Das Leben und Überleben einer Zelle wird auf verschiedenen Ebenen streng reguliert. Diese Ebenen sind eng miteinander verknüpft: (i) Signalwege leiten extrazelluläre Signale in den Zellkern, wo (ii) Genregulation sie zu Proteinen übersetzt, und (iii) Proteine kontrollieren metabolische Funktionen, die Nährstoffe zu Energie und zellulären Bausteinen konvertieren. Diese Systeme sind hochkomplex und werden oft nur einzeln betrachtet. Systembiologie ist ein interdisziplinäres Forschungsgebiet, das Methoden anbietet, um Informationen aus heutigen Hochdurchsatz-Experimenttechnologien zu extrahieren. Diese Methoden können effektiv sein, um die vorgenannten Systeme einzeln oder im Ganzen zu untersuchen. In dieser Doktorarbeit wende ich Methoden an, um Signalwege und Zellmetabolismus zu erforschen, und ich präsentiere neue Arbeitsabläufe für das Modellieren und Analysieren dieser Systeme. Beide Methoden sind auf großskalige Netzwerkrekonstruktionen fokussiert. Da die Erhältlichkeit von xperimentellen Daten eines der größten Probleme der Systembiologie darstellt, befassen sich die Methoden explizit mit dem Umgang mit Wissenslücken. Sie werden auf den Snf1 Signalweg und den Metabolismus von Hefezellen angewendet und vermitteln neue Erkenntnisse über diesen Modellorganismus. Des Weiteren präsentiert diese Arbeit eine eingehende Analyse vom metabolischen Reprogrammieren in Darmkrebszellen, welche bisher unbekannte Zusammenhänge von metabolischer Funktionalität und Onkogenen beinhaltet. Zum Abschluss stelle ich unseren Vorschlag für ein standardisiertes Datenaustauschformat vor, welches seinen Schwerpunkt auf Datentabellen der Systembiologie legt. Zusammenfassend behandelt diese Doktorarbeit die Signalwege und den Metabolismus von Zellen, inklusive neuer Modellierabläufe und biologischer Erkenntnisse. Diese Erkenntnisse werden in den Kontext unseres aktuellen Wissensstandes gesetzt und darauf aufbauend werden neue potentielle Ansatzpunkte für Experimente vorgeschlagen. / Cellular life is governed on different layers of regulation, which are tightly interconnected: (i) Signalling pathways transmit extracellular signals to the cells’ nucleus, where (ii) gene regulation translates these signals into proteins, and (iii) proteins control metabolic functions, which convert nutrients to energy and cell building blocks. Due to the complexity of each of these systems, they are often analysed individually or only partially. Systems Biology is an interdisciplinary field of research that offers techniques to harvest the information of todays high-throughput experiments. These techniques can be powerful approaches to investigate the aforementioned regulatory layers of a cell either individually or as a whole. In this thesis, I am employing means of Systems Biology to explore signalling pathways and metabolism, and I provide novel workflows for modelling and exploring these systems. Both workflows are focussed on accurate large-scale network reconstructions of the target system. Since one of the major problems in Systems Biology is the availability of experimental data, the workflows put emphasis on the handling of knowledge gaps. They are applied on the Snf1 pathway and metabolism in yeast and provide new findings about this model organism. Furthermore, this thesis presents an in-depth analysis of metabolic reprogramming in colorectal cancer cells, which yields previously unknown coherences of metabolic function and oncogenes. Finally, I am presenting a proposal for a standardised data format in Systems Biology, which is based on data tables. In summary, this thesis comprises works on signalling pathways and cell metabolism, which includes novel modelling workflows and new biological findings, analyses their impact on the scientific state of the art, and proposes directions for new experimental targets.

