Le taux de mortalité associé aux infections par le S. aureus résistant à la méticilline (SARM) est le plus élevé parmi les infections staphylococciques. Le SARM est aussi la plus fréquente des bactéries multirésistantes, plaçant souvent le médecin en situation d'impasse thérapeutique. Une meilleure compréhension des phénomènes de résistance aux antibiotiques permettrait de mieux détecter ces bactéries et concevoir de nouveaux antibiotiques. Dans ce contexte, ces travaux de thèse visaient à étudier le phénotype de résistance à la méticilline de S. aureus par une approche métabolomique.La première partie de ce travail a été dédiée au développement des outils nécessaires aux analyses métabolomiques globales de S. aureus. Ces développements ont impliqué la mise place d'un protocole de préparation d'échantillon spécifique et multi-étapes, de méthodes de chromatographie liquide couplée à la spectrométrie de masse à haute résolution (LC/HRMS) et l'implémentation d'une base de données spectrales dédiée.Dans ces conditions, nous avons pu extraire et identifier jusqu'à 210 métabolites intracellulaires, ce qui représente la plus large couverture métabolique de S. aureus obtenue à ce jour. Dans un second temps, nous avons appliqué l'approche développée à l'étude de souches SARM et de souches sensibles à la méticilline (SASM). Après optimisation rigoureuse des conditions de culture, l'étude approfondie de 24 souches cliniques nous a permis de dégager une signature métabolique du phénotype de la résistance à la méticilline, signant notamment l'implication des voies de biosynthèse de la paroi et de la capsule bactérienne.Le séquençage complet du génome de ces 24 souches combiné à l'étude complémentaire de 16 souches de SARM de fonds génétiques différents nous a ensuite permis de souligner la pertinence de cette signature métabolique vis-à-vis du phénotype étudié. Enfin, l'étude de souches isogéniques en présence d'antibiotique a également confirmé l'implication de la synthèse de la paroi dans la distinction SARM/SASM. / The mortality rate associated with Methicillin resistant S. aureus (MRSA) is the highest among the staphylococcal infections. A better understanding of antibiotic resistance phenomena would lead to better detect these bacteria and design new antibiotics. In this context, this PhD work aimed to study the MRSA phenotype by using a metabolomics approach. The first part of this work has been dedicated to developing a global metabolomic analysis of S. aureus: i.e., the setting up of a reliable sample preparation protocol, the development of liquid chromatography-high resolution mass spectrometry methods, and the implementation of a dedicated spectral database. Under these conditions, 210 metabolites have been extracted and identified in bacterial cell extracts, which is the widest metabolic coverage of S. aureus obtained to date.Secondly, we have performed a metabolomic study of MRSA and methicillin susceptible S. Aureus (MSSA) strains. The analysis of 24 clinical strains has allowed us to highlight a metabolic signature of MRSA phenotype of which metabolites involved in bacterial wall and capsule biosynthesis pathways were part. The complete genome sequencing of these 24 strains combined with the complementary study of 16 MRSA strains of different genetic backgrounds have reinforced the relevance of this metabolic signature. Finally, the study of isogenic strains in the presence of an antibiotic has confirmed the involvement of metabolites of the wall and capsule biosynthesis pathways in the distinction of MRSA/MSSA phenotypes.

Acid whey, a byproduct of Greek yogurt production, has little commercial value due to its low protein content and is also environmentally harmful when disposed of as waste. However, as a product of microbial fermentation, acid whey could be a rich source of beneficial metabolites associated with fermented foods. This study increases understanding of acid whey composition by providing a complete metabolomic profile of acid whey. Commercial and lab-made Greek yogurts, prepared with three different bacterial culture combinations, were evaluated. Samples of unfermented yogurt mix and cultured whey from each batch were analyzed. Ultra-high-performance liquid chromatography/tandem mass spectrometry metabolomics were employed to separate and identify 477 metabolites, including many with potential health benefits similar to those provided by yogurt, such as creatine and acetylcarnitine. Examples of other metabolites identified in the acid whey include beneficial phospholipids (1,2-dilinoleoyl-sn-glycero-3-phosphocholine) and sphingolipids; compounds with neuroprotective (glycerophosphorylcholine) or cardiovascular (betaine) benefits; antimicrobial compounds (benzoic acid), and anti-inflammatory compounds (citrulline). Compared to uncultured controls, acid whey showed decreases in some metabolites associated with microbial metabolism and increases in others. Metabolite production was significantly affected by combinations of culturing organisms, and production location. Differences between lab-made and commercial samples could be caused by different starting ingredients, or environmental factors or both.

