Le déoxynivalénol (DON) est une mycotoxine qui contamine la plupart des cultures de céréales dans toutes les régions du monde. Capable de résister aux procédés de transformation subies par les céréales, le DON peut se retrouver alors, à l'état de contaminants dans les matières premières (céréales) ainsi que dans les denrées alimentaires transformées destinées à l'Homme (pâtes, pain, bières) et à l'animal (granulés) à des concentrations supérieures aux limites règlementaires. Malgré les efforts de recherche pour caractériser les multiples aspects de l’impact d’une contamination par le DON, les effets bactériologiques de cette mycotoxine n’étaient pas encore documentés chez l’Homme. L'Agence Nationale de Sécurité Sanitaire (Anses), dans le cadre de sa mission de protection du consommateur, a donc souhaité évaluer l'impact d'une contamination au DON sur le microbiote intestinal humain (MIH). Dans cette étude, nous avons d’abord évalué la cinétique du DON chez le porc et chez le rat, puis nous avons utilisé un modèle de rats à flore humanisée pour évaluer l'impact d'une exposition sub-chronique de la mycotoxine sur la composition du MIH. Le DON est un contaminant rapidement distribué et éliminé. Au sein du tractus digestif, il entraine des changements bactériologiques significatifs chez certains principaux groupes bactériens composant le MIH. Cette étude apporte des données complémentaires à l’analyse du risque lié à l’exposition du DON chez l’Homme et montre l’intérêt des modèles animaux étudiés dans des scénarii particuliers d’exposition au DON. / Deoxynivalenol (DON) is one of the most prevalent mycotoxins present in cereal crops worldwide. Able to withstand the transformation process undergone by grains, DON can be found as contaminant in raw materials (cereals) and in processed food for humans (pasta, bread, beer) and animals (grains) at concentrations upper the control limits. Despite research efforts to characterize various aspects of the impact of DON contamination, the microbiological effects of DON were not documented in humans. The French agency for food, environmental and occupational health and safety (Anses), as part of its mission to protect the consumer, wanted to assess the impact of DON contamination on the Human Gut Microbiota (HGM). In this study, we first evaluated the kinetics of DON in pigs and rats, and then we assessed the impact of an oral subchronic exposure of deoxynivalenol on the composition of HGM in a human microbiota-associated rats model. DON is a contaminant rapidly distributed and eliminated. In gut, DON leads to significant changes in some of the main bacterial groups of the HGM. This study provides additional data to analyze the risk exposure of DON in humans and shows the interest of these animal models in studies dealing with particular scenarios of DON exposure.

Abstract Obesity and dyslipidemia are major risk factors for type 2 diabetes, cardiovascular diseases (CVD), cancer, and musculoskeletal disorders. In prevention, the major goal is to limit calorie consumption and to reduce LDL-C and triglyceride. Dietary fiber (DF) intake is inversely related to body weight gain, insulin resistance, dyslipidemia, and CVD. This thesis investigated the effects of the DFs polydextrose (PDX) and lignin-rich insoluble residue (INS) from brewer’s spent grain (BSG) on lipid metabolism and obesity in diet-induced obese mice. In study 1, PDX was investigated on lipid metabolism in Western-diet-fed mice. We found that PDX reduced fasting plasma cholesterol and triglyceride, food intake, and increased bacteria such as Allobaculum, Bifidobacterium and Coriobacteriaceae in the gut. These changes in the gut microbiota with PDX were associated with downregulation of the genes Fiaf, Dgat1 and Cd36, and upregulation of Fxr in the intestine. We suggest that the hypolipidemic effect of PDX is exerted via diet-induced modification of gut microbiota and gene expression. In study II, INS from BSG was studied for its degradation products in mice fed with a fiber-deficient diet. We found that INS was partially degraded by