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No. of bitstreams: 1 TeseCRS.pdf: 3506039 bytes, checksum: 68f4c5fe1c6ae3672adcedf3450e2f31 (MD5) Previous issue date: 2015-08-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The genus Salmonella spp. has been extensively investigated because these bacteria are important pathogens that frequently cause severe diseases and gastrointestinal infections in humans and animals. Moreover, in recent years, Salmonella has called attention due to the excellent results in the production and in vivo delivery of various substances with potential application in Vaccinology. However, there is still little information available concerning aspects of its metabolism, which hampers both the development of new attenuated strains and the large-scale production of live cells and cellular components. Thus, this work aimed to study the S. typhimurium LT2 metabolism, using traditional and innovative approaches to investigate different carbon sources as well as different bioreactor operation modes and aeration conditions (aerobic and anaerobic). Results obtained in batch and chemostat cultivations indicated that S. typhimurium metabolism differs significantly from E. coli metabolism, closely related bacteria species with regard to the central carbon metabolism. The main difference observed between these bacteria was the high level of acetate production exhibited by S. typhimurium LT2 cells, which, differently from E. coli, occurred even at the lowest dilution rate evaluated. Currently, genome scale metabolic models are important tools for better understanding the phenotypic behavior of many organisms. Therefore the model STM_v1.0 reconstructed for S. typhimurium LT2 was evaluated, comparing experimental data, obtained in chemostat cultivations, with model predictions. Since this model was derived from E. coli model, the simulated results for biomass formation were overestimated and, consequently, predicted acetate fluxes were lower than those obtained experimentally. Therefore, to obtain experimental data useful to improve the model and to reach a better comprehension of S. typhimurium metabolism, the technique of metabolic flux analysis using isotopic labeled substrate was adopted, allowing determination of the fluxes for the main pathways of central carbon metabolism of Salmonella. This analysis revealed different preferred metabolic pathways depending on the specific growth rate. At the lowest dilution rate evaluated, D = 0.24 h-1, glucose was catabolized predominantly by the pentose phosphate and glycolysis pathways, while at the dilution rate of 0.48 h-1, the major pathway of glucose oxidation was Entner-Doudoroff. In addition, a relatively high flux through the citric acid cycle at the higher dilution rate studied was observed. / Bactérias do gênero Salmonella spp. são extensivamente estudadas por serem importantes patógenos, causando frequentemente graves doenças e infecções gastrointestinais em humanos e animais. Além disso, nos últimos anos, estas bactérias vêm ganhando um destaque ainda maior na área da biotecnologia por apresentarem ótimos resultados na produção e veiculação in vivo de diversas substâncias com fins vacinais. No entanto, ainda há poucas informações a respeito de seu metabolismo, dificultando tanto o desenvolvimento de novas linhagens atenuadas, como também a produção em larga escala de células vivas e de componentes celulares. Neste sentido, este trabalho se propôs a estudar o metabolismo de S. typhimurium LT2, utilizando inicialmente abordagens tradicionais para investigar seu comportamento na presença de diferentes fontes de carbono, em diferentes modos de operação de biorreator e de aeração (aeróbias e anaeróbias). Os resultados obtidos em cultivos em batelada e em quimiostatos evidenciaram que o metabolismo da S. typhimurium difere bastante do metabolismo da E. coli, espécies consideradas semelhantes com relação ao metabolismo do carbono central. A principal diferença observada entre essas duas bactérias foi a elevada produção de acetato pelas células de S. typhimurium LT2, mesmo em baixas velocidades de crescimento nas quais este metabólito não é produzido por diversas estirpes de E. coli. Atualmente, modelos metabólicos em escala genômica são ferramentas importantes para que o comportamento do fenótipo de diversos organismos sejam melhor compreendidos. Assim, avaliou-se o modelo STM_v1.0 reconstruído para S. typhimurium LT2, comparando-se dados obtidos experimentalmente, em quimiostatos, e os preditos pelo modelo. No entanto, como este modelo foi baseado no modelo da E. coli, os resultados simulados para produção de biomassa foram superestimados e, consequentemente, os fluxos de acetato foram inferiores aos obtidos experimentalmente. Sendo assim, para se obter dados experimentais úteis para aprimorar o modelo e para uma compreensão maior do metabolismo de S. typhimurium, utilizou-se a técnica de análise dos fluxos metabólicos com substrato isotopicamente marcado, permitindo a determinação dos fluxos das principais vias do metabolismo do carbono central da bactéria em estudo. Essa análise revelou diferenças na utilização das vias metabólicas em função da velocidade específica de crescimento, sendo que na menor taxa de diluição avaliada, D = 0,24 h-1, a glicose foi predominantemente catabolizada pelas vias pentose fosfato e glicólise, enquanto na taxa de diluição de 0,48 h-1, a via principal de oxidação da glicose foi a Entner- Doudoroff. Além disso, também observou-se um fluxo relativamente maior na via do ciclo do ácido cítrico na maior taxa de diluição estudada.

