The prevalence of overweight and obesity have risen to epidemic proportions. Overweight and obesity are prominent risk factors for the development of chronic disease including diabetes, cardiovascular disease and cancer. Especially pronounced in women of reproductive age and children, the obesity epidemic represents a major threat to global health. Maternal obesity is a key predictor of childhood obesity and diseases of metabolic origin in adulthood. Previous work has demonstrated that the exposure to early life adversity, in the context of maternal obesity, is associated with an increased risk of metabolic disease and obesity in the offspring later in life. Although the mechanisms outlining the relationship between maternal and offspring obesity remain unclear, the intestinal microbiota has come forth as a promising area of research. To understand the factors involved in the maternal intestinal microbial shifts with healthy pregnancy, the preliminary study focused on investigating whether female sex-steroid hormones mediate maternal intestinal microbial shifts in non-pregnant, regularly cycling female mice. We have identified that intestinal microbial shifts are not associated with sex-steroid hormone fluctuations. The second study examined whether maternal intestinal microbial shifts that occur during obese pregnancy were associated with altered inflammatory signaling and function of the maternal intestine and placenta at a critical period of development; embryonic (E) day 14.5. Females fed a high fat diet (HFD) were significantly heavier at mating and throughout gestation compared to CON. At E14.5, High fat (HF) dams displayed increased adiposity, hyperglycemia, hyperinsulinemia, hyperleptinemia and were insulin resistant. Pregnancy and maternal obesity resulted in shifts in the maternal intestinal microbiota, where the most significant increase in microbial relative abundance was exhibited by the mucin degrading genus, Akkermansia. At E14.5, maternal intestinal microbial shifts were associated with higher maternal intestinal NFκB activity in all sections of the maternal intestine, most notably in the maternal colon. Maternal obesity was associated with increased Muc5ac mRNA levels and a modest increase in CD3+ T cells in the maternal colon at E14.5. However, maternal intestinal permeability was unchanged between groups. In the placenta, mRNA levels of key signaling components in the pro-inflammatory toll-like receptor 4 (TLR4) pathway; TRAF6, NFκB and potent pro-inflammatory cytokine TNF-α were increased and in HF females. Maternal obesity was associated with an increase in CD3+ T cells in the junctional zone (JZ), but not in the labyrinth zone (LZ) of the placenta at E14.5. These findings were associated with increased mRNA levels of critical nutrient transporters; glucose transporter 1 (GLUT1) and sodium-coupled neutral amino acid transporter 2 (SNAT2) and a modest increase in glucose transporter 3 (GLUT3) in HF placentae compared to CON. These data identify the mechanistic signaling pathways and cell types involved in modulating the intrauterine environment, thus contributing to the current literature devoted to the investigation of the developmental origins of obesity. / Thesis / Master of Science (MSc)

Gut microbiota has been shown to regulate the growth and development of the central and enteric nervous systems (CNS and ENS) after birth. There is ample evidence to suggest that intestinal bacteria can influence behavior of the host through both immune and immune-independent mechanisms. Gut-microbiota-regulated CNS structural changes are focused in the limbic system, at centres associated with memory, social and emotional behaviour. Several studies using germ-free (GF) and specific pathogen free (SPF) mice demonstrated microbial influence on behaviour development accompanied by neurochemical changes in the brain. Higher exploratory and lower anxiety-like behavior was found in GF mice compared to SPF mice with lower central expression of neurotrophins, such as nerve growth factor and BDNF. The mechanisms by which the microbiota influences behavior are unknown but could be immune-mediated, neural, or humoral in origin. In this study I investigated the role of immune system maturation on mouse behaviour after bacterial colonization. I showed that mono-colonization of GF mice with E. coli normalizes behaviour similar to colonization with complex microbiota (SPF and ASF) and the continuous presence of bacteria is not required to maintain this normal behaviour. I also showed that innate immunity through the MyD88/Ticam pathway is crucial for the development of normal behaviour and that multiple innate immunity and neuronal genes are involved in this process. Together these results suggest that bacterial colonization primes and matures the innate immunity and development of the central nervous system ultimately leading to normal behaviour. I believe that bacterial colonization at birth is not only important for the epithelial barrier function, gut homeostasis, and immune functions, but also for the development of normal behaviour. Altered immune priming during the postnatal period due to abnormal microbial colonization may have wider implications when considering psychiatric disorders in humans. / Thesis / Doctor of Philosophy (PhD)