gut microbiota and contributed to the phenolic pool. The major metabolite in mouse urine was 4-methylcatechol, a degradation product of lignin. In study III, the effects of INS from BSG were studied on lipid metabolism and obesity in high-fat diet-fed mice. INS showed hypocholesterolemic effects, reduced body weight and hepatic steatosis, and increased bacterial diversity, Clostridium leptum, and Bacteroides. INS increased bile acid excretion in the feces and upregulated the genes Srebp2, Hmgcr, Ldlr, Cyp7a1, Pparα, Fxr and Pxr in the liver. The present results suggest that INS from BSG induced beneficial systemic changes via bile acid and gut microbiota. In study IV, PDX was investigated for food intake and appetite-related parameters in healthy and overweight females in an acute study. A midmorning preload of 12.5 g PDX reduced hunger by 31.4% during satiation period while there was no significant change in energy intake compared to placebo. In addition, PDX lowered plasma insulin significantly, by 15.7%, and increased GLP-1 by 39.9%. PDX may reduce appetite, but a larger trial would be needed. / Tiivistelmä Liikalihavuus ja rasvatasapainon häiriöt ovat riskitekijöitä sydän- ja verisuonisairauksien, tyypin 2 diabeteksen, syövän sekä luuston ja lihaksiston sairauksien kehittymiseen. Näiden sairauksien ehkäisyssä pääasiallisena tavoitteena on vähentää energiansaantia, LDL-kolesterolia ja triglyseridejä. Ruoan ravintokuitujen saannin on osoitettu olevan yhteydessä painon ja plasman rasvatasojen laskuun sekä sydän- ja verisuonisairauksien vähenemiseen. Tässä tutkimuksessa selvitettiin ravintokuitu polydekstroosin (PDX) ja viljanjyvien prosessoinnista ylijäävän (BSG, brewer’s spent grain) ligniinipitoisen liukenemattoman sivutuotteen (INS) merkitystä rasva-aineenvaihduntaan ja aineenvaihduntasairauksiin liikalihavilla hiirillä. Tutkimuksessa I tarkasteltiin ravintokuitu PDX:n vaikutusta rasvojen aineenvaihduntaan länsimaisella ruokavaliolla ruokituilla hiirillä. Tutkimus osoitti, että ruokavalioon lisätty PDX alensi plasman kolesteroli- ja triglyseriditasoja paastossa sekä hillitsi ravinnonottoa ja lisäsi Allobaculum-, Bifidobacterium- ja Coriobacteriaceae-suolistobaktereja. Nämä suolistomikrobiston muutokset ovat yhteydessä Fiaf, Dgat1 ja Cd36 -geenien ilmentymistasojen laskuun ja Fxr -geenin ilmentymistason nousuun PDX-lisäruokittujen hiirien suolistossa. PDX:n hypolipideeminen vaikutus näyttäisi välittyvän ruokavaliosta johtuvan suoliston geenien ilmentymisen ja suolistomikrobiston muuttumisen kautta. Tutkimuksessa II tarkasteltiin runsaasti ligniiniä sisältävän INS:n hajoamistuotteiden vaikutusta aineenvaihduntaan hiirillä, joiden ruokavaliossa on vähemmän kuitua. Tutkimuksessa havaittiin, että suolistomikrobit hajottivat ravintokuitu INS:n osittain fenoliyhdisteiksi verenkiertoon. INS lisäsi virtsassa 4-metyylikatekolin määrää, joka on ligniinin hajoamistuote. Tutkimuksessa III tarkasteltiin INS-lisäyksen vaikutusta rasva-aineenvaihduntaan ja liikalihavuuteen korkearasvapitoisella ruokavaliolla ruokituilla hiirillä. Tulokset osoittivat, että INS-lisäys ruokavalioon alensi kolesterolia ja eläimen painoa sekä vähensi maksan rasvoittumista ja lisäsi vallitsevien bakteerien monimuotoisuutta, Clostridium leptum- ja Bacteroides -bakteereja. INS lisäsi sappihappojen erittymistä ulosteeseen ja Srebp2, Hmgcr, Ldlr, Cyp7a1, Pparα, Fxr ja Pxr -geenien ilmentymistä maksassa. Tuloksemme osoittivat, että BSG-ylijäämätuotteesta saatu ligniinipitoinen INS sai aikaan hyödyllisiä systeemisiä vaikutuksia suoliston mikrobiston ja sappihappojen muutosten kautta. Tutkimuksessa IV tarkasteltiin PDX:n vaikutusta ravinnonottoon ja ruokahaluun vaikuttaviin muuttujiin normaalipainoisilla ja liikalihavilla naisilla akuutissa tutkimuksessa. Tulosten mukaan ravintokuitu PDX:n nauttiminen aamiaisella vähensi näläntunnetta (31,4 %) seuraavalla aterioinnilla, kun taas plasebolla ei ollut vaikutusta. Lisäksi PDX alensi merkitsevästi insuliinitasoa (15,7 %) ja nosti GLP-1-tasoa (39,9 %). PDX vaikuttaisi vähentävän ruokahalua, mutta lisätutkimuksia tarvitaan.

