Insulin resistance and obesity are major public health concerns. The impact of diet and genetics on insulin resistance and obesity is well accepted. Additionally, the gut microbiota has been shown to influence obesity and metabolic disorders. However, much remains to be understood about the role of gut microbiota in the development of insulin resistance and obesity. We utilized a mouse model lacking PAS kinase, a protein involved in cellular metabolism, in order to better understand the relationship between diet, genetics and the gut microbiota. Previous research has shown that mice lacking PAS kinase were protected from the effects of a high fat diet, gaining less weight and showing a better response to insulin. Surprisingly, when PAS-kinase deficient mice were placed on a western-style, high fat, high sugar (HFHS) diet, they became obese and had an impaired response to insulin, much like wild type mice on the same diet. Mutant mice did, however, show more resistance to the effects of the unhealthy diet in one aspect-they maintained normal levels of claudin-1 in the colon, suggesting that they were less likely to develop excessive gut permeability (leaky gut). While significant differences in gut microbial composition were seen in response to the HFHS diet, with shifts in the ratio of Firmicutes/Bacteroidetes and increases in the levels of Actinobacteria, none of the differences correlated with genotype. Unexpectedly, however, within the mice on the HFHS diet and regardless of genotype, the composition of the gut microbiota diverged into two clusters. The mice in one cluster showed more resistance to obesity and their glucose response was like that of wild type mice on a healthy normal chow diet (NCD), while mice in the other cluster showed more weight gain and impaired glucose response. No similar gut microbiota divergence occurred in mice on the NCD, suggesting that the HFHS diet made mice vulnerable to (but did not cause) the development of a harmful gut microbiota, whereas the healthy NCD protected against spontaneous harmful shifts in the composition of the gut microbiota.

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Au cours des dernières années, l’immunothérapie a révolutionné le paysage en oncologie. L’anti-CTLA-4 a montré son efficacité sur la survie globale des patients atteints de mélanome métastatique. Cependant, ce traitement présente des limites à son utilisation telles que l'efficacité clinique obtenu chez seulement 20% des patients et la survenue fréquente de colites pouvant être sévères. La recherche de biomarqueurs prédictifs de réponse clinique et/ou de développement de toxicité devient maintenant un enjeu majeur pour sélectionner les patients pouvant avoir un bénéfice à l’utilisation de ces traitements. En partant de l’observation que les colites induites par l’anti-CTLA-4 présentent des similitudes avec les maladies inflammatoires chroniques de l'intestin, nous avons émis l’hypothèse de l’existence d’un microbiote intestinal associé à une dysrégulation du système immunitaire pouvant prédire la réponse clinique et/ou la survenue d’une colite induite par l’anti-CTLA-4. Nous avons montré dans une cohorte de patients atteints de mélanome métastatique et traités par ipilimumab, qu'un microbiote intestinal enrichi en Faecalibacterium et autres Firmicutes est associé à une meilleure survie globale et sans progression ainsi qu'un risque accru de développer une colite. Les patients avec une flore enrichie en Firmicutes présentent également après traitement par ipilimumab, une activation lymphocytaire plus efficace. Par la suite, nous nous sommes intéressés aux métabolites issus du microbiote fécal et leur implication dans la réponse à l'anti-CTLA-4. Le butyrate est le principal métabolite produit par les Firmicutes. Nous avons observé chez la souris, une inhibition de l'efficacité anti-tumorale de l'anti-CTLA-4 lorsqu'elles étaient supplémentées en butyrate. In vivo, nous avons montré que le butyrate inhibe la surexpression sur les cellules dendritiques, des molécules CD80 et CD86 (molécules B7) induite par l'anti-CTLA-4. Cette immaturité des cellules dendritiques entraine un défaut d'activation des lymphocytes T spécifiques d'antigènes dépendant de l'axe CD28/B7 réduisant ainsi l'efficacité anti-tumorale. Chez l'Homme, nous avons valider cette hypothèse en montrant qu'une concentration sérique élevée en butyrate est associée à une diminution de la survie globale et sans progression comparativement aux patients avec un faible niveau de butyrate sérique.Ces travaux mettent en évidence le lien entre la composition du microbiote et les réponses immunologiques au blocage du CTLA-4. Ils apportent une explication sur un lien indirect via le butyrate entre la composition du microbiote intestinal et la réponse anti-tumorale aux immunothérapies. / In the last years, immunotherapy has revolutionized the landscape in oncology. The efficacy of anti-CTLA-4 has been demonstrated by improving overall survival of patients with metastatic melanoma. However, this treatment has limitations to its use such as the clinical efficacy obtained in only 20% of patients and the high incidence of severe colitis. Predictive biomarkers of clinical response and / or toxicity development are mandatory for a better selection of patients who will benefit from this treatment. Based on the observation that anti-CTLA-4-induced colitis has similarities with inflammatory bowel disease, we hypothesized that the gut microbiota associated with dysregulation of the immune system may predict the clinical response and / or occurrence of anti-CTLA-4-induced colitis.In a cohort of patients with metastatic melanoma treated with ipilimumab, we have shown that a gut microbiota enriched with Faecalibacterium and other Firmicutes is associated with a better of overall and progression-free survival as well as an increased risk of developing colitis. Firmicutes-driven microbiota is also associated with an improvement in lymphocyte T activation after ipilimumab treatment. Subsequently, we were interested in microbial metabolites and their involvement in the clinical response to anti-CTLA-4. Butyrate is the main metabolite produced by the Firmicutes. In mice, we observed an inhibition of anti-tumor effect of anti-CTLA-4 in butyrate-supplemented mice. In vivo, we have shown that butyrate inhibits the overexpression on dendritic cells, of CD80 and CD86 molecules (B7molecules) induced by anti-CTLA-4. This immaturity of the dendritic cells leads to a poor signaling of CD28 / B7 axis and activation of antigen-specific T-cells, thereby reducing anti-tumor efficacy. In humans, we validated this hypothesis by showing that a high serum concentration of butyrate is associated with decreased overall and progression-free survival compared to patients with low serum butyrate levels.This studie highlights the link between the composition of gut microbiota and the immunological responses to CTLA-4 blockade. They provide an explanation of an indirect link via butyrate, between the composition of the gut microbiota and the anti-tumor response to immunotherapies.

