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Le pangénome est l’ensemble des gènes uniques retrouvé chez une espèce. Dans le cas des espèces procaryotes, notamment celles qui sont présentes dans le microbiote intestinal humain, la variation du contenu en gène est caractérisée par des événements de gain de gènes principalement par transfert horizontal de gènes (THG) et de perte de gène. Cette variation du contenu en gène peut être plus rapide que le taux de mutation et permettre aux microbes de s’adapter rapidement à des pressions sélectives. Cela justifie donc l’étude de l’évolution pangénomique des procaryotes sur des échelles de temps humaines qui sont considérées comme étant courtes du point de vue évolutif, par exemple de l’ordre de quelques années. La plupart des études sur ce sujet impliquent des espèces relativement distantes qui ont divergé depuis des millions d’années. De plus, l'équilibre des forces évolutives majeures impliquées, telles que le THG, la sélection, la dérive génétique et les mutations, n’est pas clairement défini et est au cœur d’un débat dans la littérature. Ce projet de maîtrise permet donc d’élargir le portrait évolutif des pangénomes de procaryotes en s’intéressant à l’évolution des gènes transférés horizontalement, aussi appelés gènes mobiles, sur de courtes échelles de temps. Pour ce faire, nous allons d’abord passer en revue la littérature pertinente en lien avec ce sujet, notamment les méthodes employées pour détecter les gènes mobiles et les modèles d’évolution pangénomique. Nous allons ensuite analyser l’évolution d’une collection de 37 853 gènes mobiles impliqués dans des THG récents détectés dans le microbiote intestinal d’individus provenant d’Amérique du Nord ou des îles Fidji. Pour détecter des signatures évolutives des forces en action, nous estimerons divers paramètres de génétique des populations à partir de l’alignement entre les lectures de séquençage métagénomique de 176 microbiotes fidjiens et cette collection de gènes mobiles. Nous expliquerons aussi l’outil de simulations évolutives que nous avons développé afin de valider et expliquer certaines de nos observations. Sans exclure la présence de pressions de sélection pour des gènes mobiles ayant des fonctions spécifiques, les données réelles et les simulations nous amènent à conclure que l’évolution des gènes mobiles sur de courtes échelles de temps peut être expliquée par un modèle d’évolution où les gènes mobiles ne sont pas largement adaptifs à leurs hôtes humains ou microbiens, contrairement à ce qui est parfois observé sur de longues échelles de temps évolutif. / The pangenome is the collection of unique genes found in a species. For prokaryotes, especially those present in the human gut microbiota, variation in gene content is characterized by gene gain through horizontal gene transfer (HGT) and gene loss. In human gut, gene content variations can occur at faster rates than mutation, which allow microbes to adapt rapidly to environmental changes. This justifies the study of the prokaryotes pangenome evolution on human time scales which are considered evolutionarily short, e.g. in the order of few years. Most studies about the evolution of prokaryotic pangenomes involve relatively distant species that have diverged since millions of years. In addition, the balance of major evolutionary forces involved, such as horizontal transfer, selection, genetic drift, and mutations, is not clearly defined and is debated in literature. This master's project therefore aims to broaden the evolutionary portrait of prokaryotic pangenome evolution by focusing on near-term evolution. To do this, we will first review the relevant literature related to this topic, including the methods used to detect mobile genes and the pangenome evolution models. We will then analyze the evolution of a pre-existing collection of 37 853 mobile genes involved in recent HGT events detected in the gut microbiota of individuals from North America and Fiji Islands. To detect evolutionary signatures of the forces in action, we will estimate various population genetics parameters from the alignment between metagenomic sequencing reads of 176 Fijian microbiomes and this collection of mobile genes. We will also explain the evolutionary simulation tool that we have developed in order to validate and explain some of our observations. While we don’t exclude the importance of selection for specific cellular functions for pangenome evolution, we found that the near-term evolution of mobile genes can be explained by a model in which mobile genes can spread selfishly without being largely adaptive to their human or microbial hosts, contrarily to what is often observed over longer evolutionary time scales.

