Este trabalho teve como objetivo propor novo método para estudar a permeabilidade de fármacos, utilizando epitélio intestinal de rã-touro (Rana catesbeiana) em método ex vivo, empregando células de Franz. Por utilizar epitélio intestinal, um material de descarte proveniente de um animal utilizado como alimento humano, pode ser considerado um método alternativo, pois não implica no sacrifício de animais. A quantidade de fármaco permeada foi determinada por método de eletroforese capilar com detecção ultravioleta e validado para os antivirais lamivudina, zidovudina e aciclovir, na presença de metoprolol e floridizina. O fármaco escolhido como modelo nos ensaios de permeabilidade foi a lamivudina. Para estabelecimento do protocolo experimental dos estudos de permeabilidade, foi proposta uma análise de variância three-way para verificar a influência na permeabilidade dos fármacos, das seguintes variáveis: secção intestinal, pH da solução de Ringer e temperatura. Foram determinados a quantidade total de fármaco permeado (Qt), o coeficiente de permeabilidade aparente (Papp) e a constante de absorção de primeira ordem (ka). A partir da análise do planejamento experimental, os efeitos das variáveis não foram significativos, exceto para a secção intestinal. Os resultados de coeficiente de permeabilidade aparente (Papp) obtidos foram de 0,09 x 10-4 cm/s para lamivudina e de 0,22 x 10-4 cm/s para o metoprolol. O valor de Papp obtido de para o metoprolol é próximo dos valores encontrados na literatura para outras técnicas. Para a lamivudina, entretanto, a diferença encontrada em comparação às células Caco-2 pode ser devida às diferentes técnicas empregadas. / This work aimed to propose a new method for studying drug permeability using frog intestinal epithelium (Rana catesbeiana) in ex vivo method, using Franz cells. By using intestinal epithelium, a disposal material from an animal used as human food, can be considered an alternative method, because it doesn\'t involve the sacrifice of animals. The amount of permeated drug was determined by capillary electrophoresis method with UV detection and validated for antiviral drugs lamivudine, zidovudine and acyclovir in the presence of metoprolol and floridizina. The drug chosen as a model in permeability studies was lamivudine. To establish the experimental protocol for the permeability studies, a three-way analysis of variance was proposed to check the influence of intestinal section, pH of Ringer\'s solution and the temperature on the permeability. Total amount of drug permeated (Qt), apparent permeability coefficient (Papp) and first-order constant absorption (ka) were determined. By the analysis of experimental design, the effects of the variables were not significant, except for intestinal section. The results of apparent permeability coefficient (Papp) obtained were 0.09 x 10-4 cm/s for lamivudine and 0.22 x 10-4 cm/s for metoprolol. The value of Papp obtained for metoprolol is quite close to the values found in literature for other methods. For lamivudine, however, the difference found in comparison to Caco-2 cells may be due to different techniques.

O objetivo deste trabalho foi analisar comparativamente alguns microrganismos que compõe a microbiota intestinal endógena de crianças eutróficas (30), com sobrepeso (24) e obesas (30) entre 3 a 11 anos, a partir de amostras fecais. Foi realizado o isolamento de espécies de Bacteroides, Parabacteroides e Clostridium; a identificação de B. fragilis e C. perfringens enterotoxigênicos; e a detecção quantitativa por PCR (SybrGreen) de B. fragilis, B.vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales e Clostridium (cluster I). As espécies C. perfringens e B. vulgatus foram as mais isoladas; nenhum isolado B. fragilis foi enterotoxigênico; todos C. perfringens foram classificados como tipo A e destes 8,7% e 12,2% possuiam os genes tpeL e netB, respectivamente. C. perfringens, C. difficile e Bifidobacterium spp. estavam em maior quantidade em crianças eutróficas, enquanto obesos e com sobrepeso apresentaram maior número de Lactobacillus spp. e Bacteroidales. / The aim of this study was to evaluate some microorganism from endogenous intestinal microbiota of normal weight (30), overweight (24) and obese (30) children between 3 and 11 years, from fecal samples. It was performed the isolation of species of Bacteroides, Parabacteroides and Clostridium; the identification of B. fragilis and C. perfringens enterotoxigenic; and the quantitative detection by PCR (SybrGreen) B. fragilis, B. vulgatus, P. distasonis, C. perfringens, C. difficile, Bifidobacterium spp., Lactobacillus spp., Bacteroidales and Clostridium (cluster I). The species C. perfringens and B. vulgatus were the most isolated; no isolated B. fragilis was enterotoxigenic; all C. perfringens were classified as type A and these 8.7% and 12.2% harbored tpeL and netB genes, respectively. C. perfringens, C. difficile and Bifidobacterium spp. were in greater quantity in normal weight children while obese and overweight showed a higher number of Lactobacillus spp. and Bacteroidales.