Cells must regulate their metabolism in order to grow, adapt to changes in nutrient availability and maintain homeostasis. Flux, or the turnover of metabolites, through the metabolic network can be regulated at the allosteric and transcriptional levels. While study of allosteric regulation is limited to biochemical examination of individual proteins, transcriptional control of metabolism can be explored at a systems level. We endeavored to elucidate transcriptional mechanisms of metabolic flux regulation in the model organism Caenorhabditis elegans (C. elegans). We also worked to create a visual tool to explore metabolic pathways that will support future efforts in the research of metabolic gene regulation. C. elegans is a small, free-living nematode that feeds on bacteria and experiences a high level of diversity in nutrient level and composition. Previously, we identified a mechanism by which the essential cofactor, vitamin B12, regulates the expression of genes involved in the degradation of propionate, referred to as B12‑mechanism‑I. This mechanism functions to prevent the toxic accumulation of propionate and requires the TFs NHR-10 and NHR-68. Using genetic screens as well as transcriptomic and metabolomic approaches, we discover a second mechanism by which vitamin B12 regulates metabolic gene expression: B12-mechanism-II. Unlike B12-mechanism-I, B12-mechanism-II is independent of propionate, requires the transcription factor NHR-114 and functions to maintain the metabolic activity of the Methionine/S-adenosylmethionine cycle in a tightly regulated regime. We also present WormPaths, an online resource that allows visualization of C. elegans metabolic pathways and enables metabolic pathway enrichment of user-uploaded transcriptomic data.

Les études portant sur le changement climatique prévoient des modifications significatives de la température pour les décennies à venir. Ces prévisions envisagent un réchauffement global qui risque de perturber le développement des plantes cultivées. Les stress thermiques affectent en effet presque tous les aspects du développement des plantes et notamment la croissance, le développement floral et donc le rendement. Les plantes doivent modifier leur métabolisme afin de prévenir les dommages causés par les stress environnementaux. La température joue un rôle important sur la phénologie, la vigueur et surtout le développement floral de la vigne. Ainsi, quelques degrés d’élévation de la température pendant la floraison peuvent entrainer la perte de la totalité des fleurs et donc des fruits. De plus, en stimulant le développement végétatif, l’augmentation de température accroit la demande en glucides, tandis que la photosynthèse nette diminue en raison de l’augmentation de la respiration en réponse à l’élévation de température. Au cours de cette étude, nous nous sommes intéressés à l’impact d’une augmentation de température sur le métabolisme carboné, acteur majeur de la floraison, et plus particulièrement aux modifications physiologiques de la feuille et de l’inflorescence de vigne. De plus, des analyses d’expression de gènes clés de la photosynthèse et du métabolisme ont permis d’améliorer la compréhension des mécanismes de distribution des glucides entre les organes végétatifs et reproducteurs lors d’une augmentation de la température. / Studies on climate change predict significant changes in temperature for next decades. The global warming could impact the crop plants development. Indeed, thermal stresses affect almost all aspects of plant development including growth, floral development and yield. Plants modify their metabolism to prevent damage caused by environmental changes. Temperature is an important factor for the phenology, the vigor and especially the floral development in grapevine. So, a few degrees increase during flowering can result in a complete flowers loss and therefore fruits. In addition, an increase of temperature stimulates vegetative development and thus rises the carbohydrates consumption, while net photosynthesis decreases due to the respiration raise. In this study, we investigated the temperature increase impacts on the carbon metabolism which has a major role in the flowering process in grapevine. We particularly focused our attention on the physiological modifications in leaves and inflorescences. at the level of the photosynthesis and the respiration with the increase of the day temperature. Moreover, expression analyzes of some key genes involved in photosynthesis and metabolism allowed to improve the understanding in the carbohydrate distribution mechanisms, between vegetative and reproductive organs, during a temperature increase.

