNAs zum Abbau von Transkripten, die eine wichtige Funktion in der arbuskul{\"a}ren Mykorrhiza und Wurzelkn{\"o}llchensymbiose besitzen. Die Ergebnisse dieser Studie liefern eine neue Grundlage f{\"u}r die Untersuchung von regulatorischen Netzwerken, die zur zellul{\"a}ren Umprogrammierung w{\"a}hrend der Interaktion zwischen Pflanzen und arbuskul{\"a}ren Mykorrhiza-Pilzen bei verschiedenen Phosphatbedingungen f{\"u}hren.}, language = {en} } @phdthesis{CuadrosInostroza2011, author = {Cuadros-Inostroza, Alvaro}, title = {Integrated analysis of transcriptome and metabolome to understand grapevine development}, address = {Potsdam}, pages = {136 S.}, year = {2011}, language = {en} } @phdthesis{CastroPrieto2011, author = {Castro Prieto, Aines del Carmen}, title = {Immunogenetics of free-ranging felids on Namibian farmlands}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-55505}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Genetic variation is crucial for the long-term survival of the species as it provides the potential for adaptive responses to environmental changes such as emerging diseases. The Major Histocompatibility Complex (MHC) is a gene family that plays a central role in the vertebrate's immune system by triggering the adaptive immune response after exposure to pathogens. MHC genes have become highly suitable molecular markers of adaptive significance. They synthesize two primary cell surface molecules namely MHC class I and class II that recognize short fragments of proteins derived respectively from intracellular (e.g. viruses) and extracellular (e.g. bacteria, protozoa, arthropods) origins and present them to immune cells. High levels of MHC polymorphism frequently observed in natural populations are interpreted as an adaptation to detect and present a wide array of rapidly evolving pathogens. This variation appears to be largely maintained by positive selection driven mainly by pathogenic selective pressures. For my doctoral research I focused on MHC I and II variation in free-ranging cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) on Namibian farmlands. Both felid species are sympatric thus subject to similar pathogenic pressures but differ in their evolutionary and demographic histories. The main aims were to investigate 1) the extent and patterns of MHC variation at the population level in both felids, 2) the association between levels of MHC variation and disease resistance in free-ranging cheetahs, and 3) the role of selection at different time scales in shaping MHC variation in both felids. Cheetahs and leopards represent the largest free-ranging carnivores in Namibia. They concentrate in unprotected areas on privately owned farmlands where domestic and other wild animals also occur and the risk of pathogen transmission is increased. Thus, knowledge on adaptive genetic variation involved in disease resistance may be pertinent to both felid species' conservation. The cheetah has been used as a classic example in conservation genetics textbooks due to overall low levels of genetic variation. Reduced variation at MHC genes has been associated with high susceptibility to infectious diseases in cheetahs. However, increased disease susceptibility has only been observed in captive cheetahs whereas recent studies in free-ranging Namibian cheetahs revealed a good health status. This raised the question whether the diversity at MHC I and II genes in free-ranging cheetahs is higher than previously reported. In this study, a total of 10 MHC I alleles and four MHC II alleles were observed in 149 individuals throughout Namibia. All alleles but one likely belong to functional MHC genes as their expression was confirmed. The observed alleles belong to four MHC I and three MHC II genes in the species as revealed by phylogenetic analyses. Signatures of historical positive selection acting on specific sites that interact directly with pathogen-derived proteins were detected in both MHC classes. Furthermore, a high genetic differentiation at MHC I was observed between Namibian cheetahs from east-central and north-central regions known to differ substantially in exposure to feline-specific viral pathogens. This suggests that the patterns of MHC I variation in the current population mirrors different pathogenic selective pressure imposed by viruses. Cheetahs showed low levels of MHC diversity compared with other mammalian species including felids, but this does not seem to influence the current immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease susceptibility. However, it cannot be ruled out that the low MHC variation might limit a prosperous immunocompetence in the case of an emerging disease scenario because none of the remaining alleles might be able to recognize a novel pathogen. In contrast to cheetahs, leopards occur in most parts of Africa being perhaps the most abundant big cat in the continent. Leopards seem to have escaped from large-scale declines due to epizootics in the past in contrast to some free-ranging large carnivore populations in Africa that have been afflicted by epizootics. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, I characterized genetic variation at MHC I and MHC II genes in free-ranging leopards from Namibia. A total of six MHC I and six MHC II sequences were detected in 25 individuals from the east-central region. The maximum number of sequences observed per individual suggests that they likely correspond to at least three MHC I and three MHC II genes. Hallmarks of MHC evolution were confirmed such as historical positive selection, recombination and trans-species polymorphism. The low MHC variation detected in Namibian leopards is not conclusive and further research is required to assess the extent of MHC variation in different areas of its geographic range. Results from this thesis will contribute to better understanding the evolutionary significance of MHC and conservation implications in free-ranging felids. Translocation of wildlife is an increasingly used management tool for conservation purposes that should be conducted carefully as it may affect the ability of the translocated animals to cope with different pathogenic selective pressures.