Frente à preocupação da saúde pública com o uso de antimicrobianos promotores de crescimento na produção animal, intensificou-se a pesquisa de estratégias alternativas aos antimicrobianos. Algumas pesquisas relacionadas ao uso de prebióticos e probióticos como aditivos têm demonstrado que estes, além de promoverem a modulação benéfica da microbiota intestinal, resultam em efeitos imunomodulatórios que permitiriam reduzir o estresse imunológico. Dessa forma, o presente estudo teve como objetivo verificar a influência de um prebiótico e de um probiótico sobre o desempenho, morfologia intestinal e parâmetros de imunidade de frangos de corte, tendo um antibiótico e um controle para comparação. Foram utilizados 960 pintos de corte, criados até 42 dias de idade sobre cama reutilizada. O delineamento era inteiramente casualizado, com 4 tratamentos: Dieta basal (DB) com Antibiótico; DB com Prebiótico; DB com Probiótico; Controle (DB) , sendo 8 repetições/tratamento. Quanto ao desempenho e, considerando-se o período total de criação, os aditivos alternativos testados não demonstraram efeito sobre o ganho de peso (GP), consumo de ração (CR) e mortalidade. Por sua vez, a conversão alimentar (CA) dos aditivos alternativos foi similar à do antibiótico, entretanto, não diferiu do controle. Com relação à imunidade, os aditivos alternativos não apresentaram efeito sobre boa parte dos parâmetros avaliados neste trabalho. Porém, para peso relativo do timo e taxa de fagocitose, o prebiótico mostrou exercer alguma influência. Além disso, ambos aditivos mostraram, ainda que apenas numericamente, uma melhor reposta vacinal contra Newcastle que os demais tratamentos aos 42 dias de idade. Por fim, não foi possível observar efeito benéfico dos aditivos alternativos testados sobre a morfologia intestinal de frangos de corte. Vale ressaltar que uma diversidade de fatores pode influenciar o efeito dos aditivos alternativos, culminando em resultados pouco conclusivos e até mesmo contraditórios. Diante deste fato e dos resultados aqui obtidos, conclui-se que os ensaios com aditivos alternativos devem continuar, já que houve evidências de seus possíveis efeitos beneficos na produção animal. / Due to the public health concerning with the use of antimicrobials as growth promoters in livestock, the number of researches of alternative strategies for antimicrobials has increased. Some researches about the use of prebiotics and probiotics as additives have demonstrated that those promote the beneficial modulation of the intestinal microbiota and have immunomodulatory effects which would allow decreasing of immunological stress. So, this study aimed to verify the influence of a prebiotic and of a probiotic on the performance, morphology intestinal and parameters of broilers immunity, comparing them with an antibiotic and a control group. In this experiment, 960 chicks were used and bread up to 42 days-old on litter previously used. The assignment was completely randomized, with 4 treatments: Basal diet (BS) with antibiotic, BS with prebiotic, BS with probiotic and control (only BS); with eight repetitions for each treatment. Concerning to the performance and taking into account the whole breeding period (42 days), the alternative additives groups did not present any effect on the weight gain (WG), feed intake (FI) and mortality. The feed conversion (FC) of the alternative additives groups was similar to that of the antibiotic one, but they had no significant difference from the control. Relatively to the immunity, the additives did not present any effect on the majority of the parameters evaluated in this work. However, for thymus relative weight and phagocytosis index, the prebiotic was shown to have some influence on. Moreover, both additives showed, although just numerically, a better response for vaccination against Newcastle disease than the others for 42 days-old broilers. Finally, it was not possible to observe any beneficial effects of the alternative additives on the broilers intestinal morphology. It is important to emphasize that several factors may influence the effects of those additives, which allow obtaining inconclusive, and even contradictory, results. Taking into account this fact and the results obtained in this work, one concludes that the essays with alternative additives must go on, since there have been evidences of their possible beneficial effects on animal production.

