@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{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{Geissler2008,
  author    = {Geißler, Katja},
  title     = {Lebensstrategien seltener Stromtalpflanzen : aut{\"o}kologische Untersuchung von Cnidium dubium, Gratiola officinalis und Juncus atratus unter besonderer Ber{\"u}cksichtigung ihrer Stressresistenz},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-17468},
  school      = {Universit{\"a}t Potsdam},
  year      = {2008},
  abstract  = {Die vorliegende Dissertation behandelt die {\"O}kologie von Cnidium dubium (Schkuhr) Thell. (Sumpf-Brenndolde), Gratiola officinalis L. (Gottes-Gnadenkraut) und Juncus atratus Krocker (Schwarze Binse), drei gef{\"a}hrdeten Arten, die als sogenannte Stromtalpflanzen in Mitteleuropa in ihrem Vorkommen eng an die Flussauen gebunden sind. Die Arbeit basiert auf verschiedenen Simulationsexperimenten und Feldstudien in der Unteren Havelniederung, einem „Feuchtgebiet von internationaler Bedeutung". Sie behandelt Themenkomplexe wie das Samenbankverhalten, die Samenkeimung, die Stickstofflimitierung, die Konkurrenzkraft, das Verhalten der Pflanzen nach einer Sommertrockenheit und nach einer Winter/Fr{\"u}hjahrs{\"u}berflutung. Ferner widmet sie sich der Populationsbiologie der Arten und dem Verhalten der Pflanzen nach besonderen St{\"o}rungsereignissen wie Mahd, Herbivorie und der Sommerflut 2002. Der Leser erf{\"a}hrt, wie die Pflanzen in verschiedenen Lebensphasen auf die auentypische Umwelt reagieren und erh{\"a}lt umfassende Einblicke in physiologische Mechanismen, die der Anpassung an die typischen Bedingungen einer mitteleurop{\"a}ischen Flussaue dienen. Eine Interpretation der Ergebnisse zeigt auf, welche der spezifischen Eigenschaften zur Gef{\"a}hrdung der drei Stromtalarten beitragen. Die Arbeit ist f{\"u}r den Arten-, Biotop- und Landschaftsschutz interessant. Dar{\"u}ber hinaus bietet sie zahlreiche Ankn{\"u}pfungspunkte zur {\"o}kophysiologischen Grundlagenforschung. Die verst{\"a}rkte Nutzung physiologischer Methoden bei der Kl{\"a}rung {\"o}kologischer Fragestellungen wird angeregt.},
  language  = {de}
}
@phdthesis{CastroMarin2007,
  author    = {Castro Marin, Inmaculada},
  title     = {Nitrate: metabolism and development},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-18827},
  school      = {Universit{\"a}t Potsdam},
  year      = {2007},
  abstract  = {The major aim of this thesis was to study the effect of nitrate on primary metabolism and in development of the model plant Arabidopsis thaliana. The present work has two separate topics. First, to investigate the GDH family, a small gene family at the interface between nitrogen and carbon metabolisms. Second, to investigate the mechanisms whereby nitrogen is regulating the transition to flowering time in Arabidopsis thaliana. To gain more insights into the regulation of primary metabolism by the functional characterization of the glutamate dehydrogenase (GDH) family, an enzyme putatively involved in the metabolism of amino acids and thus suggested to play different and essential roles in carbon and nitrogen metabolism in plants, knock out mutants and transgenic plants carrying RNA interference construct were generated and characterized. The effect of silencing GDH on carbon and nitrogen metabolisms was investigated, especially the level of carbohydrates and the amino acid pool were further analysed. It has been shown that GDH expression is regulated by light and/or sugar status therefore, phenotypic and metabolic analysis were developed in plants grown at different points of the diurnal rhythm and in response to an extended night period. In addition, we are interested in the effect of nutrient availability in the transition from vegetative growth to flowering and especially in nitrate as a metabolite that triggers widespread and coordinated changes in metabolism and development. Nutrient availability has a dramatic effect on flowering time, with a marked delay of flowering when nitrate is supplied (Stitt, 1999). The use of different mutants and transgenic plants impaired in flowering signalling pathways was crucial to evaluate the impact of different nitrate concentrations on flowering time and to better understand the interaction of nitrate-dependent signals with other main flowering signalling pathways. Plants were grown on glutamine as a constitutive source of nitrogen, and the nitrate supply varied. Low nitrate led to earlier flowering. The response to nitrate is accentuated in short days and in the CONSTANS deficient co2 mutant, whereas long days or overexpression of CONSTANS overrides the nitrate response. These results indicate that nitrates acts downstream of the known flowering signalling pathways for photoperiod, autonomy, vernalization and gibberellic acid. Global analyses of gene expression of two independent flowering systems, a light impaired mutant (co2tt4) and a constitutive over-expresser of the potent repressor of flowering (35S::FLC), were to be investigated under two different concentrations of nitrate in order to identify candidate genes that may be involved in the regulation of flowering time by nitrate.},
