Au cours des dernières décennies, le régime alimentaire a subi une transition sans précédent, avec une augmentation de la consommation de protéines, de lipides et de glucides simples, et la diminution des apports en fibres. Par ailleurs, au-delà de la composition, la structure des aliments joue un rôle essentiel sur les cinétiques de digestibilité et la biodisponibilité des nutriments, modulant ainsi leur accessibilité pour microbiote dans le côlon. L’impact de la structure d’une matrice alimentaire complexe, formée de protéines et de lipides, sur le microbiote a été analysé de façon intégrée et a montré in vivo que la structure seule, dans le contexte d’un régime équilibré, pouvait altérer la composition du microbiote dans les zones distales et proximales que sont l’iléon et le cæcum. L’émulsion de protéines natives en phase liquide continue avec de fines gouttelettes protéolipidiques a arboré des protéines moins digestibles que l’émulsion de protéines dénaturées, en phase gélifiée, solide, avec de grandes gouttelettes. D’autre part, les lipides de l’émulsion solide étaient, à l’inverse, moins digestibles. Les protéines non digérées de l’émulsion liquide ont favorisé in vivo, les communautés de Lactobacillus et de Copprococcus tout en activant plus fortement les métabolismes de protéolyse. Inversement, les communautés de Bifidobacterium et d’Akkermansia muciniphila ont vu leurs abondances augmenter chez les rats consommant l’émulsion solide. Le deuxième objectif de ce travail de thèse a alors été d'analyser la capacité d'espèces prévalentes du microbiote intestinal humain à métaboliser des protéines non digérées. Nous avons montré, par le suivi des cinétiques de croissance et des productions de métabolites spécifiques, que les protéines du lait étaient une source d'énergie pour B. caccae, P. distasonis, B. longum et B. cocccoides en milieu pauvre ainsi qu'en milieu riche. Dans ces mêmes conditions, le transcriptome de B. caccae a montré la sur-expression de gènes codant pour des peptidases de specifités différentes, pour la production d'indoles, de GABA et de fimbriae. Ces travaux apportent des informations nouvelles sur l'impact de la structure sur l'écosystème digestif, et ouvre des portes pour le développement de nouveaux aliments. / Over the past decades, diet in developed countries has undergone an unprecedented transition, with increased intakes of protein, fat and high glycemic index carbohydrates. The first goal of this PhD work was to investigate how, beyond its composition, the food structure itself could play a part in nutrient digestibility and bioavailability, and consequently modulate the microbiota. We showed in vivo that the structure of proteino-lipidic emulsions modulated peptides transporters, and protein fermentation. The native proteins emulsion in a continuous liquid phase, with fine proteolipid droplets, was less digestible and led to more protein fermentation. It modified the gut microbiota composition in the distal and proximal intestinal sections and increased Lactobacillus and Coprococcus communities. A second in vivo study, using 15N labelled emulsions allowed us to disentangle the digestibility from the transit time effect. The second objective of the PhD was to characterize the capacity of prevalent human gut bacterial species to use undigested proteins as energy source. By monitoring growth kinetics and the production of specific metabolites, we showed that B. caccae, P. distasonis, B. longum et B. cocccoides could use whey protein as energy source. In addition we measured in B. caccae transcriptome, the over-expression of genes encoding for distinct peptidases, but also of GABA and indole pathways, and fimbriae biosynthesis. These data provide new insights on the relationships between food structure and the digestive ecosystem and could lead to the design of new functional food.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19260号 / 理博第4115号 / 新制||理||1592(附属図書館) / 32262 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24111号 / 農博第2516号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5402(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 北島 薫, 教授 井鷺 裕司, 准教授 東樹 宏和 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the most costly virus to the swine industry, worldwide. This study explored the application of deep sequencing techniques to understand better the virus-host interaction. On the virus side, PRRSV exists as a quasispecies. The first application of deep sequencing was to investigate amino acid substitutions in hypervariable regions during acute infection and after virus rebound. The appearance and disappearance of mutations, especially the generation of a new N-glycosylation site in GP5, indicated they are likely the result of immune selection. The second application of deep sequencing was to investigate the quasispecies makeup in pigs with severe combined immunodeficiency (SCID) that lack B and T cells. The results showed the same pattern of amino acid substitutions in SCID and normal littermates and no different mutations were identified between SCID and normal littermates. This suggests the mutations that appear during the early stages of infection are the product of the virus becoming adapted to replication in pigs. The third application of deep sequencing was to investigate the locations of recombination events between GFP-expressing PRRSV infectious clones. The results identified different cross-over occurred within three conserved regions between EGFP and GFPm genes. And finally, the fourth goal was applied to develop a set of sequencing tools for analyzing the host antibody repertoire. A simple method was developed to amplify swine VDJ repertoires. Shared and abundant VDJ sequences that are likely expressed by PRRSV-activated B cells were determined in pigs that had different neutralization activities. These sequences are potentially correlated with different antibody responses.