Les inhibiteurs des protéases à sérine (Serpins) constituent une classe d'enzymes très peu étudiée chez les bactéries. Dans ce travail de thèse nous nous sommes intéressés à l'étude des serpins provenant du microbiote intestinal et l'investigation de leur potentiel anti-inflammatoire pour le traitement des maladies inflammatoires chroniques de l'intestin (MICI) chez l'homme. Pour cela nous avons identifié les serpins provenant du microbiote intestinal humain et analysé leur diversité ainsi que leur distribution entre les individus malades et sains. Ces données nous ont permis d'isoler les serpins significativement associées aux MICI. La purification de quarte d'entre elles nous a amené à démontrer qu'elles inhibent les protéases humaines impliquées dans les MICI. L'analyse biochimique et cinétique approfondie de ces protéines a montré qu'elles possèdent des propriétés originales notamment leur efficacité d'inhibition élevée. L'étude de l'effet protecteur de trois serpins chez un modèle animal de colite a démontré pour la première fois l'efficacité des serpins in vivo démontrant ainsi leur potentiel thérapeutique. / Serine protease inhibitors (Serpins) are a class of proteins that reamin poorly studied in bacteria. In this thesis we are interested in the study of serpins originating from the intestinal microbiota and the investigation of their anti-inflammatory potential for the treatment of inflammatory bowel diseases (IBD) in humans. For this we have identified serpins from the human gut microbiota and analyzed their diversity as well as their distribution between healthy and IBD patients. These data allowed isolating serpins significantly associated with IBD. The purification of four of them led us to demonstrate that they inhibit human proteases involved in IBD. Biochemical and kinetic analysis of these proteins showed that they exhibit original properties, in particular their high inhibition efficiency. The study of the protective effect of three serpins in an animal model of colitis demonstrated for the first time the efficacy of serpins in vivo demonstrating thus their therapeutic potential.

Many studies show the ability of gut microbes to modulate the anti-tumour immune response by direct triggering the immune cells or by bacterial metabolites. Interestingly bacteria may even migrate to the tumour tissue and orchestrate the immune response on site. These anti-tumour effects can be improved by the administration of immune checkpoint inhibitors (ICI). Notably, some microbial effects occur only in the presence of ICI. On the contrary, microbiota may also promote tumour growth and negatively impact the effects of ICI therapy. We have disrupted the gut microbiota homeostasis by antibiotics (ATB) to study the effects of gut microbiota on the ICI. This disturbance led surprisingly to reduced tumour growth and enhanced pro-inflammatory immune response not only in the gut but also within the tumour tissue, where especially IFN-γ orchestrated the anti-tumour immune response. Importantly the anti-tumour immune response could be transferred through colonisation of germ-free mice by ATB-changed gut microbiota if concomitantly anti- programmed cell death protein 1 (αPD-1) monoclonal antibody was administrated. These mice had elevated levels of segmented filamentous bacteria (SFB), which induced systemic immune response with increased expression of IL-17 and elevated amounts of Th 17 cells,...

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.

No description available.