L’obésité et la maladie de Crohn (MC) sont deux pathologies inflammatoires chroniques caractérisées par un développement de la masse grasse viscérale et une dysbiose. L’activité physique (AP) impactant positivement ces deux paramètres, elle apparait donc comme une stratégie thérapeutique prometteuse dans la prise en charge de ces patients. Dans ce contexte, l’objectif de ce travail était d’étudier l’effet de l’AP sur l’interaction microbiote-tissu adipeux. Sur un versant clinique, nos résultats ont confirmé l’efficacité de l’entrainement intermittent de haute intensité (HIIT) pour diminuer le tissu adipeux viscéral chez des sujets en surpoids et/ou obèses. Sur un modèle d’obésité génétique (rat Zucker), nos travaux ont montré que l’entraînement de type HIIT diminuait la masse grasse totale et viscérale mais cela indépendamment du microbiote intestinal. L’étude de la voie de la lipolyse a montré un effet anti-lipolytique du HIIT dans le tissu adipeux sous-cutané, pouvant ainsi partiellement expliquer la diminution du tissu adipeux viscéral. En outre, le HIIT améliore, pour une moindre durée de pratique (vs. l’entraînement continu d’intensité modérée), la tolérance au glucose et le statut inflammatoire. Dans un modèle d’inflammation intestinale mimant la MC, nous avons également mis en évidence que l’AP spontanée augmentait l’expression des protéines des jonctions serrées pouvant participer à la réduction de l'endotoxémie métabolique. En parallèle, l’AP spontanée favorise les bactéries bénéfiques pour la santé et augmente les niveaux de butyrate dans les selles. Ces adaptations participent à la réduction du tissu adipeux viscéral mésentérique caractérisant la MC. En conclusion, l’AP, à travers différentes modalités d’exercice, se révèle comme une « thérapie » attractive et innovante dans la prévention et/ou la prise en charge de ceux pathologies inflammatoires chroniques. / Obesity and Crohn's disease (CD) are two chronic inflammatory diseases characterized by development of visceral fat mass and dysbiosis. Physical activity (PA) has a positive impact on these two parameters. Consequently, PA appears as a promising therapeutic strategy for the management of these patients. In this context, the objective of this work was to study the effect of PA on the microbiota-adipose tissue cross-talk. Our clinical results confirmed the effectiveness of high intensity intermittent training (HIIT) to reduce visceral adipose tissue in overweight and/or obese volunteers. Then, using an animal model of genetic obesity (Zucker rats), we found that HIIT decreases total and visceral fat mass, independently of gut microbiota. Analysis of the lipolysis pathway showed an anti-lipolytic effect of HIIT in the subcutaneous adipose tissue, and this could explain the decrease in visceral adipose tissue. In addition, compared with continuous moderate intensity training, HIIT improved glucose tolerance and the inflammatory status despite the shorter exercise duration. Finally, in an animal model of CD, we found that spontaneous PA increased the expression of tight junction proteins, contributing to the reduction of metabolic endotoxemia. Concomitantly, spontaneous PA promoted healthy bacteria in the colon and increased fecal butyrate levels. These adaptations limited the expansion of mesenteric visceral adipose tissue, a typical CD feature. In conclusion, PA, through different exercise modalities, appears as an attractive and innovative « therapy » for these two chronic inflammatory diseases.

Introdução: A Síndrome do Intestino Curto (SIC) é definida como um conjunto de sinais e sintomas conseqüentes de alterações nutricionais e metabólicas secundária a insuficiência intestinal funcional e/ou orgânica, como nos casos de grandes ressecções do intestino delgado. Ressecções intestinais eliminam sítios de colonização, alteram a área de absorção e, o uso freqüente de antibióticos devido a infecções recorrentes, presença de alimentos não digeridos no cólon, trânsito acelerado e diarréia, nestes mesmos pacientes, podem contribuir para a alteração da microbiota intestinal. Objetivo: Caracterizar a microbiota intestinal de pacientes portadores da Síndrome do Intestino Curto atendidos na Unidade Metabólica e do HCFMRP/USP. Casuística e Métodos: Foram recrutados os pacientes portadores de síndrome do intestino curto atendidos na Unidade Metabólica do HCFMRP/USP (uso de terapia nutricional parenteral). Para o grupo controle foram recrutados indivíduos sadios e eutróficos da comunidade e pareados segundo sexo e idade. Foram avaliadas amostras de fezes e exames bioquímicos, estes últimos foram somente dos pacientes. A avaliação do consumo alimentar foi feita por meio do inquérito Diário Alimenta e a avaliação do estado nutricional a partir de medidas antropométricas. Para detecção de cepas patogênicas foram realizados cultivos e testes bioquímicos específicos em meio aeróbio para determinação de espécies da família Enterobacteriaceae. Em cada cepa de E. coli isolada foram aplicados anti-soros para determinação de possível patogenicidade. Metodologia molecular também foi utilizada para determinação do perfil da microbiota intestinal bacteriana: sequenciamento de bibliotecas de DNAr 16S e PCR para detecção de genes característicos de cepas patogênicas de E. coli. Resultados: Verificou-se a presença de subnutrição protéico-calórica no grupo Paciente mesmo com terapia nutricional para recuperação deste estado nutricional. Quanto a microbiota, observou-se maior diversidade de Gram negativas, porém menor quantidade por grama de fezes da família Enterobacteriaceae em pacientes com SIC. Porém a análise molecular mostrou a manutenção na proporção de espécies bacterianas, equivalente a microbiota intestinal saudável. Conclusão: Apesar da subnutrição, retirada maciça do intestino delgado, uso freqüente de antibióticos, depressão do sistema imune, presença de alimentos não digeridos no trato gastrintestinal e transito intestinal acelerado, a relação e proporção entre as espécies bacterianas intestinais permanecem similares à normalidade. / Introduction The short bowel syndrome (SBS) is defined as a set of signs and symptoms resulting from nutritional and metabolic changes after major small bowel resections. Intestinal resections eliminate colonization sites and alter the absorption area. Besides, the frequent use of antibiotics due to recurrent infections, the presence of undigested food remaining in the intestinal tract, rapid transit and diarrhea in these same patients may contribute to the change of intestinal microbiota. Objective: To characterize the intestinal microbiota of patients with short bowel syndrome treated at the Metabolic Unit and HCFMRP / USP. Materials and Methods: We recruited all the patients with short bowel syndrome treated at the Metabolic Unit of HCFMRP / USP (use of parenteral nutrition therapy). The control group was composed by healthy individuals matched by age and sex. Samples of feces and biochemical tests from the patient group were evaluated. The control group had no biochemical tests, only feces samples to be analysed. The nutritional status was evaluated through anthropometric measurements and the assessment of food intake through food diary survey. The pathogenic strains from Enterobacteriaceae were detected by cultivation and specific biochemical tests in aerobic environment. In each strain of isolated E. coli antisera were applied for determination of pathogenicity. PCR analysis was also used to determine the profile of intestinal bacterial microbiota: sequencing libraries of 16S rDNA and PCR for detection of genes characteristic of pathogenic strains of E. coli. Results: Although receiving periodic parenteral nutrition the Patient group had protein-calorie malnutrition. In regards to the microbiota there was greater diversity, but lower concentration of the family Enterobacteriaceae in patients with SBS. However, the molecular analysis showed the a proportion of bacterial species equivalent to the intestinal flora from the control group. Conclusion: Although the protein-calorie malnutrition, massive resection of the small intestine, frequent use of antibiotics, immune system depression, presence of undigested food in the gastrointestinal tract and rapid intestinal transit rate, the ratio and the proportion of the intestinal bacterial species remain similar to normal.