A possível indução de resistência bacteriana devido a inclusão de antibióticos, e a pressão dos consumidores por produtos de qualidade, levaram a proibição do uso dos mesmos na alimentação animal. Diante destes acontecimentos, as buscas por alternativas como os prebióticos, probióticos e simbióticos em substituição aos antibióticos vêm sendo bastante enfatizados na alimentação animal pois, estes, além de proporcionarem uma modulação benéfica da microbiota intestinal, atuam na diminuição do estresse imunológico. Portanto, o presente estudo teve como objetivo verificar a influencia de prebiótico, probiótico e simbiótico sobre o desempenho, morfologia intestinal e imunidade de frangos de corte em substituição ao antibiótico. Foram utilizados 1400 pintos de corte, criados até 42 dias de idade, em um delineamento inteiramente casualizado, com 5 tratamentos: Controle; Probiótico; Prebiótico; Simbiótico (prebiótico + probiótico); Antibiótico, 7 repetições/tratamento com 40 aves cada. Quanto ao desempenho, considerando o período total de criação e nas condições em que o experimento foi conduzido, não foi possível mostrar influência dos aditivos testados nos parâmetros zootécnicos avaliados. Já para imunidade, observou-se um efeito de prebiótico para resposta vacinal contra a doença de Newcastle aos 28 dias e um efeito de prebiótico para peso relativo de baço. Com relação à morfologia intestinal observou-se que o probiótico, quando adicionado à dieta, apresentou uma menor altura de vilo de jejuno e um efeito de interação de forma antagônica para altura de vilo do íleo. Além disso, no parâmetro de profundidade de cripta, verificou-se um efeito de interação de forma antagônica para o duodeno e um efeito de probiótico no jejuno, sendo que o probiótico, ao ser adicionado à dieta apresentou menor valor. Por fim a presença de prebiótico na dieta aumentou o número de células caliciformes tanto no duodeno como no jejuno. / The market pressures, since consumers require products with high quality, and the possible inducement for the bacterian resistance due to the inclusion of antibiotics as well, have caused the prohibition of the referred antibiotics in animal alimentation. Considering this event, alternatives like prebiotics, probiotics and symbiotics have been emphasized in animal alimentation because they provide intestinal microbiota benefits and less immunologic stress. Therefore, this study evaluated the influence of prebiotic, probiotic and symbiotic in broiler performance, as well as in their intestinal morphology and immunity in antibiotics replacement. 1400 day-old broiler chicks were used during 42 days, in a randomized block design, with 5 treatments: Control, Probiotic, Prebiotic, Symbiotic (prebiotic + probiotic) and Antibiotic. There were 7 repetition with 40 chicks each. Regarding the performance, there was no influence from the tested additives, considering the zoothecnics parameters studied. Considering the immunity, there was a prebiotic effect for the antibodies response against Newcastle disease on the 28th day, and a prebiotic effect to the spleen weigh. Regarding the intestinal morphology, it was observed that when the probiotic was added to the diet, it showed a shorter jejunum villus and an antagonic interaction effect for ileum villus height. Besides, there was antagonic interaction effect for the duodenum crypt depth and a probiotic effect in the jejunum. Still considering the jejunum, it was observed that when the probiotic was added to the diet, it shows lower results. The prebiotic presence in the diet also shows the increase of the caliciform cells number in both duodenum and jejunum.

A obstrução intestinal mecânica representa uma condição de urgência, necessitando diagnóstico precoce e terapêutica adequada, em virtude do seu elevado grau de morbidade e de mortalidade. Desta forma, o objetivo deste estudo foi avaliar o efeito da N-acetilcisteína associada ao Ringer lactato ou à solução salina hipertônica na resposta inflamatória, histologia e translocação bacteriana em modelo experimental de obstrução e isquemia intestinal. Para tanto, Foram constituídos quatro grupos experimentais, com 10 ratos Wistar em cada, além do grupo de referência: OI - Submetidos a obstrução e isquemia intestinal e enterectomia com anastomose intestinal, sem reanimação volêmica; RL - Submetidos a obstrução e isquemia intestinal, reanimação volêmica com Ringer lactato (32ml/kg, i.v., em 10 minutos) e enterectomia com anastomose intestinal; RLNAC - Submetidos a obstrução e isquemia intestinal, reanimação volêmica com Ringer lactato associado a NAC (32ml/kg + 150 mg/kg i.v. em 10 minutos) e enterectomia com anastomose intestinal; SHNAC - Submetidos a obstrução