Le microbiote intestinal est constitué d’un écosystème complexe de microorganismes appartenant à différents règnes. Cependant, la majorité de ces microorganismes sont d’origine bactérienne. Par conséquent, de nombreuses études, y compris la présente, se concentrent sur l’étude des communautés bactériennes. Les microorganismes ont développé une relation mutualiste avec le corps humain et agissent de plusieurs manières sur sa santé. Une perturbation du microbiote intestinal, nommée dysbiose, est reliée au développement d’une multitude de problèmes de santé chez diverses espèces animales. La transplantation de microbiote fécal suscite l’intérêt dans le domaine de la médecine vétérinaire. La préparation et l’entreposage affectent la composition et la viabilité bactérienne des fèces destinées à la transplantation de microbiote fécal (TMF). Jusqu’à présent, il demeure l’absence d’un protocole vétérinaire pour effectuer la préparation et l’entreposage des transplants fécaux canins. Par conséquent, l’objectif de cette étude était de comparer la viabilité bactérienne d’échantillons fécaux en présence et en absence d’oxygène et d’effectuer la congélation à l’aide de deux cryoprotecteurs différents. Les hypothèses de ce projet étaient les suivantes : la préparation des échantillons en absence d’oxygène préservera la viabilité bactérienne, l’utilisation d’un cryoprotecteur contenant des antioxydants pour la congélation générera le meilleur taux de viabilité, et le microbiote de chaque individu n’aura pas la même capacité à résister aux effets de la préparation et de l’entreposage. Les fèces de 10 chiens en santé ont été collectées et immédiatement transférées à l’intérieur d’une chambre anaérobique. Des aliquotes de 1,8 g ont été diluées dans 7,2 ml d’un cryoprotecteur contenant du glycérol à 10% (Gly) ou des antioxydants (Cryo). Les échantillons ont été homogénéisés et filtrés en condition aérobique (Ae) et en condition anaérobique (An) à l’intérieur d’une chambre anaérobique, simulant la préparation de la TMF. Les échantillons ont été congelés à -20 °C durant 90 jours (F) pour l’évaluation des effets de l’entreposage. La viabilité bactérienne des échantillons a été déterminée à l’aide de la cytométrie de flux. L’analyse de la composition bactérienne chez les 10 donneurs de matières fécales a été réalisée par le séquençage de la région V4 du gène de l’ARNr 16S à l’aide de la plateforme Illumina MiSeq. Les échantillons non congelés, préparés en absence d’oxygène et dilués avec Cryo présentaient les plus grands taux de viabilité (66,78 %) par rapport aux autres groupes (p < 0,05). Les échantillons exposés à l’oxygène avaient une viabilité bactérienne inférieure (p < 0,01). Toutefois, les échantillons dilués avec Cryo présentaient une viabilité plus élevée (65,26 %) que les échantillons dilués dans Gly (55,20 % ; p < 0,001) en présence d’oxygène. La viabilité bactérienne a diminué en raison de la congélation des échantillons (p < 0,001). L’ensemble des échantillons frais avaient une viabilité médiane de 62,23 % et, à la suite de la congélation, elle était de 22,68 %. Cependant, les échantillons congelés à l’aide de glycérol avaient une viabilité plus élevée (30,61 % ; p < 0,001). Le genre Prevotella était fortement corrélé à la viabilité (R = 0,731 ; p < 0,05, R = 0,756 ; p < 0,05, R = 0,834 ; p < 0,01, R = 0,752 ; p < 0,05). Ces résultats indiquent que la viabilité bactérienne est optimale lors de l’utilisation de matières fécales en absence d’oxygène et lors d’une dilution à l’aide d’un cryoprotecteur contenant des antioxydants. La congélation a significativement réduit la viabilité bactérienne, mais le glycérol semble mieux préserver les bactéries. La présence de certaines espèces plus résistantes et l’impact de la composition du microbiote sur l’efficacité de la TMF nécessitent une enquête plus approfondie. / The intestinal microbiota is made up of a complex ecosystem of microorganisms belonging to different kingdoms. However, bacterial cells are much more numerous. Therefore, many studies, including the present one, focus on the study of bacterial communities. Microorganisms have developed a mutualistic relationship with the animal body and act in several ways on its health. A disturbance of the intestinal microbiota, called dysbiosis, is linked to the development of a multitude of health problems in various animal species. There is an emerging interest in the transplantation of fecal microbiota in veterinary medicine. Preparation and storage affect the quality of transplants intended for faecal microbiota transplantation (FMT). Considering the absence of a protocol in veterinary medicine, the objective of this study was to optimize