The effect of gluten-free diet on β-cell residual capacity, immune function and gut microbiome in children with newly diagnosed type 1. diabetes Abstract The pathophysiology of the onset and progression of type 1 diabetes (T1D) is not fully understood. Gluten has a proinflammatory effect on the immune system and is therefore considered as one of the factors affecting the onset and progression of T1D. The aim of the thesis is to allow a complex insight into the role of the GFD on the residual β-cell capacity, T1D control, gut microbiome, gut permeability, subtypes of immune cells and the effect of gut microbiome transfer into germ-free non-obese diabetic (NOD) mice on the incidence of diabetes. On the group of 45 children with T1D (26 intervention group, 19 control group) we proved the association of the GFD with slower decrease of β-cell residual capacity (the difference in the trend of C-peptide decrease 409 pmol/l/year; p = 0,04) and lower HbA1c (by 7,8 mmol/mol; p=0,02). We also described the changes in the gut bacteria that were differentially abundant after the administration of the GFD and the changes in abundance of the regulatory and effector immune cells. We showed there was no change in the gut permeability with respect to the study group. We also proved that the transfer of human gut microbiota...

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<p>  Fetal Alcohol Syndrome (FAS) is the most serious form of Fetal Alcohol Spectrum Disorders (FASD) and the most prevalent neurodevelopmental disorder in North America. Patients with FAS may exhibit cognitive problems with working memory, manipulating information, and reduced executive functioning. Additionally, previous studies exhibited that stress responses are affected by prenatal alcohol consumption Gut microbiota compositions can also influence stress responses and memory, as several studies have shown strong relationships between the enteric gut system and the brain. However, few studies have examined how prenatal alcohol exposure’s effects on the gut microbiome and neuroinflammatory responses. For this study, pregnant HsdBlu:LE Long Evans rats were treated with either a dry diet, liquid diet, or liquid diet with alcohol. On day 28 and 42 after birth, three male and three female adolescent pups from each treatment group had their gut microbiome (fecal samples) analyzed through 16S rRNA amplicon sequencing. Brain histology staining of the cortex and hippocampus regions was also done to evaluate changes in the CNS through microglial counts and morphology analysis. There were no significant differences in alpha diversity of the fecal microbiome between groups of pups based on prenatal alcohol exposure (PAE), sex, age, the interaction of PAE and sex, or in the morphology of cortex microglia. However, analysis of beta diversity using Bray-Curtis dissimilarity and weighted UniFrac suggested distinct microbial communities between the treatment groups based on PAE and the interaction of PAE, sex, and the interaction between PAE and sex. Microglial count comparisons by PAE or sex were only statistically different in the cortex (p ≤ 0.005). The significance of this study suggests that there are some associations between the gut microbiome and CNS inflammation in rats with PAE. Based on these findings, 11 future studies may implement therapeutics such as antibiotics or probiotics to mitigate cognitive or neural symptoms of FASD affected individuals. </p>

Les communautés bactériennes sont constituées d’un grand éventail d’espèces pouvant interagir entre elles dans des environnements spatialement hétérogènes tels que le sol, les plantes ou l'intestin humain. À quel point ces interactions stimulent ou entravent la diversité du microbiome demeure inconnu. Historiquement, deux hypothèses ont été proposées pour expliquer comment les interactions interespèces pourraient influencer la diversité. L’hypothèse ‘l’écologie contrôle’ (EC) prédit une relation négative, dans laquelle l'évolution ou la migration de nouvelles espèces est freinée à mesure que les niches se saturent. En revanche, l’hypothèse ‘la diversité engendre la diversité’ (DBD) prédit une relation positive, où la diversité existante favorise l'accumulation d'une plus grande diversité à travers des interactions telles que la construction de niche. De nombreuses études ont investigué ces modèles chez les vertébrés ou les plantes, et certaines les ont testés sur des bactéries en culture ; mais le modèle qui régit les communautés bactériennes naturelles demeure inconnu. En utilisant les données du gène ARN ribosomique 16S provenant d’un large éventail de microbiomes, j'ai montré une relation positive générale entre la diversité des taxons et la diversité des communautés de niveaux taxonomiques plus élevés. Cette observation est conforme à l’hypothèse du DBD, mais cette tendance positive plafonne à des niveaux élevés de diversité en raison des limites physiques de la niche. Ensuite, j'ai observé que le modèle DBD restait valide à une résolution plus fine, en analysant la variation génétique intra espèce dans les métagénomes des microbiomes intestinaux humains. Conformément au DBD, j'ai observé que le polymorphisme génétique ainsi que le nombre de souches intra espèces étaient positivement corrélés avec la diversité Shannon de la communauté. Dans le chapitre 3, j'ai examiné les interactions antagonistes entre V. cholerae et ses phages virulents et la manière dont ces interactions affectaient le cours de l’infection et la diversité génétique de V. cholerae chez les patients infectés. J'ai quantifié les abondances relatives de V. cholerae et des phages virulents associés