The aim of this thesis is to obtain a better understanding of mutation rates within as well as between the genomes of humans and chimpanzees using data generated by high throughput sequencers. I will start with a review of the field and an overview of the technologies and protocols used to generate and analyse high throughput sequencing data. I apply some of the discussed techniques to show that there is evidence of a selective advantage of pathogenic de novo mutations in the Fibroblast Growth Factor Receptor 3 gene in the male germ line of humans. Furthermore, I use some of the methods to generate a map of genome-wide sequence variation in Western chimpanzees. Ever since Darwin [Darwin, 1871] and Huxley [Huxley, 1863] postulated more than a century ago that African great apes are our closest living evolutionary relatives, the study of chimpanzee individuals is of great scientific interest from an evolutionary point of view, as comparisons between the genomes of human and chimpanzee offer the potential to help to understand the molecular basis for similarities and differences between the two species. I use the generated data to explore the breadth of the nucleotide diversity in the chimpanzee genome in order to shed light on whether or not the local variation in mutation rate has been conserved since the divergence of the two species and to place human nucleotide diversity into perspective with an evolutionary closely related species. I explore the relationship of nucleotide diversity in chimpanzees with specific large-scale genome features to reveal a number of highly significant correlations which explain over 40% of the observed variation. I use data from the 1000 Genomes Project to examine the occurrence of ancestral polymorphisms shared between human and chimpanzee on a genome-wide scale. These ancestral polymorphisms do not only influence fine-scale divergence rates across the genome in very closely related species, they are also good candidates for regions under balancing selection and thus, they are a useful tool to study long-time population demographics and speciation. Using these variants, I postulate that long-term balancing selection may be more common than previously believed. I conclude with a discussion of the results contained in the body of the thesis and suggest a number of areas for future research.

In this thesis, I investigate the genetics of neuropsychiatric disorders by analysing large data sets derived from high-throughput experiments, using novel comparative genomics approaches. In the first project, I explore characteristics of rare, de novo copy number variants identified among autism patients by employing various bioinformatics resources including Mouse Genome Informatics phenotypes, Gene Ontology terms, and protein-protein interactions. I describe how I objectively identified a number of mouse model phenotypes that are significantly associated with autism, and that provide insight into the aetiologies for both copy number deletions and duplications. In the second project, I investigate the genetics of obsessive-compulsive disorder by resequencing genomic regions of human case-control cohorts and the best spontaneous disease model organisms, namely dogs with canine compulsive disorder, and breed-matched controls. Targeted sequencing experiments yielded a large number of high-quality genetic variants in both humans and dogs. I prioritised variants and genes using case- control comparisons and functional annotations such as types of mutation, evolutionary conservation status and regulatory marks. In turn, I generated several hypotheses that are experimentally tractable. Replication of these findings in a larger cohort is necessary, although it lies beyond the scope of this thesis. Results from both projects indicate that the analytical frameworks employed in this thesis could be profitably applied to other neuropsychiatric disorders.