Prostate cancer is the most common cancer among men. The purpose of this quantitative, meta-analysis study was to examine one-carbon metabolism gene polymorphisms in a group of genes to determine their association with prostate cancer risk. The genetic epidemiology theory provided the framework for the study. The data collected were from published articles. From over 2,800 individual studies, 20 articles were retained for results and data abstraction, following the title, abstract screen, and full text screening in the second phase. The data were analyzed by a meta-analysis statistical method, combining the results from selected studies to estimate the overall association. According to study results by the adjusted p-values of fixed model, there was a significant association between decreased risk of prostate cancer and the variant of Allele T, Genotype TT, and the recessive model of C667T polymorphism. In the random model, the adjusted p-values show a significant association between decreased risk of prostate cancer, the variant of Genotype TT, and recessive model. There was an increased risk of prostate cancer in A1298C polymorphism by adjusted p-value on the variant of Genotype AC, in the fixed model. This study leads to positive social change by providing information on an optimization surveillance strategy to ensure valid screening test for prostate disease reporting. Future studies with a greater number of samples are needed, including gene-gene and gene-environment interaction to verify study results.

Die vorliegende Arbeit untersucht die Rolle des Metabolismus in der Entstehung und Progression des Hepatozellulären Karzinoms (HZK). Der Schwerpunkt der Studie liegt auf Veränderungen zentraler Stoffwechselwege, unter anderem der Glykolyse, der Gluconeogenese, des Citratzyklus und anderer Prozesse des Zellstoffwechsels. Umfassende Multiomikanalysen, wie etwa Proteomik, Metabolomik und gezielte Genomsequenzierung wurden angewandt, um in vivo die Mechanismen der HZK Entstehung zu verstehen. Es wurden zwei Systeme untersucht: das ASV-B Mausmodell und klinische Patientenproben. Die Kohorte bestehend aus Biopsien und Resektaten von 95 Patienten umfasste 47 Fälle von HZK und 48 Fälle ohne HZK. Das Proteom des Mausmodells und der Patientenkohorte zeigen eine deutliche Herabregulierung wesentlicher Energie bereitstellender Kreisläufe im HZK: Glykogenstoffwechsel, de novo Synthese von Glukose, Glutaminaufnahme in den Citratzyklus, des weiteren sind 60% der Enzyme des Citratzyklus, und des Transports von Pyruvat in Mitochondrien im HZK herabreguliert. In dieser Arbeit wurde ein Isoformenwechsel auf mehreren Ebenen des zentralen Kohlenstoffmetabolismus gezeigt. Sowohl das Mausmodell, als auch die Gewebeproben von HZK-Patienten weisen Isoformenwechsel der Phosphoglyzeratmutasen und der Pyruvatkinasen auf. Die Hauptmerkmale finden sich sowohl in Modellmäusen, als auch in Patienten, und stellen so einen universalen metabolomischen Fingerabdruck des HZK dar. Darüber hinaus demonstriert diese Studie, dass die Proteomanalyse von bioptischen Material ein aussagekräftiges und ausreichendes molekular-diagnostisches Instrument für die Krebsforschung ist: die Proteomanalyse von Lebermaterial erlaubt die Unterscheidung von Tumorgewebe und tumorfreien Proben und die Dokumentation des Krankheitsverlaufs. / The present work evaluates the role of metabolism in development and progression of hepatocellular carcinoma (HCC). This study focuses on changes of central metabolic pathways, including glycolysis, gluconeogenesis, tricarboxylic acid (TCA) cycle and other processes involved in cellular metabolism and known to be dysregulated during cancer formation. Comprehensive multiomics analyses, such as proteomics, metabolomics and targeted genome sequencing, were applied in order to better understand HCC developmental mechanisms in vivo. Two main systems were studied: the ASV-B mouse model and clinical samples from human patients. The human cohort was composed of biopsy and surgery material from 95 patients: 47 HCC and 48 non-HCC. Proteomic data from both mice and humans show a clear downregulation of the main energy-producing pathways in HCC. Glycogen metabolism, de novo glucose synthesis, glutamine uptake to the TCA cycle, approximately 60% of enzymes of TCA cycle, and transport of pyruvate to mitochondria are downregulated in HCC. An isoform switch at various levels of central carbon metabolism was demonstrated in this work. Both mice and humans with HCC reveal isoform switches at the level of phosphoglycerate mutases and pyruvate kinases. The key features are found in both mouse and human, showing a universal metabolic HCC fingerprint. This study also demonstrates that proteomic analysis of the bioptate material is a strong and sufficient molecular diagnostic tool for research in cancer: the proteomic analysis of liver material allows the distinction of tumor samples from non-tumor samples and also to track the level of disease progression. Targeted genome sequencing revealed that no clear distinction between cancer and precancerous conditions could be made exclusively from the mutation analysis. Human metabolomic data remains inconclusive, possibly due to the different sources of tissue samples.