e isquemia intestinal, reanimação volêmica com solução salina hipertônica a 7,5% associado com NAC (4ml/kg + 150 mg/kg i.v., em 10 minutos) e enterectomia com anastomose intestinal. Grupo Referência (n=5): Animais anestesiados, submetidos a coleta de materiais para cultura e histologia e sacrificados por exsanguinação. Os animais receberam uma associação anestésica de cetamina e xilazina intramuscular em membro posterior direito, na dose de 60mg/kg e 10mg/kg, respectivamente. Decorridas 24 h do tratamento, a eutanásia foi realizada por exanguinação, sob anestesia, após a coleta dos tecidos / Mechanical intestinal obstruction represents a condition of urgency, necessary early diagnosis and appropriate therapy, due to their high degree of morbidity and mortality. In this way, the objective of this study was to evaluate the effect of N-acetylcysteine associated with lactated Ringer\'s or hypertonic saline solution in the inflammatory response, histology and translocation in an experimental model of intestinal obstruction and ischemia. For Four experimental groups were constituted with 10 Wistar rats each, in addition to the reference group: OI - submitted to obstruction and ischemia intestinal and enterectomy with intestinal anastomosis, without volume resuscitation; RL - Undergoing intestinal obstruction and ischemia, volume resuscitation with Ringer\'s lactate (32ml / kg, i.v., within 10 minutes) and anastomosis enterectomy intestinal; RLNAC - Undergoing obstruction and intestinal ischemia, resuscitation with lactated Ringer\'s lactating NAC (32 ml / kg + 150 mg / kg i.v. in 10 minutes) and enterectomy with intestinal anastomosis; SHNAC - Submitted to obstruction and intestinal ischemia, volume resuscitation with saline solution hypertension at 7.5% associated with CAP (4 ml / kg + 150 mg / kg i.v., in 10 minutes) and enterectomy with intestinal anastomosis. Reference Group (n = 5): Anesthetized animals, submitted to collection of materials for culture and histology and sacrificed by exsanguination. The animals received a anesthetic association of ketamine and intramuscular xylazine in limb posterior right, at the dose of 60mg / kg and 10mg / kg, respectively. After 24 h of treatment, euthanasia was performed by exsanguination, under anesthesia, after collection of tissues

Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-05-17T15:27:41Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-05-22T11:42:00Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-05-22T11:42:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-03 / Os fungos têm um papel complexo no trato intestinal, influenciando diretamente na saúde e na doença e sua disbiose pode contribuir para a obesidade. O objetivo deste estudo foi avaliar a diversidade de fungos da microbiota intestinal humana entre indivíduos eutróficos, com sobrepeso e obesos. Foram coletados espécimes fecais de 72 indivíduos adultos e análises dependentes e independentes de cultivo foram realizadas para avaliar os fungos presentes. As leveduras foram identificadas pela técnica de ionização e dessorção a laser assistida por matriz acoplada a analisador do tipo espectro de massa por tempo de vôo (MALDI-TOF MS) e os fungos filamentosos por microcultivo. A contagem média de fungos filamentosos e leveduras cultiváveis foi de 1,58 Log10 UFC/g de fezes. Diferenças significativas no nível populacional dos fungos filamentosos foi observado entre os grupos dos indivíduos eutróficos e obesos. Trinta e quatro gêneros de fungos foram identificados. O filo predominante foi Ascomycota com 29 gêneros e/ou espécies diferentes, seguido por Zigomycota e Basidiomycota. O fungo mais isolado nos indivíduos eutróficos foi Paecylomyces sp. e nos indivíduos obesos Penicillium sp. Os indivíduos eutróficos apresentam uma diversidade ligeiramente maior de fungos filamentosos do que os indivíduos obesos. Os indivíduos do sexo feminino tiveram um maior número de fungos diferentes quando comparados aos do sexo masculino. A análise de eletroforese em gel de gradiente desnaturante (DGGE) mostrou agrupamento dos indivíduos eutróficos e com sobrepeso em um cluster e dos indivíduos obesos em outro cluster distinto, embora a riqueza tenha sido baixa nos três grupos. A análise de hibridização in situ florescente (FISH) demonstrou maior densidade relativa de C. albicans no grupo obeso quando comparado ao eutrófico e pela análise de reação da polimerase em cadeia quantitativa (qPCR) um número maior de cópias de DNA de fungos e do filo Ascomycota nos indivíduos obesos quando comparado aos indivíduos com sobrepeso e obesos. Foi verificada correlação positiva entre os fungos e os parâmetros antropométricos colesterol