Cette thèse est consacrée à l'étude de systèmes d'équations aux dérivées partielles. En particulier nous nous intéressons à des systèmes issus de la mécanique des fluides avec contraintes qui permettent de décrire de manière continue en temps et en espace des quantités physiques telles que la densité ou la vitesse. Dans ce cadre nous construisons des modèles pour la biologie : modélisation de la croissance d'un biofilm de micro-algues et modélisation du gros intestin et de sa couche de mucus. Ces modèles sont ensuite testés numériquement à l'aide de schémas numériques spécifiquement élaborés pour ces modèles. Cette thèse est complétée par une étude numérique du modèle d'Aw-Rascle avec contrainte pour le trafic routier / This thesis is devoted to the study of partial differential equation systems. In particular, we are interested in constrained systems coming from the fluid mechanics field which allow to describe, in time and space, physical quantities such as density or speed. In this context we build models for biology: modeling of the growth of micro-algae biofilms and modeling of the large intestine and its mucus layer. These models are then tested numerically using numerical schemes specifically developed for these models. This thesis is supplemented with a numerical study of Aw-Rascle model with constraint for road traffic

<p>It is well known that colonic microbiota is influenced by both intrinsic and extrinsic factors; out of all these, diet plays a major role. The traditional human diet has typically been high in overall dietary fiber intake, due its inherent presence in plant-derived foods. However, over the years, dietary patterns have transitioned into a low-fiber Westernized diet. This diet is increasingly implicated in colonic diseases. Dietary fiber consumption is known to increase microbial diversity, yet the mechanisms are still unclear. This is partially true because dietary fiber as a category is composed of a wide variety of structures, which may have divergent effects on the gut microbiome. The food industry has extracted, isolated, refined and purified non-digestible carbohydrates and, in some cases, modified them for improved function, which may influence their interaction with the gut microbiome. This study was developed in two phases: we first hypothesized that glucans produced by different processes were structurally distinct and that these fine structural differences in glucans would govern microbial responses to the polymers. To test this hypothesis, we first determined the structural characteristics of the glucans by gas chromatography and mass spectrometry, which revealed substantial structural differences among the glucans with respect to size and linkage patterns, consequently categorizing the glucans by structure (i.e., mixed linkage α-glucans, resistant maltodextrins, and polydextroses). The second study involved the <i>in vitro </i>fecal fermentation of these commercially available soluble glucans which are uniformly composed of glucose linked into different structural arrangements. We further hypothesized that each glucan would select for different microbiota and that there would be glucan-specific general responses across microbiomes. We were able to identify a variety of idiosyncratic metabolic patterns as well as differential organisms selecting for specific glucan structures. Although there were associations with glucan classes at the family level (e.g., <i>Bacteriodaceae </i>and <i>Lachnospiraceae </i>were discriminants of the resistant maltodextrins and polydextroses respectively), associations with glucans across individual species within these families varied. These findings suggest that microbiome responses to structurally distinct glucans depend upon both fine glucan structure and community context, and community metabolic phenotypes emerge from the interaction of the two. These findings are relevant to the food industry as they may enable optimization of synthesis to generate chemical structures that select for specific organisms and/or improve overall gut health.</p>

Abstract available in the Current Developments in Nutrition.