Le cancer du pancréas est l’un des plus chimiorésistants, avec un taux de survie sur 5 ans inférieur à 5%. La chimiorésistance pourrait être due à la présence de cellules initiatrices de tumeur (TICs), une petite sous-population des cellules tumorales possédant la capacité de régénérer une nouvelle tumeur. Il a été démontré que la metformine cible les TICs par un mécanisme non élucidé. Il est connu que la metformine affecte le métabolisme du carbone. Il a également été démontré que le métabolisme du carbone, plus précisément la glycine décarboxylase (GLDC), est à la fois nécessaire et suffisant à l’acquisition de propriétés d’initiation tumorale. Nous proposons que la metformine cible les cellules initiatrices de tumeur en affectant le métabolisme du carbone. Nous avons utilisé des lignées cellulaires dérivées d’un modèle murin de cancer du pancréas pour comparer l’expression génique de lésions bénignes versus malignes. Les cellules malignes surexpriment Gldc. La metformine diminue l’expression de Gldc, et la surexpression de Gldc diminue la sensibilité à la metformine dans un essai de sphères tumorales. La metformine induit une augmentation du ratio NADP+/NADPH, et la surexpression de Gldc empêche cette augmentation. Nous proposons que la metformine diminue l’expression de Gldc, ce qui cause une diminution du flux du métabolisme du carbone, et donc une diminution de la production de NADPH par ce dernier. L’augmentation du ratio NADP+/NADPH inhibe la synthèse des acides gras et la régénération de la glutathione, ce qui pourrait expliquer la diminution de la formation de sphères tumorales sous traitement metformine. / Pancreatic cancer is one of the most chemoresistant cancers, with a 5-year survival rate lesser than 5%. Chemoresistance might be due to the presence of tumor-initiating cells (TICs), a small subpopulation of tumor cells with stem-like characteristics which possess the unique ability to self-renew and to generate a new tumor. Metformin has been shown to affect TICs in various cancer types, but the mechanism through which it does so is unclear. It is known that metformin affects one-carbon metabolism. It has also been shown that one-carbon metabolism, more precisely the glycine decarboxylase (GLDC) enzyme, is both necessary and sufficient to the acquisition of tumor-initiating properties. Considering this, we propose that metformin affects TICs by targeting one-carbon metabolism. Using cell lines derived from a genetically engineered mouse model of pancreatic cancer, we compared gene expression data from cells derived from benign pancreatic neoplasia with cells derived from pancreatic ductal adenocarcinoma (PDAC), and found that PDAC cells exhibited a dramatic increase in Gldc expression. Metformin treatment decreases Gldc expression in PDAC cell lines, and Gldc overexpression greatly decreases metformin sensitivity in a tumor sphere assay. Metformin induces an increase in NADP+/NADPH ratio, which is rescued by Gldc overexpression. We propose a model in which metformin decreases Gldc expression, which causes reduced flux through mitochondrial one-carbon metabolism. This results in decreased NADPH production by this pathway. This increase in NADP+/NADPH ratio impairs fatty acid biosynthesis and glutathione regeneration. Together these effects might explain the decrease of tumor sphere formation under metformin treatment.