Résumé indisponible / The objective of this thesis focuses on the study and the development of bioinformatics platforms and tools on the grid. The second objective is to develop applications in molecular epidemiology and metagenomics based on these tools and platforms. Based on the studies of existing bioinformatics platforms and tools, we propose our solution: a platform and a portal for molecular epidemiology and high throughput metagenomics on the grid. The main idea of ​​our platform is to simplify the submission of jobs to the grid via the pilots jobs (jobs generic that can control and launch many real tasks) and the PULL model (tasks are retrieved and executed automatically). There are other platforms that have similar approaches but our platform focuses on the simplicity and the saving time for the submission of jobs. Bioinformatics tools chosen to deploy the platform are popular tools that can be used in many bioinformatics analyses. We apply a workflow engine in the platform so that users can make the analysis easier. Our platform can be seen as a generalized system that can be applied to both the epidemiological surveillance and metagenomics of which two use cases are deployed and tested on the grid. The first use case is used to monitor bird flu. The approach of this application is to federate data sequences of influenza viruses and provide a portal with tools on the grid to analyze these data. The second use case is used to apply the power of the grid in the analysis of high throughput sequencing of amplicon sequences. In this case, we prove the efficiency of the grid by using our platform to gridifier an existing application, which has much less performance than the gridified version.

Les ARN (Acide RiboNucléique) remplissent de nombreuses fonctions clés dans le vivant. Ils peuvent être support de l'information génétique, régulateurs de celle-ci. Visualiser ces molécules au sein d'une cellule représenterait une étape importante vers une meilleure compréhension de la régulation de l'expression des gènes. Les ARN fluorogènes tels que Spinach et Mango sont des outils extrêmement prometteurs pour atteindre cet objectif. Cependant ces deux ARN fluorogènes présentent une brillance limitée. La Compartimentation in vitro assistée par microfluidique (µCIV) est un outil très prometteur dont notre groupe a démontré l’efficacité pour l’évolution d’ARN. Dans le cadre de cette thèse, la µCIV a été adaptée à la sélection d'aptamères d'ARN fluorogènes pour en améliorer les propriétés (surtout la brillance). De plus, l’utilisation conjointe du séquençage haut débit a permis l’optimisation très rapide et semi-automatisée à la fois d’aptamères mais aussi de biosenseurs fluorogènes. Ainsi, cette thèse a permis de mettre en place et d’exploiter des technologies de criblage robustes pour la découverte de nouveaux aptamères d'ARN et de biosenseurs. / RNA is a key molecule in gene expression and its regulation. Therefore, being able to monitor RNA through live-cell imaging would represent an important step toward a better understanding of gene expression regulation. RNA-based fluorogenic modules are extremely promising tools to reach this goal. To this end, two light-up RNA aptamers (Spinach and Mango) display attractive properties but they suffer from a limited brightness. Since previous work in the group demonstrated the possibility to evolve RNA using microfluidic-assisted in vitro compartmentalization (µIVC), this technology appeared to be well suited to improve light-up aptamers properties by an evolution strategy. Therefore, the µIVC procedure was adapted to fluorogenic RNA aptamers to improve their properties (especially the brightness). Finally, using µIVC in tandem with high-throughput sequencing (NGS) allowed further developing the technology into a more integrated and semi-automatized approach in which RNAs and biosensors are selected by µIVC screening and the best variants identified by a bioinformatics process upon NGS analysis. To summarize, this thesis allowed establishing robust µIVC screening workflows for the discovery of novel efficient light-up RNA aptamers as well as metabolites biosensors.