dans plus de 300 métagénomes provenant de selles de patients atteints de choléra, tout en tenant compte de leur exposition aux antibiotiques. Les phages et les antibiotiques ont supprimé V. cholerae et ont été associés à une déshydratation légère chez les patients. J'ai également investigué les mécanismes de défense contre les phages dans V. cholerae et découvert que les éléments connus de résistance aux phages (integrative conjugative elements, ICEs) étaient associés à de faibles rapports phage: V. cholerae. J’ai pu montrer aussi que lorsque les ICEs ne sont pas détectés, la résistance aux phages semble être acquise par l’accumulation de mutations ponctuelles non synonymes. Mes résultats valident que les phages virulents sont un facteur qui protège contre le choléra tout en sélectionnant la résistance dans le génome de V. cholerae. / Bacterial communities harbor a broad range of species interacting within spatially heterogeneous environments such as soil, plants or the human gut. The extent to which these interactions drive or impede microbiome diversity is not well understood. Historically, two hypotheses have been suggested to explain how species interactions could influence diversity. The ‘Ecological Controls’ (EC) hypothesis predicts a negative relationship, where the evolution or migration of novel species is constrained as niches become filled. In contrast, the ‘Diversity Begets Diversity’ (DBD) hypothesis predicts a positive relationship, with existing diversity promoting the accumulation of further diversity via niche construction and other interactions. Many studies investigated these models in vertebrates or plants, some focused on cultured bacteria, but we still lack insights into how natural communities are assembled in the context of these two hypotheses. Using 16S RNA gene amplicon data across a broad range of microbiomes, I showed a general positive relationship between taxa diversity and community diversity at higher taxonomic levels, consistent with DBD. Due to niche’ limits, this positive trend plateaus at high levels of community diversity. Then, I found that DBD holds at a finer resolution by analyzing intra-species strain and nucleotide variation in sampled metagenomes from human gut microbiomes. Consistent with DBD, I observed that both intra-species polymorphism and strain number were positively correlated with community Shannon diversity. In Chapter 3, I investigated the antagonistic interactions between V. cholerae and its virulent phages and how these interactions affect the course of the infection and the within V. cholerae genetic diversity in natural infections. I quantified relative abundances of Vibrio cholerae (Vc) and associated phages in 300 metagenomes from cholera patients stool, while accounting for antibiotic exposure. Both phages and antibiotics suppressed V. cholerae and were inversely associated with severe dehydration. I also looked at V. cholerae phage-defense mechanisms and found that known phage-resistance elements (integrative conjugative elements, ICEs) were associated with lower phage:V. cholerae ratios. In the absence of detectable ICEs, phages selected for nonsynonymous point mutations in the V. cholerae genome. My findings validate that phages may protect against severe cholera while also selecting for resistance in the V. cholerae genome within infected patients.

Colorectal cancer (CRC) is one of the most common and deadly cancers in the western world. The incidence of CRC shows the tendency to rise with the increase of obesity, which is caused by current increase in fat intake, suggesting the correlation between CRC and high-fat diet (HFD). HFD-induced obesity causes gut inflammation which is also noticed in inflammatory bowel diseases (IBD) and CRC and can be seen as an important factor in CRC development. Moreover, it has been demonstrated, that while both sexes are at risk of developing CRC, men have higher incidence compared to women, showing the protective effect of estrogen. In addition, since gut microbiome is first to respond to colon inflammation, we hypothesized, that intestinal estrogen signaling could contribute to reduced initiation and progression of colon cancer by modifying the microbiota composition. For that, two experiments with two different mouse models were conducted. First part of the study concentrated on the effect of (HFD, 60%) and different estrogenic ligands (17-β estradiol, and DPN) on microbiota. Bioinformatics analysis on whole genome sequencing (WGS) data and qPCR validation were used as the methods. Here we found that estrogenic ligands achieved restoration of close-to-normal microflora after significant change initiated by HFD. We also found that microbiome in males showed stronger reaction to HFD than female microbiome, implying protective actions in females. Furthermore, the effect of ligands also proved to be stronger in males. Second part of the study concentrated on the effect of estrogen receptor β (ERβ) on microbiota for which ERβ knockout mice were used in addition to cancerogenic AOM/DSS treatment. Bioinformatics analysis on WGS data was used as the method. We found that female mice were more affected by AOM/DSS treatment compared to males, especially the mice with knockout gene. The genotype alone, however, resulted in very few differences. In summary, this project shows the effect of HFD, estrogen and ERβ expression on gut microbiota diversity. It shows that microbiome of male mice is more susceptible to dietary changes and estrogen supplementation. Likewise, it demonstrates, that the microbiome of females reacts strongly to combination of carcinogenic treatment and lack of iERβ.