Le fer est un micronutriment important pour la croissance et le développement des plantes. Il agit comme cofacteur pour plusieurs enzymes et il est important pour des processus tels que la photosynthèse et la respiration. Souvent, le Fe dans le sol n’est pas bio-disponible pour la plante. Les plantes ont développé des stratégies pour solubiliser le Fe du sol pour le rendre disponible et assimilable pour elles. Il y a deux stratégies, la première est caractéristique des dicotylédones et la seconde est caractéristique des monocotylédones. Le modèle utilisé dans cette étude est une culture cellulaire de Solanum tuberosum. Une partie de la recherche effectuée a permis la mesure d’activité et d’expression relative de certaines enzymes impliquées dans le métabolisme énergétique et la fourniture de précurseurs pour la synthèse d’ADN : la Nucléoside diphosphate kinase, la Ribonucléotide reductase, la Glucose 6-phosphate déshydrogénase et la 6-Phosphogluconate déshydrogénase dans les cellules en présence ou en absence de Fe. Chez certains organismes, la déficience en Fe est associée à une perte de croissance qui est souvent liée à une diminution de la synthèse d’ADN. Chez les cultures de cellules de S. tuberosum, les résultats indiquent que la différence de biomasse observée entre les traitements n’est pas due à une variation de l’activité ou l’expression relative d’une de ces enzymes. En effet, aucune variation significative n’a été détectée entre les traitements (+/- Fe) pour l’activité ni l’expression relative de ces enzymes. Une autre partie de la recherche a permis d’évaluer l’activité des voies métaboliques impliquées dans la stratégie 1 utilisée par S. tuberosum. Cette stratégie consomme des métabolites énergétiques: de l’ATP pour solubiliser le Fe et du pouvoir réducteur (NAD(P)H), pour réduire le Fe3+ en Fe2+. Des études de flux métaboliques ont été faites afin d’étudier les remaniements du métabolisme carboné en déficience en Fe chez S. tuberosum. Ces études ont démontré une baisse du régime dans les différentes voies du métabolisme énergétique dans les cellules déficientes en Fe, notamment dans le flux glycolytique et le flux de C à travers la phosphoenolpyruvate carboxylase. En déficience de Fe il y aurait donc une dépression du métabolisme chez S. tuberosum qui permettrait à la cellule de ralentir son métabolisme pour maintenir sa vitalité. En plus des flux, les niveaux de pyridines nucléotides ont été mesurés puisque ceux-ci servent à réduire le Fe dans la stratégie 1. Les résultats démontrent des niveaux élevés des formes réduites de ces métabolites en déficience de Fe. L’ensemble des résultats obtenus indiquent qu’en déficience de Fe, il y a une baisse du métabolisme permettant à la cellule de s’adapter et survivre au stress. / Iron is an important micronutrient for plant growth and development. It participates as a cofactor for several enzymes and is important for processes such as photosynthesis and respiration. Often soil Fe is not bioavailable to the plant. Plants have developed strategies to solubilize the Fe in the soil to make it available and easy to assimilate. There are two strategies, the first is characteristic of dicotyledones and the second is characteristic of monocotyledones. The model used in these studies is a cell culture of Solanum tubersoum. A first part of the research involved the study of expression and activity of enzymes required in energy metabolism and the provision of precursors for DNA synthesis: Nucleoside dehydrogenase, Ribonucleotide reductase, Glucose 6-phohate dehydrogenase and 6-Phosphogluconate dehydrogenase. In several organisms, Fe deficiency induces a loss of biomass which is often associated with a decrease in DNA synthesis. In S. tuberosum cell cultures, the results indicate that the loss of biomass observed in Fe deficiency is not linked to a change in the activity or relative expression of these enzymes. Indeed, no significant changes were detected between treatments (+/- Fe) for activity or relative expression. In another part of the research, we evaluated the activity of the metabolism pathways involved in strategy 1, which is used by S. tuberosum. This strategy consumes energetic metabolites: ATP to solubilize Fe and reducing power (NAD(P)H) to reduce the Fe3+ to Fe2+. Metabolic flux studies were done to investigate the alterations of carbon metabolism during Fe deficiency in S. tuberosum. These studies demonstrated that in Fe deficient cells, there is a decrease in the fluxes of some pathways of energy metabolism. Particularly, in the glycolytic flux and the anaplerotic flux of PEPC. Under Fe deficiency there would be a depression of metabolism in S. tuberosum which would allow the cell to slow its metabolism to maintain its vitality. In addition to the fluxes, the levels of pyridine nucleotides were measured since they serve to reduce Fe in the strategy 1. The results show an increase in the reduced forms of these metabolites during Fe deficiency. All results together point out that during Fe deficiency the metabolism decreases, allowing the cell to survive and adapt to the stress.