Pancreatic cancer is a malignant disease with an unfavorable prognosis. Currently, the diagnosis of pancreatic cancer is based mainly on CT, MR and EUS imaging methods, because a reliable biomarker of pancreatic cancer is not available. It is considered a success in the current clinical practice if patients suitable for surgical resection can be selected, because in this group the overall survival is slightly better than in the group where surgery is not technically possible. In the first part, the presented dissertation focuses on testing of already established imaging diagnostic methods used in common practice, i.e. EUS and EUS FNA, as well as modern examination possibilities, such as contrast endosonography, and the comparison of the accuracy of the methods used. The dominant role of the EUS FNA was confirmed. In the next phase of the work, the methodological procedure of EUS FNA collection and the preprocessing of pancreatic tissue samples was tested with regard to the quantity and quality for epigenetic examination and further testing using the prognostic role of KRAS and miR-21. It has been suggested that cytological smears are the most suitable source of biological material for DNA and miRNA analysis, where non-tumor tissue contamination is low. Although the prognostic role of KRAS is...

Motivation: Alzheimer’s Disease (AD) is a highly prevalent, neurodegenerative disease which causes gradual cognitive decline. As documented in the literature, evi- dence has recently mounted for the role of metabolic dysfunction in AD. Metabolomic data has therefore been increasingly used in AD studies. Metabolomic disease studies often suffer from small sample sizes and inflated false discovery rates. It is therefore of great importance to identify algorithms best suited for the inference of metabolic networks from small cohort disease studies. For future benchmarking, and for the development of new metabolic network inference methods, it is similarly important to identify appropriate performance measures for small sample sizes. Results: The performances of 13 different network inference algorithms, includ- ing correlation-based, regression-based, information theoretic, and hybrid methods, were assessed through benchmarking and structural network analyses. Benchmark- ing was performed on simulated data with known structures across six sample sizes using three different summative performance measures: area under the Receiver Op- erating Characteristic Curve, area under the Precision Recall Curve, and Matthews Correlation Coefficient. Structural analyses (commonly applied in disease studies), including betweenness, closeness, and eigenvector centrality were applied to simu- lated data. Differential network analysis was additionally applied to experimental AD data. Based on the performance measure benchmarking and network analysis results, I identified Probabilistic Context Likelihood Relatedness of Correlation with Biweight Midcorrelation (PCLRCb) (a novel variation of the PCLRC algorithm) to be best suited for the prediction of metabolic networks from small-cohort disease studies. Additionally, I identified Matthews Correlation Coefficient as the best mea- sure with which to evaluate the performance of metabolic network inference methods across small sample sizes. / Graduate

No description available.

Mercury (Hg) is a ubiquitous environmental contaminant, which is capable of global atmospheric transport. As a result, even the most pristine aquatic ecosystems are affected by atmospheric Hg deposition, following which microbial transformation yield organic Hg forms, the most concerning of which is methylmercury (MeHg). Methylmercury is capable of bioaccumulation and biomagnification in food webs, resulting in potentially toxic body burdens due to regular dietary exposure in long-lived organisms at higher trophic levels. It is also a molecular mimic of some endogenous amino acids, providing a route of transfer from mother to offspring via large amino acid transporters. Exposure during neurodevelopment can lead to serious, irreversible neurological dysfunction, associated with a variety of cognitive and motor abnormalities across species. The present studies evaluate the effects of maternally-transferred dietary MeHg, at environmentally relevant concentrations on early life stage fathead minnows (Pimephales promelas). Embryos were collected from adult fatheads exposed to one of three diets with varying concentrations of MeHg for 30 days. Adult reproductive metrics were also monitored over the course of the study, with results indicating no effects on spawning frequency, clutch size, or total egg output. In embryos, Hg concentration was a function of female diet and the duration (number of days) of female exposure. Offspring spawned in tanks administered the low Hg diet displayed altered embryonic movement patterns (hyperactivity), decreased time to hatch, decreased mean larval size, and alterations to several metabolite abundances when compared with controls. Significantly altered metabolites include those associated with cellular energetics, fatty acid metabolism, and polyamine synthesis, indicating current environmental exposure scenarios are sufficient to disrupt important cellular pathways. Dysregulation of the dopaminergic system of embryos is also characterized, and may be a possible mechanism by which hyperactive behaviors are observed in these embryos. Offspring from tanks administered the high Hg diet exhibited delayed hatching, increased mortality, and physiological abnormalities. Brain tissue of exposed adults from the low diet were dissected into regions, and also evaluated for alterations in dopamine cycling. Collectively, these results indicate current exposure scenarios in North American lakes and rivers are sufficient to cause reductions in fitness and survival of early life stage fish. The potential for community structure impacts exists, as sensitive individuals and species become disproportionately affected by chronic, low-level MeHg exposure.

No description available.

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.