bacterial viability during the preparation and storage of canine fecal transplants. The hypotheses of this project were that the preparation of samples in the absence of oxygen will preserve bacterial viability, that the use of a cryoprotectant containing antioxidants for freezing will yield the best viability rate and the microbiota of individuals does not have the same ability to withstand the effects of preparation and storage. Feces from ten healthy dogs were collected, and immediately transferred inside an anaerobic chamber. Aliquots of 1.8 g were diluted in 7.2 ml of a cryoprotectant containing glycerol (Gly) or antioxidants (Cryo). The samples were homogenized and filtered, simulating the TMF preparation. To evaluate the impact of oxygen on bacterial viability, the procedures were performed outside (Ae) and inside (An) the anaerobic chamber. Samples were frozen at -20°C for 90 days (F) to evaluate effect of freezing. The bacterial viability of samples was determined using flow cytometry. Analysis of the bacterial composition was performed by sequencing the V4 region of the 16S rRNA gene, using the Illumina MiSeq platform. Fresh samples prepared under anaerobiosis and diluted with Cryo had the highest viability (66.78%) compared to the other groups (p < 0.05). Bacterial viability was affected by oxygen (p < 0.01) but solutions prepared with Cryo had higher viability (65.26%) than samples diluted in Gly (55.20%; p < 0.001). Freezing decreased bacterial viability from 62.23% to 22.68% (p < 0.001). However, samples frozen using glycerol showed higher viability (30.61%; p < 0.001). The genus Prevotella was strongly correlated with viability (R = 0.731; p < 0.05, R = 0.756; p < 0.05, R = 0.834; p < 0.01, R = 0.752; p < 0.05). These results show that bacterial viability is optimal when preparing feces under anaerobic conditions and using a cryoprotectant containing antioxidants. If freezing is necessary, glycerol seems to preserve the bacteria better. The presence of some more resilient species and the impact of microbiota composition on the efficacy of TMF requires further investigation.

<p>The gut and brain are involved in a bi-directional communication system, referred to as the gut-brain axis. While it has been established that antimicrobials induce dysbiosis in the gut, which further disrupts immune and metabolic homeostasis, research on brain and behaviour development is becoming a topic of interest. We propose that alterations via antibiotics at the level of the gut microbiota impacts the gut-brain axis. The primary interest of this thesis is to understand the effects that antibiotics have on brain and behaviour development in conjunction with changes in the immune system and metabolism using the antibiotic mouse model. Mice treated with antibiotics revealed behavioural differences in the open field apparatus and three-chamber social behaviour apparatus, but not in the elevated plus maze and auditory fear conditionings enclosures. Evaluation of intestinal permeability revealed that female Balb/C mice administered a combination of bacitracin, neomycin and primaricin and another group administered a combination of ampicillin, neomycin and primaricin showed reduced intestinal permeability. Furthermore, the immune system condition was evaluated using flow cytometric analysis of spleens, which revealed no effect of treatment on immune cell profiles in CD1 mice treated with ampicillin. Evaluation of serum cytokine levels showed minimal differences in Balb/C and C57Bl/6 mice treated with antibiotics. Body weight and water and food consumption were evaluated in mice administered antibiotics. Weight loss differences were observed in two groups of female Balb/C mice, with the first group administered bacitracin, neomycin and primaricin and the second group administered ampicillin , neomycin and primaricin. Antibiotic treatment dependent differences in water and food consumption were observed. Serum insulin and leptin level investigation revealed that female Balb/C mice administered ampicillin, neomycin and primaricin had reduced serum insulin levels compared to strain matched controls. These findings indicate that antibiotic treatment impact metabolic function. This pilot study using antibiotic treated mouse models provides insight on the microbiota’s effects on the gut-brain axis, which can help to potentially identify methods of preventing gut microbiota mediated pathology in humans.</p> / Master of Science (MSc)