Tuberkulose ist auch heute noch eine der bedrohlichsten Infektionskrankheiten weltweit, verantwortlich für über 1.5 Millionen Todesfälle jährlich. Diese „Erfolgsgeschichte“ ihres Erregers Mycobacterium tuberculosis ist dabei wesentlich durch einen extrem flexiblen Stoffwechsel bestimmt, der dem Bakterium das Wachstum unter den restriktiven Bedingungen der menschlichen Wirtszelle erlaubt. Diese Arbeit erkundet die Flexibilität des zentralen Kohlenstoffmetabolismus in Mykobakterien mit Hilfe mathematischer Modellierungsansätze, ergänzt durch die Integration von qualitativ hochwertigen experimentellen Daten. Ausgehend von einem Überblick über die metabolische Landschaft des zentralen Kohlenstoffmetabolismus, erhöht sich Schritt für Schritt die Detailtiefe bis hin zur genauen Analyse spezieller infektionsrelevanter metabolischer Wege. Die Verknüpfung des zentralen Kohlenstoffmetabolismus zu umgebenden Stoffwechsel- und Biosynthesewegen wird systematisch offen gelegt, als Voraussetzung für eine thermodynamische Charakterisierung des Systems, welche die Glykolyse als limitierenden Stoffwechselweg unter verschiedenen Wachstumsbedingungen charakterisiert. Basierend auf Protein- und Metabolitdaten im Fleißgleichgewicht, erlaubt eine neu vorgestellte Methode die Vorhersage regulatorischer Punkte für den metabolischen Übergang zwischen verschiedenen Kohlenstoffquellen. Abschließend wird mit Hilfe thermodynamisch-kinetischer Modellierung das Zusammenspiel zweier Stoffwechselwege mechanistisch erklärt, welche den robusten Abbau einer intrazellulären Kohlenstoffquelle ermöglichen. Durch die Entwicklung neuer Modellierungstechniken in Kombination mit hochauflösenden experimentellen Daten, trägt diese Arbeit zum besseren Verständnis der kontextabhängigen Flexibilität des mycobakteriellen Stoffwechsels bei, einem vielversprechenden Angriffspunkt für die Entwicklung neuer Medikamente gegen Tuberkulose. / Tuberculosis remains one of the major global health threats responsible for over 1.5 million deaths each year. This ’success story’ of the causative agent Mycobacterium tuberculosis is thereby closely linked to a flexible metabolism, allowing growth despite the restrictive conditions within the human host. In this thesis, the flexibility of the mycobacterial central carbon metabolism is explored by modeling approaches integrating high-quality experimental data. The analyses zoom in from a network based view to the detailed functionalities of individual, virulence relevant pathways. The interconnection of the central carbon metabolism to the remaining metabolic network is charted as a prerequisite to characterize its thermodynamic landscape, debunking glycolysis as bottleneck in different nutritional conditions. Based on steady state metabolomics and proteomics data, regulatory sites for the metabolic transition between different carbon sources are predicted by a novel method. Finally, the flexible interplay between two seemingly redundant pathways for the catabolism of an in vivo-like carbon source is explained mechanistically by means of thermodynamic-kinetic modeling. By employing novel modeling methods in combination with high-resolution experimental data, this work adds to the mechanistic understanding of the context dependent flexibility of mycobacterial metabolism, an important target for the development of novel drugs in the battle against tuberculosis.

La phosphorylation oxydative est un processus majeur du métabolisme énergétique qui est catalysée par les enzymes de la chaîne respiratoire (OXPHOS), localisées dans la membrane interne des mitochondries. Sa dérégulation est souvent associée à des pathologies, par exemple aux maladies mitochondriales et neurodégénératives. La régulation de la phosphorylation oxydative par la phosphorylation reste encore peu comprise et peu étudiée. Pourtant, la phosphorylation est une des modifications post-traductionnelles les plus répandues dans la cellule, régulant de nombreux aspects de la vie cellulaire et dont l’altération est associée à des pathologies au niveau cellulaire (Alzheimer, Parkinson, cancer). Concernant la phosphorylation oxydative, il est à noter que quelques sites de phosphorylation des complexes respiratoires, en particulier du complexe IV, ont été montrés comme ayant un effet sur leur stabilité et/ou leur activité. Toutefois la connaissance du phosphoprotéome mitochondrial n’est pas suffisamment documentée à ce jour pour identifier les différents rôles que pourraient jouer la phosphorylation au niveau de la mitochondrie et en particulier, de la chaîne respiratoire. Dans la première partie de la thèse, nous nous sommes intéressés à l’analyse du phosphoprotéome mitochondrial de Saccharomyces cerevisiae dans trois conditions de culture : respiratoire (YLAC), respiro-fermentaire (YPGalA) et fermentaire (YPGA). Nous avons quantifiés près de 300 sites de phosphorylation dans la mitochondrie, dont 90 ont un niveau de phosphorylation variable selon le substrat. Les données que nous avons obtenues constituent une base pour l’analyse de la phosphorylation mitochondriale et de la compréhension de son mécanisme. Les sites de phosphorylation de la voie métabolique énergie sont ceux présentant le plus de variation de leur niveau de phosphorylation. La localisation des résidus phosphorylés sur la structure des complexes respiratoires nous a permis d’émettre des hypothèses sur le rôle de ces résidus. Afin de normaliser la quantité des résidus phosphorylés dans les trois conditions de culture, nous avons aussi quantifié le protéome mitochondrial dans les trois conditions de culture. Ceci nous a permis d’argumenter en faveur d’un