Das mitochondriale Genom der Pflanze weist mit einer heterogenen Population linearer, häufig auch verzweigten und zusätzlichen kleineren, zirkulären Molekülen eine komplexe Struktur auf. Um Einblicke in die mitochondrialen Genkopienzahl und deren Regulation sowohl unter normalen als auch unter Stressbedingungen zu erhalten, wurde die Kopienzahl pro Zelle vier repräsentativer Gene mittels qRT-PCR und Durchflusszytometrie ermittelt. Die Bestimmung der mitochondrialen Genkopienzahl in unterschiedlichen Spezies sowie in Organen der Modellpflanze Arabidopsis thaliana zeigte, dass die Kopienzahl mitochondrialer Gene sich nicht nur in den unterschiedlichen Spezies, sondern auch zwischen den unterschiedlichen Organen unterschied, wobei die höchsten Werte in der Wurzelspitze erreicht wurden. In Arabidopsis Keimlingen, welche zur Unterdrückung der plastidären Translation auf Spectinomycin-haltigem Medium angezogen wurden, wurde im Vergleich zu Kontrollpflanzen ein dreifacher Anstieg der Genkopienzahl festgestellt. Dieser Effekt erwies sich als spezifisch für Blatt- bzw. Kotyledonengewebe und warr unabhängig vom Licht. Mutanten mit Defekten in der Respiration zeigten ebenfalls erhöhte Genkopienzahlen, die durch Anzucht der Pflanzen auf Spectinomycin noch erhöht werden konnten. Dieses Ergebnis legt ein komplexes, regulatorisches Netzwerk nahe, in welchem sowohl Respiration als auch Photosynthese die Aufrechterhaltung einer stabilen Genkopienzahl innerhalb der Pflanzenzelle beeinflussen. Die Untersuchungen einer Spectinomycin-behandelter mt-GFP Arabidopsis Pflanzenlinie mittels CLSM zeigten einen Stillstand der Motilität der Mitochondrien in den epidermalen Zellen der weißen Kotyledonen, obwohl eine TEM Analyse eine normale, interne Morphologie ergab. Weitere Untersuchungen führten zu der Schlussfolgerung, dass es auch hier die Stärke der plastidären Beeinträchtigung, welche zu einem gelb-weißen Phänotyp führt, für den Arrest der Mobilität verantwortlich ist. / The plant mitochondrial genome has a complex structure. It exists in the form of a heterogeneous population of linear, often branched molecules with smaller than genome-size circular molecules being present in low abundance. In order to study the mitochondrial genome abundance and its regulation in plants under both standard and stress conditions, we determined the gene copy number of four representative mitochondrial genes using quantitative real-time PCR and flow-cytometry. Determination of mitochondrial gene copy number in different plant species and in organs of the model plant Arabidopsis thaliana showed that the copy number of the four investigated genes varied between species and also between different organs, having the highest values in the root tips. The growth of Arabidopsis seedlings on MS medium containing spectinomycin (a plastid translation inhibitor) led to a three-fold increase in the copy number in white versus green seedlings, an effect that is leaf/cotyledon specific and light-independent. Respiration deficient mutants also showed an increase in the gene copy number, this effect being further amplified when the mutants were grown on spectinomycin. The data suggest a complex regulatory network in which both photosynthesis and respiration influence the maintenance of a stable mitochondrial gene copy number within plant cells. CLSM investigations of a spectinomycin-treated mt-GFP line showed that in epidermal cells of white cotyledons most of the mitochondria are not motile with TEM analysis presenting normal internal morphology. Further investigations led to the conclusion that the threshold level of chloroplast impairment that leads to a motility arrest is represented by the appearance of a yellow-white cotyledon phenotype. These results point to a new regulatory mechanism of mitochondrial dynamics that is directly influenced by impaired chloroplast development under standard growth conditions.

The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.

No description available.

No description available.