Bacterial colonization of the gastrointestinal (GI) tract takes place during the perinatal period, thus coinciding with a critical window of enteric nervous system (ENS) development. Previous work has found that the myenteric plexus of germ free (GF) mice exhibits structural and functional aberrancies in the early postnatal period as compared to specific pathogen free (SPF) and altered Schaedler flora (ASF) mice. These early life disruptions in ENS development in GF mice compared to SPF mice, and more specifically ASF mice, support the notion that a simple intestinal flora is sufficient for directing perinatal ENS development. It has previously been believed that the intrauterine environment during fetal development is sterile. Recent evidence showing successful isolation of microbial communities from embryonic cord blood and newborn meconium that are not of maternal origin suggests that the intrauterine environment is not sterile and is unique to the fetus. Coinciding with this timeline of fetal microbial colonization is the development of the ENS through a population of precursors known as enteric neural crest derived cells (ENCDCs). The prenatal period is characterized by rapid expansion and differentiation of ENCDCs into the many enteric neuron subtypes that comprise the ENS. Terminal differentiation of ENCDCs continues into the early postnatal period. In the current study, we tested the hypothesis that ENCDCs interact directly with microbial products during ENS development. Further, these ENCDC-bacterial product interactions influence the proliferation, apoptosis, and chemical coding of enteric neuron precursors. These objectives were carried out in an in vitro model of ENCDCs isolated from the prenatal period that was established for the first time in our lab using immunoselection. Further, this model was characterized at key timepoints for proliferation, apoptosis, and differentiation. Our results are suggestive of direct ENCDC interactions with lipopolysaccharide (LPS), a TLR4 ligand, and flagellin, a TLR5 ligand, in stimulating ENCDC proliferation and differentiation into early born neurons of nitrergic and serotonergic subtypes. Peptidoglycan derivatives, muramyl dipeptide (MDP) and ƴ-D-Glu-mDAP (iE-DAP), ligands for NOD2 and NOD1 respectively, appear to mainly stimulate differentiation into nitrergic neurons, and possibly serotonergic neurons. The lack of apoptosis in all conditions is consistent with the notion that apoptosis is not an important characteristic of ENCDC maturation and ENS development. Finally, the lack of significance for differentiation into dopaminergic neurons could be further evidence of their late born nature, which has previously been reported to be stimulated by serotonin after the emergence of serotonergic neurons. / Thesis / Master of Science (MSc)

Des études ont récemment mis en évidence le rôle des lipopolysaccharides (LPS) des bactéries à Gram négatif de la flore intestinale dans le processus de l’inflammation conduisant à l’obésité et au diabète de type 2.Le présent travail est réalisé dans le cadre d’une collaboration entre les équipes du Dr. Caroff (U. Paris-Sud, Orsay) et du Pr. Zhao (U. Jiao Tong, Shanghai). Les expériences présentées ont été réalisées lors de séjours dans les deux laboratoires.Il a été démontré en Chine que des bactéries Sulfato-réductrices (SRB) à Gram négatif étaient présentes en plus forte proportion dans la flore intestinale chez les souris suivant un régime gras. Les mêmes résultats ont été observés chez l'homme. L’hypothèse selon laquelle des SRB seraient à l’origine de grandes quantités d’endotoxines chez les obèses et les patients diabétiques a été émise. Plusieurs souches de SRB isolées de la flore intestinale humaine d’un sujet sain et d’un sujet diabétique ont été cultivées en Chine. Des études de relation structure/activité des LPS isolés de ces bactéries ont été réalisées dans le laboratoire Français pour déterminer leur rôle dans le développement des maladies métaboliques. Les souches isolées des deux sujets ont pu être classées dans le genre Desulfovibrio. Les LPS correspondants ont été extraits et purifiés par des méthodes mises au point dans l’équipe d’Orsay. La structure chimique a été élucidée par les méthodes suivantes : Electrophorèse, Chromatographie sur couche mince, Chromatographie en phase gazeuse et Spectrométrie de masse MALDI. C’est ainsi que des spectres de masse ont été obtenus et que la structure des lipides A, principes actifs des LPS, isolés de SRB a été décrite pour la première fois. Les activités biologiques testées (TNFα, IL-6) varient en fonction du nombre d’acides gras présents. Les LPS de SRB du patient sain ont une structure variable (Smooth versus Rough) en fonction de la quantité de fer présent dans le milieu, et ceux isolés du patient diabétique présentent des structures atypiques qui ne sont pas toutes inflamogènes. Une