métabolisme respiro-fermentaire en YPGalA, question encore largement discutée à ce jour. Enfin, cette première étude quantitative du protéome et phosphoprotéome mitochondrial constitue une avancée dans l’étude de la régulation de la mitochondrie par la phosphorylation. Elle peut notamment apporter des informations applicables à l’étude du cancer : en effet, les cellules saines ont un métabolisme respiratoire tandis que les cellules tumorales, dérégulées, ont un métabolisme fermentaire. La seconde partie de la thèse concerne l’analyse du rôle de deux sous-unités du complexe IV de la chaîne respiratoire : les sous-unités Cox12p et Cox13p, encore peu étudiées à ce jour. De plus, deux sites de phosphorylation ont été identifiés sur la sous-unité Cox12p. Dans un premier temps, nous nous sommes intéressés au rôle de ces sous-unités, notamment au niveau de l’assemblage et de l’activité du complexe IV, en analysant des mutants Δcox12, Δcox13 et Δcox12Δcox13. Dans un deuxième temps, nous nous sommes intéressés au rôle des deux sites de phosphorylation de Cox12p : Ser7 et ser82. Nous avons généré les mutants phosphomimétiques de ces deux résidus et étudié leurs effets sur la stabilité et/ou l’activité du complexe IV. Cette seconde étude nous a notamment permis d’identifier un rôle de Cox12p sur la stabilité du complexe et un rôle de Cox13p dans sa dimérisation. La phosphorylation de Cox12p au niveau de la Ser7 semble aussi déstabiliser le complexe IV. De plus, la phosphorylation de la Ser7 et de la Ser82 semblent influencer l’interaction du cytochrome c avec le complexe IV. Cette hypothèse reste à vérifier mais est pertinente du fait de la proximité de Cox12p avec Cox2p, qui porte le lieu de fixation du cytochrome c. / Mitochondria are the powerhouses of cells, providing energy in the form of adenosine triphosphate (ATP). The synthesis of ATP is achieved by oxidative phosphorylation (OXPHOS), a process catalyzed by the respiratory chain, which is located in the inner membrane of mitochondria. Deregulation of OXPHOS is often associated to diseases. Deregulation is particularly observed in mitochondrial diseases and neurodegenerative diseases, but regulation of respiration by phosphorylation is still poorly understood.However, phosphorylation is one of the most frequent post-translational modifications in the cell, modulating most processes, and defects at a cellular level are observed in some diseases (Alzheimer, Parkinson, cancer). Moreover, some phosphorylation sites have been identified in the respiratory complexes, particularly in the complex IV; some of them have an effect on the stability and/or activity of the complex, but we still lack a comprehensive study about mitochondrial phosphoproteome. Such analysis would be necessary to extend the role of phosphorylation in the regulation of mitochondrial functions in general, and in the regulation of the respiratory chain in particular.In the first part of this thesis, we focused on the analysis of the mitochondrial phosphoproteome of Saccharomyces cerevisiae. We studied the mitochondrial phosphoproteome in three growth conditions: in the respiratory condition (YLAC), in the fermentable condition (YPGA) and in an intermediate one (YPGalA). We quantified around 300 mitochondrial phosphorylation sites in which 90 displayed a different level of phosphorylation according to the substrate. This study is a first step towards understanding mitochondrial phosphorylation and its mechanism. Phosphorylation sites with varying levels of phosphorylation according to their conditions are mostly located on proteins involved in energy metabolism. We localized the phosphosites on the structure of the respiratory complexes when it was possible. This allowed us to make hypotheses on the role of these residues. In order to normalize the quantity of phosphorylation sites in the three growth conditions, we also studied the mitochondrial proteome in the three conditions. These results helped us to understand the energetic metabolism of galactose, which is surely intermediate between respiration and fementation, a question still debated nowadays.Finally this proteomic and phosphoproteomic study is a step forward in the comprehension of regulation of mitochondria by phosphorylation. These results can be used as a model to study cancer cells because they display a deregulation in the energetic metabolism: normal cells display respiratory metabolism whereas cancer cells exhibit fermentable metabolism.The second part of this thesis was the study of two subunits of complex IV of the respiratory chain: Cox12p and Cox13p, which had been poorly studied. Moreover, two phosphorylation sites had been identified in the subunit Cox12p. First we were interested in the role of these two proteins, thus we compared the mitochondria of mutants Δcox12, Δcox13 et Δcox12Δcox13 with wild-type mitochondria. We particularly focused on the assembly and the activity of complex IV. Secondly, we analyzed the role of the two phosphosites of Cox12p: Ser7 and Ser82. We generated phosphomimetic mutants of these two residues and observed their effects on the stability and/or activity of complex IV.All of these results allowed us to identify a role of Cox12p in the stability of complex IV and a role of Cox13p in the dimerization of complex IV. Phosphorylation of Ser7 of Cox12p seemed to destabilize the complex. Moreover phosphorylation of both Ser7 and Ser82 of Cox12p seemed to modify the interaction between cytochrome c and complex IV; this hypothesis remains to be tested but is relevant according to the proximity between Cox12p and the subunit Cox2p, where the cytochrome c interacts.