<div> <div> <div> <p>As a major part of the dietary fiber classification, plant polysaccharides often have chemically complex structures which may differ by genera and species, and perhaps even by genotype and growing environment. Arabinoxylans from cereal cell walls are known to differently impact human gut microbiota composition and fermentation metabolites due to variability in chemical structure, though specificities of structure to these functions are not known at the level of genotype ́ environment. In the first study, corn bran arabinoxylan (CAX) extracted from 4 genotypes ́ 3 growing years at the Purdue Agronomy Farm was compared in human fecal fermentations to test the hypotheses that, 1) CAXs extracted from brans from different corn genotypes and grown over different years (environments) show distinct structures, and 2) these cause differences in gut microbiota response and fermentation metabolites. Monosaccharides and linkage analysis revealed that CAXs had different structures and the differences were genotype-specific, but not significantly due to environment. PCA analysis revealed that both short chain fatty acid production and the microbial community shifted also in a genotype-specific way. Thus, small structural changes, in terms of sugar and linkage compositions, cause significant changes in fermentation response showing very high specificity of structure to gut microbiota function. </p> <p>Insoluble fermentable cell wall matrix fibers have been shown to support beneficial butyrogenic Clostridia, but have restricted use in food products due to their insoluble character.</p></div></div> </div> <div> <div> <div> <p>In the second study, a soluble fiber matrix was developed that exhibited a similar fermentation effect as fermentable insoluble fiber matrices. Low arabinose/xylose ratio CAX was extracted with two concentrations of sodium hydroxide to give soluble polymers with relatively low and high residual ferulic acid (CAX-LFA and CAX-HFA). After laccase treatment to make diferulate crosslinks, soluble matrices were formed with average size of 3.5 to 4.5 mer. In vitro human fecal fermentation of CAX-LFA, CAX-HFA, soluble crosslinked ~3.5 mer CAX-LFA (SCCAX- LFA), and ~4.5 mer SCCAX-HFA revealed that the SCCAX matrices had slower fermentation property and higher butyrate proportion in SCCAX-HFA. 16S rRNA gene sequencing showed that SCCAX-HFA promoted OTUs associated with butyrate production including Unassigned Ruminococcaceae, Unassigned Blautia, Fecalibacterium prausnitzii, and Unassigned Clostridium. This is the first work showing the fabrication of soluble crosslinked fiber matrices that favors growth of butyrogenic bacteria. </p> <p>Moreover, these same SCCAXs exhibited an interesting gel forming property on simple pH reduction, which is similar in gelling property to low acyl gellan gum, though is differently readily soluble in water. Both of the SCCAXs formed gels at pH 2, with SCCAX-HFA forming the stronger gel. Gels showed shear-thinning behavior and a thermal and pH reversible property. A gel forming mechanism was proposed involving noncovalent crosslinking including hydrogen bonds and hydrophobic interaction among the SCCAX complexes. This mechanism was supported by structural characterization of SCCAX complexes using a Zeta-sizer and FT-IR spectroscopy. SCCAX-HFA could be used in low sugar gels and has the above property of promoting butyrogenic bacteria in the gut. </p> <p>In conclusion, gut microbiota responds differentially to CAXs with various fine structures. This probably due to dietary fiber-gut microbiota relationships have been evolved over time to be highly specific. Forming soluble fiber matrices could be a good strategy to promote butyrogenic bacteria and improve gut health, in a readily usable form in beverages.</p></div></div></div>

The gut microbiome and its metabolic processes are dynamic systems. Surprisingly, our understanding of gut microbiome dynamics is limited. Here we report a metaproteomic workflow that involves protein stable isotope probing (protein-SIP) and identification/quantification of partially labeled peptides. We also developed a package, which we call MetaProfiler, that corrects for false identifications and performs phylogenetic and time series analysis for the study of microbiome dynamics. From the stool sample of five mice that were fed with 15-N hydrolysate from Ralstonia eutropha, we identified 15,297 non-redundant unlabeled peptides of which 10,839 of their heavy counterparts were quantified. These peptides revealed incorporation profiles over time that were different between and within taxa, as well as between and within clusters of orthologous groups (COGs). Our study helps unravel the complex dynamics of protein synthesis and bacterial dynamics in the mouse gut microbiome.

Time-restricted eating is a fasting diet where the food intake is restricted to a short, typically eight-hour, window each day. It is associated with health benefits such as weight loss, improved sleep, protection against cognitive disorders, and improved cognitive function. The cognitive effects of time-restricted eating have primarily been explained by the production of ketogenesis – an alternative energy source produced when calories are restricted – and anti-inflammatory cytokines. The gut microbiota is the trillions of microorganisms inhabiting the intestinal tract and has also been associated with improved mental health through communication via the gut-brain axis. This review aims to investigate whether changes in the microbiota may mediate the effect of time-restricted eating on cognitive function. Studies investigating the effect of time-restricted eating on the microbiota were systematically reviewed. The results indicate that time-restricted eating may alter the microbiome composition and increase butyrate-producing bacteria. Butyrate is a short-chain fatty acid associated with the expression of genes involved in neural development and the reduction of neuroinflammation. Limited by the few studies reviewed, the results may indicate a possible link between time-restricted eating and cognitive function via the microbiota, although more research is needed.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).