total, LDL, triglicerídeos, hemoglobina, HOMA-IR, HOMA-β, insulina, glicose sérica, creatinina e porcentagem de fibras e carboidratos da dieta. Outros estudos são necessários para melhor compreender a interrelação entre a micobiota do intestino e a obesidade. Futuramente, este conhecimento poderá ser utilizado na modulação da micobiota intestinal e no tratamento da obesidade. / Fungi have a complex role in the intestinal tract, directly influencing health and disease and potential dysbiosis could contribute to obesity. The aim of this study was to investigate the fungal diversity of human gut microbiota among eutrophic, overweight, and obese individuals and to understand the gut microbial ecology shifts between healthy and obese individuals. Stool samples of 72 adult individuals were collected and dependent and independent cultive approach were performed to evaluate the fungi. The yeasts were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the filamentous fungi were identified by microculture. The mean count of filamentous fungi and yeast was 1.58 log10 CFU/g of feces. Significant differences in the population level of the filamentous fungi was observed within eutrophic and obese groups. Overall, 34 genera were identified. The predominant phylum was Ascomycota with 20 different genera, followed by Basidiomycota and Zygomycota. The most prevalent specie was Paecylomyces sp. in euthrophic individuals and Penicillium sp. in obese individuals. The results of the eutrophic individuals suggest a slightly higher diversity of fungi within these individuals. Female individuals had a greater diversity of fungal types compared to males. The denaturing gradient gel electrophoresis (DGGE) analysis showed the grouping of eutrophic and overweight individuals in one cluster and of obese individuals in another cluster, although richness was low in all three groups. The fluorescence in situ hybridization (FISH) analysis showed a higher relative density of C. albicans in the obese group when compared to eutrophic and by the quantitative real time PCR (qPCR) a larger number of DNA copies of fungi and the phylum Ascomycota in obese individuals when compared to overweight and obese individuals. There was a positive correlation between fungi and the anthropometric parameters, total cholesterol, LDL, triglycerides, hemoglobin, HOMA-IR, HOMA-β, insulin, serum glucose, creatinine and percentage of dietary fibers and carbohydrates. Other studies are needed to better understand the causal relationship between gut mycobiota and obesity. This knowledge could be used in modulating the gut mycobiota and identifying future obesity treatments.

Les systèmes d'élevage des petits ruminants au pâturage sont les plus répandus en zone tropicale humide mais l'exposition aux strongles gastro-intestinaux entraîne des pertes importantes de production. L'objectif de ce travail est d'évaluer un système de gestion mixte du pâturage associant diverses espèces animales permettant à priori d'accroître les performances via la réduction du parasitisme et!ou une meilleure alimentation. L'étude de la littérature a conduit à une méta-analyse confirmant de meilleures performances individuelles ou à l'hectare pour les ovins en mixte. Préalablement un dispositif visant à vérifier la non-transmission des larves d'Haemonchus contortus des caprinsaux génisses Créoles a été validé. Un second dispositif avec des chevrettes conduites en mixte, infestées ou pas par Haemonchus comortus (Ml et MnI) ont été comparées à des chevrettes contrôles infestées ou non (CI et CnI). Ce dispositif a été conduit pendant 2 ans avec des mesures du couvert pâturé, des mesures individuelles des quantités ingérées, de la digestibilité, du parasitisme et de la croissance. De plus fortes croissances ont étéobservées en pâturage mixte que les chevrettes soient infestées ou pas (43.25 et 31.68 glj pour MDI et MI vs. 32.44 et 17.91 glj) avec une moindre biomasse. Les mesures d'ingestion et d'ingéré digéré ont été corrélées aux croissances des chevrettes contrairement aux variables parasitaires et met en évidence le rôle de l'alimentation et de la résilience dans le bénéfice du pâturage mixte. Une autre gestion intégrée du pâturage a été étudiée basée sur l'utilisation du vermicompost et s' ant sur les mêmes leviers d'action: l'alimentation et le itisme / Systems of small ruminant grazing are most prevalent in the humid tropics but this environment exposes animals to gastro-intestinal with production losses. The objective ofthis thesis is to evaluate a system ofmixed management ofthe pasture combining various animal species, which allows a priori to increase animal performance by reducing the parasitism and/or better nutrition. The study of literature bas led