}, language = {en} } @phdthesis{CamaraMattosMartins2011, author = {Camara Mattos Martins, Marina}, title = {What are the downstream targets of trehalose-6-phosphate signalling in plants?}, address = {Potsdam}, pages = {164 S.}, year = {2011}, language = {en} } @phdthesis{Borwankar2011, author = {Borwankar, Tejas}, title = {Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1}, address = {Potsdam}, pages = {120 S.}, year = {2011}, language = {en} } @phdthesis{Baumgartner2011, author = {Baumgartner, Jens}, title = {Nucleation and Growth of Magnetite Nanoparticles under Biomimetric Conditions}, address = {Potsdam}, pages = {102 S.}, year = {2011}, language = {en} } @phdthesis{Bauer2011, author = {Bauer, Barbara}, title = {The relevance of species traits for predicting the dynamics of diverse plankton communities}, address = {Potsdam}, pages = {190 S.}, year = {2011}, language = {en} } @phdthesis{Bandholtz2011, author = {Bandholtz, Sebastian}, title = {Entwicklung von Peptid-Analoga f{\"u}r die Rezeptor-vermittelte Tumordiagnostik}, address = {Potsdam}, pages = {133 S.}, year = {2011}, language = {de} } @phdthesis{Athikomrattanakul2011, author = {Athikomrattanakul, Umporn}, title = {Development and characterization of molecularly imprinted polymers as binding elements against nitrofurantoin}, address = {Potsdam}, pages = {97 S.}, year = {2011}, language = {en} } @phdthesis{AndradeLinares2011, author = {Andrade Linares, Diana Roc{\´i}o}, title = {Characterization of tomato root-endophytic fungi and analysis of their effects on plant development, on fruit yield and quality and on interaction with the pathogen Verticillium dahliae}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-51375}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Non-mycorrhizal fungal endophytes are able to colonize internally roots without causing visible disease symptoms establishing neutral or mutualistic associations with plants. These fungi known as non-clavicipitaceous endophytes have a broad host range of monocot and eudicot plants and are highly diverse. Some of them promote plant growth and confer increased abiotic-stress tolerance and disease resistance. According to such possible effects on host plants, it was aimed to isolate and to characterize native fungal root endophytes from tomato (Lycopersicon esculentum Mill.) and to analyze their effects on plant development, plant resistance and fruit yield and quality together with the model endophyte Piriformospora indica. Fifty one new fungal strains were isolated from desinfected tomato roots of four different crop sites in Colombia. These isolates were roughly characterized and fourteen potential endophytes were further analyzed concerning their taxonomy, their root colonization capacity and their impact on plant growth. Sequencing of the ITS region from the ribosomal RNA gene cluster and in-depth morphological characterisation revealed that they correspond to different phylogenetic groups among the phylum Ascomycota. Nine different morphotypes were described including six dark septate endophytes (DSE) that did not correspond to the Phialocephala group. Detailed confocal microscopy analysis showed various colonization patterns of the endophytes inside the roots ranging from epidermal penetration to hyphal growth through the cortex. Tomato pot experiments under glass house conditions showed that they differentially affect plant growth depending on colonization time and inoculum concentration. Three new isolates (two unknown fungal endophyte DSE48, DSE49 and one identified as Leptodontidium orchidicola) with neutral or positiv effects were selected and tested in several experiments for their influence on vegetative growth, fruit yield and quality and their ability to diminish the impact of the pathogen Verticillium dahliae on tomato plants. Although plant growth promotion by all three fungi was observed in young plants, vegetative growth parameters were not affected after 22 weeks of cultivation except a reproducible increase of root diameter by the endophyte DSE49. Additionally, L. orchidicola increased biomass and glucose content of tomato fruits, but only at an early date of harvest and at a certain level of root colonization. Concerning bioprotective effects, the endophytes DSE49 and L. orchidicola decreased significantly disease symptoms caused by the pathogen V. dahliae, but only at a low dosis of the pathogen. In order to analyze, if the model root endophytic fungus Piriformospora indica could be suitable for application in production systems, its impact on tomato was evaluated. Similarly to the new fungal isolates, significant differences for vegetative growth parameters were only observable in young plants and, but protection against V. dahliae could be seen in one experiment also at high dosage of the pathogen. As the DSE L. orchidicola, P. indica increased the number and biomass of marketable tomatoes only at the beginning of fruit setting, but this did not lead to a significant higher total yield. If the effects on growth are due to a better nutrition of the plant with mineral element was analyzed in barley in comparison to the arbuscular mycorrhizal fungus Glomus mosseae. While the mycorrhizal fungus increased nitrogen and phosphate uptake of the plant, no such effect was observed for P. indica. In summary this work shows that many different fungal endophytes can be also isolated from roots of crops and, that these isolates can have positive effects on early plant development. This does, however, not lead to an increase in total yield or in improvement of fruit quality of tomatoes under greenhouse conditions.}, language = {en} } @phdthesis{Andorf2011, author = {Andorf, Sandra}, title = {A systems biological approach towards the molecular basis of heterosis in Arabidopsis thaliana}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-51173}, school = {Universit{\"a}t Potsdam}, year = {2011}, abstract = {Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood. In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis. Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon. In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes. This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis. In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches.}, language = {en} }