Le méthane, puissant gaz à effet de serre (GES), est produit dans le rumen des bovins par la fermentation microbienne anaérobie des aliments. Cette production est également responsable d’une perte d’énergie pour l’animal représentant 6 % à 8 % de l’apport alimentaire. Pour ces raisons, plusieurs travaux de recherches sont entrepris pour réduire ces émissions, en jouant sur la composition des aliments ou sur l’utilisation d’additifs alimentaires. Actuellement, la mesure des émissions de méthane se fait par différentes techniques, qui présentent des inconvénients, notamment le coût ou encore la difficulté d’application à grande échelle sur le terrain. De ce fait, de nombreuses recherches sont menées pour trouver des méthodes alternatives ou « proxy » pour la mesure indirecte du méthane. Mon travail de thèse a pour objectif de rechercher des marqueurs associés à la production de méthane chez la vache laitière, par une approche métabolomique multiplateforme. Ces marqueurs, une fois validés, pourront être dosés par des méthodes simples de laboratoire et appliqués à grande échelle pour évaluer les émissions de méthane. Étant donné le caractère exploratoire de l’approche et pour maximiser nos chances de réussite, nous avons combiné l’utilisation d’un inhibiteur spécifique de la méthanogènèse et l’analyse des profils métaboliques de quatre matrices (le lait, le plasma, le contenu ruminal et les urines)  avec différentes plateformes analytiques complémentaires : la résonnance magnétique nucléaire et la chromatographie liquide couplée à la spectrométrie de masse. Les échantillons biologiques proviennent d’une expérimentation animale utilisant 25 vaches Holstein primipares, et séparées en deux groupes suivant la présence ou non de l’additif anti-méthane dans la ration. Nous avons mesuré une diminution des émissions de méthane de 22,7% chez les animaux ayant reçu l’additif. Les analyses statistiques multivariées ont montré des profils métaboliques différents entre les 2 groupes d’animaux dans le plasma (27 métabolites discriminants) et le lait (16 métabolites discriminants). Les travaux sur les urines et le contenu ruminal sont en cours. L’analyse des réseaux métaboliques a mis en évidence plusieurs voies métaboliques impactées par la réduction des émissions de méthane impliquées dans le métabolisme énergétique ou encore dans celui des acides-aminés. / Ruminants produce significant amount of enteric methane, which is a major contributor of greenhouse gas emissions from agricultural origin. This production also results in a loss of 6 to 8% of the energy present in the diet. The reduction of enteric methane emissions from ruminants is an active area of research that requires the use of expensive, constraining measurement methods such as respiratory chambers or the use of a tracer gas that are difficult to use outside experimental farms. Therefore, there is a need of alternative, non-invasive measurement methods that can be used on large number of animals. In this work, we used an open metabolomic approach for identifying potential metabolic biomarkers associated with the production of methane in dairy cows. These discriminant metabolites, once validated, may be used for monitoring methane emissions under field conditions. To maximize the chance of finding such biomarkers, we used a multiplatform metabolomics approach (nuclear magnetic resonance and mass spectrometry) to better cover the metabolome and analyzed four biological matrices, namely milk, plasma, ruminal fluid and urine. We carried out a study involving 25 primiparous Holstein dairy cows that were divided into two groups fed a diet with or without a specific anti-methanogenic compound. We measured 22.7% reduction of methane emissions in the Treated group compared to the Control group. We identified 27 discriminant metabolites in plasma and 16 discriminant metabolites in milk. Metabolic network analysis highlighted pathways involved in energy and amino acids metabolism suggesting a general effect on the host animal induced by a reduction in methanogenesis.

Dans un marché globalisé où 40% du vin consommé est importé, le contrôle de la traçabilité est un enjeu majeur de la filière viti-vinicole. L'authentification du vin est le processus pouvant faire appel à différentes méthodes analytiques devant pouvoir contrôler trois paramètres fondamentaux : l’origine géographique, le cépage et le millésime. La spectroscopie RMN 1H quantitative (RMNq) est aujourd'hui considérée comme un outil très prometteur pour l'étude de l'authenticité des vins. Lors de cette thèse, une technique de dosage de 40 composés majoritaires des vins par RMN 1H a été développée. Elle permet l’acquisition d’une information riche et complexe en une seule analyse non-spécifique. La capacité de cette technique à authentifier un vin a été démontrée suite à une collaboration avec le Château Mouton-Rothschild, par comparaisons avec des analyses officielles réalisées par la DGDDI et DGCCRF de Pessac (SCL). La détermination d’un seuil de conformité a été établie en prenant en compte l’évolution naturelle des vins en bouteille. Une étude de caractérisation des vins rouges de Bordeaux a été menée. La singularité de ces vins a été observée par comparaison avec d’autres vins français, mettant en évidence des métabolites caractéristiques des vins de Bordeaux. Les résultats fournissent une description globale du potentiel de la RMN 1H pour l’authentification des vins. / In a globalized market where 40% of the wine consumed is imported, traceability control is a major challenge for the wine industry. Wine authentication is the process that can use different analytical methods able to control three fundamental parameters: geographical origin, grape variety and vintage. Quantitative 1H NMR spectroscopy (qNMR) is now considered as a very promising tool for studying wine authenticity. During this thesis, a technique was developed for the determination of 40 major wine compounds by 1H NMR. It allows the acquisition of rich and complex information in a single non-specific analysis. The ability of this technique to authenticate a wine has been demonstrated following a collaboration with Château Mouton-Rothschild, by comparison with official analyses carried out by the DGDDI and DGCCRF of Pessac (SCL).The determination of a compliance threshold has been established by taking into account the natural evolution of bottled wines. A characterization study of Bordeaux red wines was carried out. The singularity of these wines was observed in comparison with other French wines, highlighting the characteristic metabolites of Bordeaux wines. The results provide a global description of the potential of 1H NMR for wine authentication.