Estudos com farinha de banana verde (FBV), rica em amido resistente, mostram efeitos positivos sobre a saciedade, resposta glicêmica e melhora do funcionamento intestinal. Entretanto, pouco se sabe sobre a capacidade da FBV em estimular seletivamente o crescimento e/ou atividades de microbiota intestinal benéfica e os efeitos fisiológicos do consumo habitual. No presente trabalho foi investigado o efeito da ingestão regular e descontinuada de FBV sobre a microbiota intestinal em voluntários saudáveis, bem como as interações com hormônios relacionados à fome e saciedade, funcionamento intestinal e homeostase da glicose. Para tanto foi realizado estudo de intervenção, duplo cego paralelo controlado com placebo, no qual voluntários saudáveis consumiram FBV ou maltodextrina, veiculadas através de sopa prontas congeladas, três vezes por semana e durante seis semanas. Os resultados evidenciaram que a FBV pode aumentar a saciedade, promover redução no aporte energético de refeições subsequentes (14%) e melhorar o funcionamento intestinal. Ao mesmo tempo reduz a secreção plasmática de insulina no jejum e o Índice HOMA2-RI em 20%, sinalizando aumento na sensibilidade à insulina. A análise da microbiota intestinal utilizando o rDNA 16S mostrou que existem dois grupos distintos de indivíduos, os quais respondem diferentemente ao consumo de FBV. O consumo de FBV por voluntários, cujo microbioma era mais abundante no gênero Prevotella, apresentou aumento de genes envolvidos em vias metabólicas relacionadas à degradação anaeróbia de carboidratos (794 Kegg orthologs, FDR=0,05), como as vias do metabolismo de amido e glicose, do butirato, propionato. Paralelamente outros genes indicaram redução de algumas vias metabólicas, incluindo a biossíntese de lipopolissacarídeos. Este mesmo grupo de voluntários apresentou gêneros microbianos positivamente relacionados com conteúdo de ácidos graxos de cadeia curta (AGCC), em padrão distinto do outro grupo de voluntários que consumiu FBV e do grupo Controle. Foi possível demonstrar que o consumo de FBV pode promover a modulação do microbioma em indivíduos saudáveis com enterótipos distintos, trazendo efeitos benéficos para a saúde humana. / Studies with Unripe Banana Flour, rich in resistant starch, shave shown positive effects on satiety, glycemic response and improved intestinal function. Nevertheless, little is known about its capacity to selectively stimulate the intestinal microbiota\'s activity, or the physiological effects of its habitual consumption. This study investigated the effects of the regular, discontinued ingestion of UBF on the intestinal microbiome in healthy volunteers, as well as effects on hormones related to satiety, intestinal function and glucose homeostasis. To achieve these goals, a double blind, parallel, placebo controlled study was designed, in which healthy volunteers ingested UBF or maltodextrin added to a standardized frozen soup meal, 3 times a week for 6 weeks. The results showed that UBF can improve satiety, promote a reduction in energy intake at subsequent meals (14%) and improve intestinal function. At the same time, it reduces plasmatic secretion of fasting insulin and e the HOMA2-RI index by 20%, signaling an increase in insulin sensitivity. The analysis of the microbiome using the 16S rDNA gene showed that there are two clusters of individuals, which respond differently to the dietary intervention. The UBF consumption by volunteers with a Prevotella dominant microbiome showed an increase in genes related to anaerobic carbohydrate degradation (794 Kegg orthologs, FDR=0,05), such as members of the starch and glucose metabolism, propanoate metabolism and butyrate metabolism. At the same time, other genes were reduced, including the biosynthesis of lipopolysaccharides. The same volunteers presented several microbial groups positively correlated with the short chain fatty acids (SCFA) present in the fecal samples analyzed. This was a distinct pattern to that observed for the remaining volunteers. We demonstrated that the consumption of UBF can promote the overall health of the human host as well as the modulation of the intestinal microbiome in healthy individuals and that this effect is dependent on the enterotype present.