Les vitamines B et les facteurs liés au métabolisme monocarboné (C1) aident à maintenir la synthèse de l'ADN, régulent l'expression des gènes, et peuvent affecter le risque de cancer. L'objectif général de cette thèse est d'étudier l'importance des biomarqueurs du C1 dans l'étiologie de trois cancers distincts qui diffèrent dans leur force d'association avec le tabagisme. Les articles inclus dans cette thèse ont été conduits au sein de deux études prospectives : l'étude prospective européenne sur la nutrition et le cancer (EPIC) et le consortium de cohortes du cancer du poumon (LC3). Dans l'article 1, nous avons étudié la relation entre les biomarqueurs du C1 et l'incidence ainsi que le pronostic du cancer de la sphère oto-rhino-laryngée (ORL) et de l'oesophage au sein de l'étude EPIC. Nous avons observé que les sujets ayant des concentrations élevées d'homocystéine avaient un risque accru de développer un cancer de la sphère ORL. Dans l'article 2, nous avons examiné la relation entre les biomarqueurs du C1 et l'incidence ainsi que le pronostic du carcinome à cellules rénales (CCR) au sein de l'étude EPIC. Nous avons constaté que les participants ayant des concentrations de vitamine B6 élevées avaient une diminution du risque de CCR avec un effet dose-réponse ainsi qu'une amélioration de la survie post-diagnostic. Dans l'article 3, nous avons étudié si les biomarqueurs du C1 sont associés au risque de cancer du poumon au sein de l'étude LC3. Dans l'ensemble, nous avons mis en exergue une faible association inverse, sans tendance claire, entre les concentrations de vitamine B6 et de folate et le risque du cancer du poumon. La principale conclusion de nos études est que les concentrations élevées de vitamine B6 sont associées à un risque plus faible de développer un CCR, et également à un meilleur pronostic chez les patients atteints de cette pathologie. Davantage d'études sont nécessaires afin d'évaluer si la vitamine B6 exerce une influence causale sur l'étiologie et la mortalité du CCR, ou si d'autres facteurs métaboliques sont impliqués / B-vitamins and factors related to one-carbon metabolism (OCM) pathway help to maintain DNA synthesis and regulate gene expression and may affect cancer risk. The overarching aim of this thesis is to investigate the importance of OCM biomarkers in the etiology of three distinct cancer sites that differed in their strength of association with smoking. Papers included in this thesis were conducted within two prospective studies, the European Prospective Investigation into nutrition and Cancer (EPIC) study and the Lung Cancer Cohort Consortium (LC3). In paper 1, we investigated if OCM biomarkers are associated with incidence and survival of cancer of the head and neck and esophagus in the EPIC study. We observed that subjects with higher concentrations of homocysteine had increased risk of developing head and neck cancer. In paper 2, we investigated if OCM biomarkers are associated with incidence and survival of renal cell carcinoma (RCC) in the EPIC study. We observed that study subjects with elevated vitamin B6 concentrations had lower risk of RCC in a dose-response fashion and improved survival following diagnosis. In paper 3, we investigated whether OCM factors are associated with lung cancer risk in the LC3 study. Overall, we observed a weak inverse association, with no clear trend, between concentrations of vitamin B6 and folate and risk of lung cancer. The most important conclusion is that elevated vitamin B6 concentrations are associated with lower risk of developing RCC, and also better prognosis among RCC cases. Further studies are warranted to evaluate if vitamin B6 exerts a causal influence on RCC etiology and mortality, or if other metabolic factors are involved