  language  = {en}
}
@phdthesis{Mubeen2018,
  author    = {Mubeen, Umarah},
  title     = {Regulation of central carbon and nitrogen metabolism by Target of Rapamycin (TOR) kinase in Chlamydomonas reinhardtii},
  school      = {Universit{\"a}t Potsdam},
  pages     = {vii, 153},
  year      = {2018},
  abstract  = {The highly conserved protein complex containing the Target of Rapamycin (TOR) kinase is known to integrate intra- and extra-cellular stimuli controlling nutrient allocation and cellular growth. This thesis describes three studies aimed to understand how TOR signaling pathway influences carbon and nitrogen metabolism in Chlamydomonas reinhardtii. The first study presents a time-resolved analysis of the molecular and physiological features across the diurnal cycle. The inhibition of TOR leads to 50\% reduction in growth followed by nonlinear delays in the cell cycle progression. The metabolomics analysis showed that the growth repression is mainly driven by differential carbon partitioning between anabolic and catabolic processes. Furthermore, the high accumulation of nitrogen-containing compounds indicated that TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression. In the second study the cause of the high accumulation of amino acids is explained. For this purpose, the effect of TOR inhibition on Chlamydomonas was examined under different growth regimes using stable 13C- and 15N-isotope labeling. The data clearly showed that an increased nitrogen uptake is induced within minutes after the inhibition of TOR. Interestingly, this increased N-influx is accompanied by increased activities of nitrogen assimilating enzymes. Accordingly, it was concluded that TOR inhibition induces de-novo amino acid synthesis in Chlamydomonas. The recognition of this novel process opened an array of questions regarding potential links between central metabolism and TOR signaling. Therefore a detailed phosphoproteomics study was conducted to identify the potential substrates of TOR pathway regulating central metabolism. Interestingly, some of the key enzymes involved in carbon metabolism as well as amino acid synthesis exhibited significant changes in the phosphosite intensities immediately after TOR inhibition. Altogether, these studies provide a) detailed insights to metabolic response of Chlamydomonas to TOR inhibition, b) identification of a novel process causing rapid upshifts in amino acid levels upon TOR inhibition and c) finally highlight potential targets of TOR signaling regulating changes in central metabolism. Further biochemical and molecular investigations could confirm these observations and advance the understanding of growth signaling in microalgae.},
  language  = {en}
}
@phdthesis{Riewe2008,
  author    = {Riewe, David},
  title     = {The relevance of adenylate levels and adenylate converting enzymes on metabolism and development of potato (Solanum tuberosum L.) tubers},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-27323},
  school      = {Universit{\"a}t Potsdam},
  year      = {2008},
  abstract  = {Adenylates are metabolites with essential function in metabolism and signaling in all living organisms. As Cofactors, they enable thermodynamically unfavorable reactions to be catalyzed enzymatically within cells. Outside the cell, adenylates are involved in signalling processes in animals and emerging evidence suggests similar signaling mechanisms in the plants' apoplast. Presumably, apoplastic apyrases are involved in this signaling by hydrolyzing the signal mediating molecules ATP and ADP to AMP. This PhD thesis focused on the role of adenylates on metabolism and development of potato (Solanum tuberosum) by using reverse genetics and biochemical approaches. To study the short and long term effect of cellular ATP and the adenylate energy charge on potato tuber metabolism, an apyrase from Escherichia coli targeted into the amyloplast was expressed inducibly and constitutively. Both approaches led to the identification of adaptations to reduced ATP/energy charge levels on the