molécule membranaire inconnue, que nous avons nommée « Glycosyl’X » était co-extraite avec les LPS. Elle joue apparemment un rôle important dans la croissance des SRB et a été étudiée après des étapes de purification complexes. Les structures et le pouvoir inflammatoire de ces molécules dont la structure varie avec les souches, et qui chélatent le fer, ont été étudiées. Elles sont de nature principalement osidique et fixées à la membrane. La proportion de ces molécules par rapport aux LPS varie avec la quantité de fer disponible dans le milieu. Un milieu riche en fer favorise la croissance des Desulfovibrio portant les Glycosyl’X qui n’ont pas de pouvoir inflammatoire eux-mêmes, mais entrent en compétition avec les LPS, modulant ainsi indirectement l’activité de ces derniers. L’augmentation du nombre de Desulfovibrio conduisant à l’augmentation des molécules Glycosyl’X pourrait aussi moduler positivement (par présentation) ou négativement (par élimination des bactéries) l’adsorption du fer dans les intestins dont l’équilibre est essentiel pour l’homéostasie métabolique.Par ailleurs, la croissance des Desulfovibrio augmente la production d’Hydrogène Sulfuré connu pour son action délétère sur les cellules. Nous favorisons l’hypothèse selon laquelle son action sur la disjonction des cellules épithéliales permettrait le passage des différents LPS relargués par la flore Gram-négative intestinale, et même des bactéries entières, vers la circulation sanguine. / Recent studies have highlighted the role of lipopolysaccharide (LPS) in the intestinal flora (gut microbiota) which could contribute to the inflammation process leading to obesity and type 2 diabetes. This thesis is part of a collaborative project between the laboratories of Dr. Caroff (U. Paris -Sud, Orsay, France) and Prof. Zhao (U. Jiao Tong , Shanghai, China). It has been shown by Pr.Zhao’s team in 2010 that the Sulfate -Reducing Bacteria (SRB) were presented in greater proportion in the intestinal mice flora following a fat diet compared to mice following a normal diet. The same results were observed in humans. The starting hypothesis was that SRB could produce a large amount of endotoxin in obese and diabetic patients and play a role in the development of metabolic diseases. Several SRB strains isolated from the human intestinal flora of a healthy subject and of a diabetic subject were grown in the Chinese laboratory. Studies of their LPS structure / activity relationships were carried out in the French laboratory. The aim of this study was to determine their roles in the development of metabolic diseases.Strains isolated from the two subjects could be classified in the Desulfovibrio genus. The corresponding LPS were extracted and purified by the methods developed in the French laboratory. The chemical structure was elucidated by the following methods: Electrophoresis, Thin layer chromatography, Gas chromatography and MALDI mass spectrometry. The mass spectra were obtained and the structure of lipid A, the active part of LPS isolated from SRB was described here for the first time. The biological activities test (TNFα, IL-6) vary depending on the number of fatty acids present in their lipid A structure. The LPS of SRB isolated from the healthy patient had a variable structure (Smooth versus Rough) depending on the amount of iron present in the medium, and those isolated from diabetic patients had atypical structures are not all inflamogenic .An unknown membrane molecule, which we named "Glycosyl'X" was co-extracted with the LPS. It apparently plays an important role in the growth of SRB was investigated after complex purification steps. The structures and the inflammatory power of these molecules variying with strains chelating iron were studied. They are mainly of glycosidic nature and linked to the bacterial membrane.The proportion of these molecules relatively to LPS varies with the amount of iron in the medium. An environment rich in iron promotes the growth of Desulfovibrio Glycosyl'X, molecules but competes with LPS and indirectly modulates the activity of the latter. The increase number of Desulfovibrio leading to increased Glycosyl'X molecules may also modulate positively (by presentation) or negatively (by killing bacteria) the absorption of iron in the intestines which balance is essential for metabolic homeostasis.Furthermore, the growth of Desulfovibrio increasing the production of Hydrogen Sulfide is known for its deleterious effects on the cells. We favor the hypothesis that its action on the separation of epithelial cells favors the passage of different LPS released by the Gram- negative of intestinal flora and even whole cell bacteria into the bloodstream.