to a meta-analysis confirming the better individual performances or calculated per ha for sheep reared mixed. The first experiment validated the non-transmission of Iarvae ofHaemonchus contortus from goats to heifers. In a second experiment, goats mixed with heifers, infested or not with Haemonchus contortus (Ml vs MnI) were compared with controls goats, reared alone, infested or not (CI and CNI). This deviee with a continuous driving was studied for two years with measurements on the sward, individual measures of intake, digestibility, parasitism and growth ofthe goats. The highest growth rates were measured with the mixed pasture as goats are infested or not (43.25 and 31.68 g / d for MN! and MI vs. 32.44 and 17,91 g / d) with lower biomass. The herbage intake and the digested herbage intake were well correlated to the growth rate, unIike parasitic variables, and highlight the role of diet in the benefit of mixed driving for goats. Therefore, the infested goats with Haemonchus, expressed greater resilience with improved growth performance compared with controls. Parallel to the study ofmixed pasture, another integrated management of grazing was studied, based on the use of vermicompost and relying on the same levers. namelv throuzh feedinz and parasitism

La période des 1000 premiers jours de vie constitue une fenêtre de sensibilité au cours de laquelle l’environnement peut moduler le développement du foetus et du nourrisson, et conditionner la santé tout au long de la vie. Les conséquences à long terme de l’environnement périnatal sur le risque de maladies intestinales sont toutefois encore peu connues. L’objectif de ce travail était de montrer l’impact de l’environnement nutritionnel précoce sur la maturation intestinale et la santé intestinale à long terme.Un retard de croissance postnatal (RCPN) était induit chez la souris FVB/NRj par augmentation de la taille des portées. Le RCPN était responsable d’un retard de maturation de l’intestin chez la souris au sevrage, en particulier de la barrière intestinale, caractérisé par une augmentation de la perméabilité intestinale, concomitante d’une désorganisation des protéines des jonctions serrées. Le microbiote intestinal était moins riche en espèces bactériennes chez la souris au sevrage en cas de RCPN et sa composition était différente, avec en particulier une proportion anormalement élevée de Parabacteroides spp, Enterococcus spp, Erysipelatoclostridium spp, Eubacterium coprostanoligenes spp, Staphylococcus spp, et Escherichia-Shigella spp, et une plus faible proportion d’espèces productrices de butyrate. L’absence de barrière intestinale efficace et la dysbiose induites par le RCPN, étaient associées à une altération de la réponse inflammatoire de l’intestin à l’âge adulte, caractérisée par une augmentation des réponses immunitaires Th1, Th17 et Treg et une plus grande susceptibilité à la colite chimiquement induite.Ce travail démontre l’importance de l’environnement nutritionnel précoce dans la programmation de la santé intestinale au cours de la vie, et conforte l’hypothèse d’une origine développementale des maladies intestinales chroniques. / The first thousand days of life are a critical time for the development of both the fetus and the infant, and can modify the risk profile for diseases in later life. However, the longterm consequences of the perinatal environment on the susceptibility to intestinal disorders have not yet been assessed. The aim of the present study was to investigate the impact of the early nutritional environment on intestinal maturation and gut health in later life.Postnatal growth restriction (PNGR) was induced in FVB/NRj mice during the suckling period by adjusting the litter size. PNGR delayed intestinal maturation in pups at weaning. PNGR was associated with a maturation delay of the intestinal barrier, characterized by an increased intestinal permeability and impaired tight junctions. At the same time, PNGR affected gut bacterial colonization. Pups with PNGR harbored a decreased bacterial diversity, higher Parabacteroides spp, Enterococcus spp, Erysipelatoclostridium spp, Eubacterium coprostanoligenes spp, Staphylococcus spp, and Escherichia-Shigella spp, and lower butyrate producers. The lack of an efficient intestinal barrier and the dysbiosis induced by PNGR were associated with an altered intestinal inflammatory response in adult mice, characterized by an increase of Th1, Th17 and Treg immune responses, and a higher susceptibility to chemically induced colitis.Our data emphasize the importance of the early nutritional environment in programming of gut health in later life, and support the hypothesis of the developmental origin of chronic intestinal disorders.