<p>Accumulating evidence from randomized-controlled, full-feeding trials suggests neutral to beneficial effects of consuming lean and unprocessed red meat within a healthy dietary pattern on cardiovascular health. It is unclear how red meat interacts with the dietary pattern and the host in mediating its health effects. The gut microbiome provides a novel perspective in understanding the diet-host relationship for its abilities to metabolize dietary components including those within red meat and influence cardiovascular health. Prior to starting our study, we identified a lack of evidence in the English literature for the effects of consuming lean red meat, in unprocessed or processed forms, on gut microbiota in the context of a controlled healthy dietary pattern. Our findings presented in this dissertation demonstrate differential effects of consuming unprocessed versus processed red meat on gut microbiota. Consistent with previous research, we observed cardiovascular improvements induced by the healthy dietary pattern independent of lean red meat intakes among young adults with apparently healthy cardiovascular profiles. Importantly, with three intervention periods, we found consistent and reproducible changes in both gut microbiota and cardiovascular risk factors when repeatedly adopting and abandoning a healthy dietary pattern. Collectively, findings in this dissertation highlight the importance of gut microbiota in potentially mediating or responding to diet-induced cardiovascular improvements. Future research should investigate the changes in the function of gut microbiota induced by healthy dietary patterns containing red meat. Studies assessing the unique food matrix of processed versus unprocessed animal- or plant-based protein-rich foods are also warranted. Additionally, dietary strategies should focus on promoting healthy dietary modifications and enhancing dietary adherence for long-term cardiovascular benefits. </p>

L’avènement des inhibiteurs du point de contrôle immunitaire (ICIs) ciblant l’axe PD-1/PD-L1 a révolutionné le traitement des patients avec un carcinome pulmonaire non à petites cellules (CPNPC). Ce mémoire consolide les conclusions de trois études distinctes visant à analyser et à améliorer l'efficacité des ICIs en monothérapie chez le CPNPC. La première section explore les bénéfices associés au durvalumab suivant une chimioradiothérapie chez les patients présentant un CPNPC localement avancé, confirmant le bénéfice de survie associé aux ICI et élargissant les perspectives émises depuis l'étude PACIFIC, y compris au niveau de la valeur prédictive du PD-L1. Dans l’optique de caractériser de nouveaux biomarqueurs d’efficacité, la deuxième section souligne le rôle crucial du microbiome intestinal dans la modulation de la réponse aux ICIs, spécifiquement au niveau de la dysbiose intestinale liée aux antibiotiques. La méta-analyse proposée confirme l’impact délétère des antibiotiques sur la survie des patients traités avec un ICI, tout particulièrement lorsque l’antibiothérapie précède l’inhibition du PD-1/PD-L1. Dans le domaine des stratégies immunomodulatrices émergentes, la troisième section explore l’impact de la cryoablation chez les patients présentant un CPNPC avec PD-L1≥50% et traités avec le pembrolizumab. Sans relever un signal d’efficacité supérieure, cette étude de phase I/II confirme la faisabilité et l’innocuité de la cryoablation, une technique permettant la libération d’antigènes tumoraux en circulation sans dénaturation thermique. Ultimement, ce mémoire propose un survol des bénéfices de survie, biomarqueurs prédictifs et stratégies immunomodulatrices liés à l’utilisation des ICIs chez les patients avec un CPNPC avec l’espoir d’optimiser les paradigmes thérapeutiques existants. / The advent of immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has revolutionized the therapeutic landscape for patients diagnosed with non-small cell lung cancer (NSCLC). This thesis consolidates the findings of three distinct studies aiming to analyze and enhance the efficacy of ICI monotherapy in NSCLC. The first section delves into the real-world use of durvalumab following chemoradiotherapy in stage III NSCLC, confirming the survival benefit associated with ICI administration in this context and broadening the insights derived from the PACIFIC study, particularly regarding the predictive value of PD-L1. With the aim of characterizing new biomarkers of efficacy, the second section sheds light on the crucial role of the gut microbiome in modulating responses to ICIs, particularly intestinal dysbiosis related to antibiotics. The meta-analysis confirms the detrimental impact of antibiotics on the overall survival of patients with advanced cancer treated with ICI monotherapy, especially when antibiotic therapy precedes PD-1/PD-L1 inhibition. In the realm of emerging immunomodulatory strategies, the third section explores the impact of cryoablation in patients with NSCLC and PD-L1≥50% treated with pembrolizumab. Although the procedure did not translate into a signal of superior efficacy, the proposed phase I/II study confirms the feasibility and safety of cryoablation, a technique allowing the release of circulating tumor antigens without heat-related denaturation. Ultimately, this thesis presents a contemporary overview of survival benefits, predictive biomarkers, and immunomodulatory strategies associated with the use of ICIs in monotherapy in patients with NSCLC, with the hope of optimizing existing therapeutic paradigms.