Die Signalwege des transformierenden Wachstumsfaktors β (TGF-β) spielen eine entscheidende Rolle bei der Zellproliferation, -migration und -apoptose durch die Aktivierung von Smad-Proteinen. Untersuchungen haben gezeigt, dass die biologischen Wirkungen des TGF-β-Signalwegs stark vom Zellkontext abhängen. In dieser Arbeit ging es darum zu verstehen, wie TGF-β-Signale Zielgene unterschiedlich regulieren können, wie unterschiedliche Dynamiken der Genexpression durch TGF-β-Signale induziert werden und auf welche Weise Smad-Proteine zu unterschiedlichen Expressionsmustern von TGF- β-Zielgenen beitragen. Der Fokus dieser Studie liegt auf den transkriptionsregulatorischen Effekten des Nodal / Activin-Liganden, der zur TGF-β-Superfamilie gehört und ein wichtiger Faktor in der frühen embryonalen Entwicklung ist. Um diese Effekte zu analysieren, habe ich kinetische Modelle entwickelt und mit den Zeitverlaufsdaten von RNA-Polymerase II (Pol II) und Smad2-Chromatin-Bindungsprofilen für die Zielgene kalibriert. Unter Verwendung des Akaike-Informationskriteriums (AIC) zur Bewertung verschiedener kinetischer Modelle stellten wir fest, dass der Nodal / Activin-Signalweg Zielgene über verschiedene Mechanismen reguliert. Im Nodal / Activin-Smad2-Signalweg spielt Smad2 für verschiedene Zielgene unterschiedliche regulatorische Rollen. Wir zeigen, wie Smad2 daran beteiligt ist, die Transkriptions- oder Abbaurate jedes Zielgens separat zu regulieren. Darüber hinaus werden eine Reihe von Merkmalen, die die Transkriptionsdynamik von Zielgenen vorhersagen können, durch logistische Regression ausgewählt. Der hier vorgestellte Ansatz liefert quantitative Beziehungen zwischen der Dynamik des Transkriptionsfaktors und den Transkriptionsantworten. Diese Arbeit bietet auch einen allgemeinen mathematischen Rahmen für die Untersuchung der Transkriptionsregulation anderer Signalwege. / Transforming growth factor-β (TGF-β) signaling pathways play a crucial role in cell proliferation, migration, and apoptosis through the activation of Smad proteins. Research has shown that the biological effects of TGF-β signaling pathway are highly cellular-context-dependent. In this thesis work, I aimed at understanding how TGF-β signaling can regulate target genes differently, how different dynamics of gene expressions are induced by TGF-β signal, and what is the role of Smad proteins in differing the profiles of target gene expression. In this study, I focused on the transcriptional responses to the Nodal/Activin ligand, which is a member of the TGF-β superfamily and a key regulator of early embryonic development. Kinetic models were developed and calibrated with the time course data of RNA polymerase II (Pol II) and Smad2 chromatin binding profiles for the target genes. Using the Akaike information criterion (AIC) to evaluate different kinetic models, we discovered that Nodal/Activin signaling regulates target genes via different mechanisms. In the Nodal/Activin-Smad2 signaling pathway, Smad2 plays different regulatory roles on different target genes. We show how Smad2 participates in regulating the transcription or degradation rate of each target gene separately. Moreover, a series of features that can predict the transcription dynamics of target genes are selected by logistic regression. The approach we present here provides quantitative relationships between transcription factor dynamics and transcriptional responses. This work also provides a general computational framework for studying the transcription regulations of other signaling pathways.

Autoinflammatorische Erkrankungen umfassen eine immer größer werdende, genetisch heterogene Gruppe von Erkrankungen mit breitem und variablem klinischen Spektrum. Aus nosologischer Perspektive wird eine strikte Abgrenzung der Autoinflammation von Autoimmunität und Immundefizienz dem aktuellen Kenntnisstand zu pathogenetischen Mechanismen nicht gerecht. Daher erscheint eine systembasierte Einteilung, die sich an den in die inflammatorischen Prozesse involvierten Signalwegen orientiert, auch im Hinblick auf das klinische Management sinnvoll. So sprechen die Inflammasomopathien in vielen Fällen auf eine Blockade des Interleukin(IL)-1β an, während die Typ-1-Interferonopathien einer Therapie mithilfe der Januskinase(JAK)-Inhibition zugänglich sind. / Autoinflammatory diseases comprise a growing genetically heterogeneous group of diseases with a broad and variable clinical spectrum. From a nosological perspective, a strict demarcation of autoinflammation from autoimmunity and immunodeficiency does not reflect the current state of knowledge on pathogenetic mechanisms. Therefore, a system-based classification according to the signalling pathways involved in the inflammatory processes, appears to be more useful also with respect to clinical management. As such, inflammasomopathies commonly respond to an interleukin 1 beta (IL-1-beta) blockade, while type 1 interferonopathies can be treated with Janus kinase (JAK) inhibition.

No description available.

No description available.

The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.