Le récepteur membranaire TGR5 (Takeda G Protein-coupled Receptor 5), est un récepteur ubiquitaire sensible aux acides biliaires. Il est exprimé dans de nombreux tissus et organes dont l’intestin (dans les cellules entéroendocrines L), la vésicule biliaire, les muscles lisses et squelettiques, le tissu adipeux brun et dans certaines cellules immunitaires. Des études menées in vitro et in vivo chez l’animal ont montré des effets bénéfiques de l’activation de TGR5 sur l’homéostasie énergétique et glucidique. Il est maintenant communément admis que les effets bénéfiques de TGR5 sur l'homéostasie du glucose sont, au moins en partie, médiés par sa capacité à promouvoir la sécrétion de l'incrétine intestinale glucagon-like peptide-1 (GLP-1) au niveau des cellules entéroendocrines L.Cependant, de récentes expériences ont montré que l’activation de TGR5 par des agonistes systémiques dans des modèles animaux peut induire des effets non souhaités tels qu’une augmentation du volume de la vésicule biliaire, des démangeaisons et des effets cardiovasculaires. Afin de s’affranchir des effets non désirés d’agonistes systémiques de TGR5, le projet s’est orienté vers le développement d’agonistes de TGR5 présentant une distribution tissulaire ciblée et limitée à l’intestin et dont la biodisponibilité orale serait très faible, voire nulle. Nous avons alors émis l’hypothèse qu’une activation de TGR5 limitée à l’épithélium intestinal sans exposition systémique permettrait d’obtenir des effets bénéfiques sur l’homéostasie du glucose via l’effet GLP-1 sécrétagogue, tout en minimisant les effets non souhaités sur d’autres tissus ou organes exprimant TGR5.A partir des études de relations structure-activités obtenues au laboratoire sur une série d’agonistes de TGR5, nous avons conçu des composés chimériques de la façon suivante : le pharmacophore responsable de l’activité sur le récepteur TGR5 est lié via un bras espaceur à des éléments structuraux appelés kinétophores qui ajustent les propriétés physicochimiques et pharmacocinétiques de nos agonistes pour limiter leur absorption intestinale. Ainsi, l’objectif de ce travail était d’obtenir des agonistes non systémiques de TGR5, puissants et originaux, exerçant leur action dans l’intestin afin de générer la preuve de concept in vivo de l’intérêt d’utiliser de tels agonistes dans le traitement du diabète de type 2.Une étude systématique de l’effet de kinétophores variés a été réalisée. Une trentaine de composés ont été synthétisés en 8 à 12 étapes permettant l’identification d’agonistes puissants et présentant des propriétés pharmacocinétiques en accord avec notre stratégie de composés topiques intestinaux. Des études in vivo ont ensuite permis de valider l’effet GLP-1 sécrétagogue de tels composés. Enfin, l’évaluation d’un des meilleurs composés dans un modèle murin de diabète nous a permis de valider l’hypothèse qu’un agoniste topique intestinal de TGR5 peut avoir un effet bénéfique sur l’homéostasie énergétique et glucidique. / The membrane receptor TGR5 (Takeda G Protein-coupled Receptor 5) is an ubiquitous receptor sensitive to bile acids. It is expressed in many tissues and organs including the intestine (in enteroendocrine L cells), the gallbladder, smooth and skeletal muscles, brown adipose tissue and in some immune cells. In vitro and in vivo studies in animals have shown beneficial effects of TGR5 activation on energy and glucose homeostasis. It is now commonly accepted that the beneficial effects of TGR5 on glucose homeostasis are, at least in part, mediated by its ability to promote the secretion of the intestinal incretin glucagon-like peptide-1 (GLP-1) in enteroendocrine L cells.However, recent experiments have shown that the activation of TGR5 by systemic agonists in animal models can induce unwanted effects such as increased gallbladder volume, itching and cardiovascular issues. In order to avoid the undesired effects of systemic agonists of TGR5, the project focused on the development of TGR5 agonists with an intestine targeted distribution and a very low oral bioavailability. Then, we hypothesized that the activation of TGR5 limited to the intestinal epithelium without systemic exposure would promote the beneficial effects on glucose homeostasis via the GLP-1 secretagogue effect, while minimizing systemic effects on other tissues or organs expressing TGR5.On the basis of structure-activity relationships on a series of TGR5 agonists developed in the laboratory, we have designed chimeric compounds as follows: the pharmacophore responsible for activity on the TGR5 receptor is bound, via a linker, at structural elements called kinetophores that fine-tune the physicochemical and pharmacokinetic properties of our agonists to limit their intestinal absorption. Thus, the aim of this work was to obtain powerful and original non-systemic TGR5 agonists acting in the intestine to generate the in vivo proof of concept of the therapeutic potential of such agonists in the treatment of type 2 diabetes.A systematic study of the effect of various kinetophores was performed. About thirty compounds have been synthesized in 8 to 12 steps allowing the identification of powerful agonists with pharmacokinetic properties in accordance with our strategy of topical intestinal compounds. In vivo studies were then used to validate the GLP-1 secretagogue effect of such compounds. Finally, evaluation of one of the best compounds in a murine model of diabetes allowed us to validate the hypothesis that a topical intestinal agonist of TGR5 can have a beneficial effect on energy and glucose homeostasis.