Inositolpolyphosphate (InsPs) bilden eine ubiquitäre Gruppe an hochphosphorylierten, intrazellulären Signalmolekülen in eukaryotischen Zellen. Trotz deren Beteiligung an unzähligen biologischen Prozessen bleibt die Detektion von InsPs (insb. einzelner Enantiomere) eine Herausforderung, da die momentan verfügbaren Analysemethoden immer noch limitiert sind. In der vorliegenden Arbeit wird die stabile Isotopenmarkierung von myo-Inositol (Ins) und InsPs in Kombination mit Kernspinresonanzspektroskopie (engl. Nuclear Magnetic Resonance spectroscopy, NMR) erkundet, um diese Lücke zu schließen. Die Abhängigkeit von NMR-Daten und chemischer Struktur erlaubte die Analyse komplexer Mixturen aus InsPs aus in vitro-Experimenten und biologischen Proben. Durch stereospezifische 13C-Markierung konnten sogar Enantiomere voneinander unterschieden werden. Mit Hilfe dieser Methode wurden mehrere InsP-Stoffwechselwege untersucht. Als Erstes wurde das menschliche, Phytase-artige Enzym MINPP1 (engl. Multiple Inositol Polyphosphate Phosphatase 1) detailliert in vitro und in lebenden Zellen charakterisiert. Dabei wurde ein bisher unbeschriebener InsP-Stoffwechselweg in menschlichen Zellen erstmals beschrieben. Als Zweites wurden InsP verdauende Bakterien aus der menschlichen Darmflora untersucht, sodass der Abbauweg von Inositolhexakisphosphat beleuchtet werden konnte. Als Drittes wurden DUSP-Enzyme (engl. Dual-Specificity Phosphatases) identifiziert und in vitro charakterisiert, die in der Lage sind, die Phosphoanhydrid-Bindung von Inositolpyrophosphaten (PP-InsPs) zu spalten. Die vorliegende Arbeit demonstriert, dass 13C-Markierung in Verbindung mit NMR ein mächtiges Werkzeug darstellt, um InsP-Stoffwechselvorgänge zu untersuchen. / Inositol polyphosphates (InsPs) comprise a ubiquitous group of densely phosphorylated intracellular messengers in eukaryotic cells. Despite their contributions to a myriad of biological processes the detection of InsPs remains challenging to this day, especially with regards to differentiating enantiomers, as the available analytical toolset is still limited. In this thesis the use of stable isotope labelling of myo-inositol (Ins) and InsPs is explored to address this shortcoming. Combining 13C-labelling and nuclear magnetic resonance spectroscopy (NMR) provides both enhanced sensitivity and makes use of NMR’s strong structure-data dependency. This enabled the deconvolution of complex mixtures of InsPs from in vitro experiments or biological samples. With stereo-specific 13C-labels InsP mixtures could be resolved to individual enantiomers. Using this technique several InsP metabolic pathways were examined. Firstly, the human phytase-like enzyme Multiple Inositol Polyphosphate Phosphatase (MINPP1) was characterized in depth in vitro and in living cells, establishing a hitherto undescribed inositol polyphosphate metabolic path in humans. Secondly, inositol phosphate digesting bacteria isolated from the human gut microbiome were investigated, shedding light on the metabolic fate of inositol hexakisphosphate in the digestive track. Thirdly, a set of Dual-Specificity Phosphatases (DUSPs) were identified to be able to hydrolyze the phosphoanhydride bond of inositol pyrophosphates (PP-InsPs) and characterized in vitro. The 13C-labelling approach of InsPs in junction with NMR represents a powerful tool for the study of inositol polyphosphate metabolism. In the thesis at hand, this method has facilitated our understanding of inositol polyphosphate pathways and it will be continuing doing so in the future in several biological contexts.