Le campagnol terrestre, Arvicola terrestris, occasionne en France, lors de ses pullulations cycliques interannuelles, d’importants dégâts aux prairies de montagne. Un groupe de travail constitué des équipes de recherche de l’Université de Franche-Comté (UFC), de l’INRA (Centre de Biologie et de Gestion des Populations) et d’organismes agricoles (Fédération Régionale de Défense contre les Organismes Nuisibles de Franche-Comté, FREDON), ont privilégié une approche « systémique » dans laquelle les interactions entre les campagnols, leur habitat (paysage, prédateurs) et les pratiques agricoles sont analysées de façon hiérarchisées (spatialement et temporellement). Un des objectifs est de mettre en évidence le plus grand nombre possible de facteurs de contrôle sur lesquels il est possible d’agir, et l'échelle à laquelle ces actions sont pertinentes. Ces études ont permis d’initier une stratégie, expérimentée avec succès, notamment en Franche-Comté et en Auvergne, et qui privilégie la lutte raisonnée. Il subsiste néanmoins des zones d’ombre relatives à la compréhension du cycle, notamment concernant les déterminants de la phase de déclin. Le rôle du cortège de pathogènes (parmi lesquels certains peuvent être transmis à l’homme) reste pour l’instant sujet de débat dans la littérature scientifique. La compréhension des facteurs clés déterminant cette phase devrait permettre aux éleveurs de mieux anticiper les impacts économiques et adopter les stratégies de contrôles des population les plus adéquates. Objectifs de la thèse (1) Tester les hypothèses des pathogènes et de la sénescence pour expliquer le déclin démographique. (2) Rechercher des indicateurs biologiques (diversité des pathogènes et/ou indicateurs immunitaires) qui permettent de prédire les phases de déclin et d’anticiper des mesures agricoles appropriées pour restaurer les prairies. (3) Evaluer le rôle de la transition entre la phase de forte densité et de déclin démographique pour l’émergence de pathogènes circulants par les populations de campagnols et responsables de maladies humaines. Méthodologie générale Des suivis de populations avec des prélèvements réguliers (mensuels) seront réalisés sur plusieurs populations (répliquats) dans la période qui encadre le déclin démographique. Des méthodes fondées sur le séquençage à haut débit (NGS : Next Generation Sequencing) pour l’épidémiologie permettent d’établir des catalogues complets des pathogènes (virus, bactéries, parasites) hébergées par les populations, et d’en mesurer les prévalences. / Context In France, during cyclic population surges, water voles, Arvicola terrestri, cause extensive damage to mountain grassland. A working group consisting of researchers from the University of Franche-Comté (UFC), INRA (Centre de Biologie et de Gestion des Populations) agricultural organizations (Fédération Régionale de Défense contre les Organismes Nuisibles de Franche-Comté, FREDON) are working on systems approach in which interactions between voles, their habitat (landscape, predators) and agricultural practices are analysed hierarchically (in space and time). One of the objectives is to highlight the largest possible number of control factors on which it is possible to act, and the scale at which these actions are relevant. These studies have helped initiate a strategy, successfully tested in Franche-Comté and in Auvergne, which promotes the integrated control of water vole populations. Nevertheless, there are still grey areas in the understanding of the cycle, particularly on the determinants of the decline phase. The role of pathogen communities (some species may even be transmitted to humans) so far remains the subject of debate in the scientific literature. The understanding of the key factors determining this phase should allow farmers to better anticipate economic impacts and to adopt optimal strategies for vole population control Objectives: (1) To test the pathogens and senescence hypotheses in order to explain the population decline. (2) To look for biological indicators (diversity of pathogens and / or immune indicators) that may predict the decline phase in order to anticipate appropriate measures to restore grasslands. (3) To assess the role of the transition between high population density phase and the decline phase for the emergence of pathogens in vole populations that may cause human diseases.General Methodology Population monitoring with regular (monthly) sampling will be made on several populations (replicates) in the period that brackets the vole population declines. Methods based on Next Generation Sequencing (NGS) makes it possible to establish extensive catalogues of pathogens (viruses, bacteria, other parasites) hosted by vole populations and to measure the prevalence.