L'épithélium intestinal se renouvelle tous les 4 à 6 jours chez les mammifères grâce aux cellules souches localisées au fond des cryptes. Le renouvellement dépend des signaux émis par le microenvironnement et requiert une phase de prolifération des cellules souches, de différenciation et d'apoptose/desquamation des cellules épithéliales. La signalisation Wnt, joue un rôle majeur dans l'homéostasie intestinale par l'action de deux gradients inversés le long de l'axe crypte/lumière ; la signalisation Wnt canonique, active au fond des cryptes, contrôle la prolifération alors que la signalisation non-canonique, active vers le haut des cryptes contrôle la différenciation. Il a été montré que ces deux voies contrôlent l'activité de la GTPase atypique RhoU/Wrch1. RhoU fait partie des GTPases qui s'activent spontanément, son activité est donc directement proportionnelle à son niveau d'expression dans la cellule. Enfin, cette GTPase atypique est sous exprimée dans de nombreuses tumeurs gastriques et colorectales.Compte tenu de ces données, nos objectifs étaient donc de caractériser les changements morphologiques induits par l'invalidation conditionnelle de RhoU dans l'épithélium intestinal murin et d'en déterminer les mécanismes d'action. Nos résultats montrent que la déplétion de RhoU n'est pas létale, cependant elle a induit une augmentation de 20% de la densité cellulaire et une désorganisation de la structure de l'épithélium dans le haut des cryptes du colon. Cette augmentation concerne aussi bien les lignages sécrétoires et absorptifs, cependant, l'absence de RhoU a induit une sur-représentation du lignage sécrétoire. Dans la lignée de tumeur colorectale DLD-1, nous avons montré que l'absence de RhoU mime le phénotype d'augmentation de la densité cellulaire observé chez la souris. L'invalidation de RhoU ne modifie pas la distribution des phases du cycle cellulaire ni de celle de la mitose, cependant, elle réduit le nombre des cellules en apoptose dans le colon des souris et dans les cellules DLD-1. L'invalidation de RhoU a réduit la signalisation Hippo et a altéré la contractilité cellulaire via une augmentation de la phosphorylation de la protéine MLC2. Des travaux récents ont montré que la diminution du niveau MLC2 phosphorylée est nécessaire pour l'activation des caspases par un stimulus apoptotique. Ceci suggère que cette perturbation de la contractilité peut être à l'origine de cette diminution de l'apoptose qui est la cause majeure responsable de ce phénotype. En conclusion, RhoU est un régulateur de l'homéostasie intestinale chez la souris via son rôle modérateur de la mort cellulaire. / In Mammals, the intestinal epithelium is renewed every 4-6 days through the stem cells located at the bottom of crypts. The renewal depends on signals from the micro-environment and requires a proliferation phase of stem cells, then a differentiation and apoptosis/desquamation phases of epithelial cells. Wnt signaling plays a major role in intestinal homeostasis by the action of two reversed gradients along the axis crypt/ lumen: canonical Wnt signaling, active in the bottom of crypts, control proliferation while non canonical signaling, active in the top of the crypts control cell differentiation. It was shown that these two pathways are regulator of the atypical GTPase RhoU/Wrch1. The RhoU protein activates spontaneously, its activity is directly proportional to its expression level in the cell and is expressed as in gastric and colorectal tumors. In view of these informations, our objectives were therefore to characterizethe morphological changes induced by conditional invalidation of RhoU in the intestinal epithelium of mice and to determine the mechanisms of action. Our results show that RhoU depletion is not lethal. However, it induces an increase of cell density (+20%) and a disruption of the epithelium structure in the top of the colonic crypts. This increase affects both absorptive and secretory lineages. However, the absence of RhoU induced over-representation of secretory lineage. In colorectal tumor cell line DLD-1, we have shown that the absence of RhoU mimics the phenotype of cell density increase observed in mice. RhoU invalidationdid not change the distribution of cell cycle phases and mitosis, however, it reduces the number of apoptotic cells in the colon of mice and in the DLD-1 cells. RhoU invalidation reduced Hippo signaling and altered cell contractility via the increase of the protein MLC2 phosphorylation. Recent work has shown that the reduction of MLC2-P level is necessary for the caspase protein activation by an apoptotic stimulus. Suggesting that the perturbation of contractility may be the cause of this apoptosis decrease which is the main cause responsible of this phenotype. Finally, RhoU is a regulator of the intestinal homeostasis in micevia its moderating role of cell death.