molecular and developmental level. These comprised a reduction of metabolites and pathway fluxes that require significant amounts of ATP, like amino acid or starch synthesis, and an activation of processes that produce ATP, like respiration and an immense increase in the surface-to-volume ratio. To identify extracellular enzymes involved in adenylate conversion, green fluorescent protein and activity localization studies in potato tissue were carried out. It was found that extracellular ATP is imported into the cell by an apoplastic enzyme complement consisting of apyrase, unspecific phosphatase, adenosine nucleosidase and an adenine transport system. By changing the expression of a potato specific apyrase via transgenic approaches, it was found that this enzyme has strong impact on plant and particular tuber development in potato. Whereas metabolite levels were hardly altered, transcript profiling of tubers with reduced apyrase activity revealed a significant upregulation of genes coding for extensins, which are associated with polar growth. The results are discussed in context of adaptive responses of plants to changes in the adenylate levels and the proposed role of apyrase in apoplastic purinergic signaling and ATP salvaging. In summary, this thesis provides insight into adenylate regulated processes within and outside non-photosynthetic plant cells.},
  language  = {en}
}
@phdthesis{RodriguezCubillos2018,
  author    = {Rodriguez Cubillos, Andres Eduardo},
  title     = {Understanding the impact of heterozygosity on metabolism, growth and hybrid necrosis within a local Arabidopsis thaliana collection site},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-416758},
  school      = {Universit{\"a}t Potsdam},
  pages     = {106},
  year      = {2018},
  abstract  = {Plants are unable to move away from unwanted environments and therefore have to locally adapt to changing conditions. Arabidopsis thaliana (Arabidopsis), a model organism in plant biology, has been able to rapidly colonize a wide spectrum of environments with different biotic and abiotic challenges. In recent years, natural variation in Arabidopsis has shown to be an excellent resource to study genes underlying adaptive traits and hybridization's impact on natural diversity. Studies on Arabidopsis hybrids have provided information on the genetic basis of hybrid incompatibilities and heterosis, as well as inheritance patterns in hybrids. However, previous studies have focused mainly on global accessions and yet much remains to be known about variation happening within a local growth habitat. In my PhD, I investigated the impact of heterozygosity at a local collection site of Arabidopsis and its role in local adaptation. I focused on two different projects, both including hybrids among Arabidopsis individuals collected around T{\"u}bingen in Southern Germany. The first project sought to understand the impact of hybridization on metabolism and growth within a local Arabidopsis collection site. For this, the inheritance patterns in primary and secondary metabolism, together with rosette size of full diallel crosses among seven parents originating from Southern Germany were analyzed. In comparison to primary metabolites, compounds from secondary metabolism were more variable and showed pronounced non-additive inheritance patterns. In addition, defense metabolites, mainly glucosinolates, displayed the highest degree of variation from the midparent values and were positively correlated with a proxy for plant size. In the second project, the role of ACCELERATED CELL DEATH 6 (ACD6) in the defense response pathway of Arabidopsis necrotic hybrids was further characterized. Allelic interactions of ACD6 have been previously linked to hybrid necrosis, both among global and local Arabidopsis accessions. Hence, I characterized the early metabolic and ionic changes induced by ACD6, together with marker gene expression assays of physiological responses linked to its activation. An upregulation of simple sugars and metabolites linked to non-enzymatic antioxidants and the TCA cycle were detected, together with putrescine and acids linked to abiotic stress responses. Senescence was found to be induced earlier in necrotic hybrids and cytoplasmic calcium signaling was unaffected in response to temperature. In parallel, GFP-tagged constructs of ACD6 were developed. This work therefore gave novel insights on the role of heterozygosity in natural variation and adaptation and expanded our current knowledge on the physiological and molecular responses associated with ACD6 activation.},
  language  = {en}
}