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.

Orientador: Paulo Câmara Marques Pereira / Banca: Maria Tereza Duenhas Monreal / Banca: Lenice do Rosário de Souza / Resumo: A microbiota intestinal normal embora bastante estável pode se alterar em condições patológicas, modificações na composição da dieta, presença de distúrbios gastrointestinais e/ou ingestão de drogas. A associação de infecção com a utilização de medicamentos dificulta a interpretação da participação desses fatores na microbiota intestinal. O objetivo do presente estudo foi avaliar a microbiota intestinal de indivíduos que sofreram acidente ocupacional com materiais biológicos e receberam anti-retrovirais. Foram estudados 23 indivíduos adultos com idade entre 18-45 anos, sendo 13 doadores de sangue, grupo controle (GC) e 10 que sofreram acidente ocupacional com material biológico e realizaram profilaxia anti-retroviral. Foram avaliados a microbiota intestinal, medidas antropométricas, exames laboratoriais (hemograma, função renal, hepática, lipidograma, glicemia, proteínas totais e frações) pré, após a medicação e 30 dias após o término da medicação. A zidovudina mais a lamivudina foi utilizada em 70% dos indivíduos associado ao nelfinavir, 20% ao efavirenz e 10% ao ritonavir. Náuseas, vômitos e diarréia estiveram presentes em 80% no segundo momento do estudo. Sobrepeso em 70%, desnutrição e eutrofia em 10%, dos indivíduos sem alteração durante o estudo. As enzimas AST, ALT, Gama-GT e triglicérides, LOL-colesterol se elevaram no segundo momento e se normalizaram 30 dias após término da medicação. Foi observada redução significativa dos três gêneros de bactérias anaeróbias avaliadas Lacfobacillus , Bifidobacferium e Bacleróides em relação ao grupo controle nos três momentos. O uso de anti-retrovirais provocou impacto significativo na microbiota intestinal dos indivíduos normais em uso de anti-retrovirais, não sendo recuperada 30 dias após o término da medicação. / Abstract: Pathological conditions, changes in diet composition, presence of gastrointestinal disorders and/or ingestion of drugs may alter the normal intestinal microbiota, regardless of its sufficient steadiness. The association of infection with the use of medicine makes the interpretation of the participation of these factors in intestinal microbiota difficult. The objective of the present study was to evaluate the intestinal microbiota from individuais injured by biological materiais in occupational accident, submitied to antiretroviral prophylaxis. 23 adult individuais with ages between 18-45 years old were studied, being 13 blood donors (control group - CG) and 10 individuais injured by biological materiais in occupational accident, submitled to antiretroviral prophylaxis. Intestinal microbiota, anthropometric measures and biochemical examinations (blood count, renal and hepatic functions, glucose and lipids blood levels, total proteins and fractions) were evaluated before, right after and 30 days after the end of medication. Zidovudine plus lamivudine were used in 70% of the individuais associated to nelfinavir, 20% to efavirenz and 10% to ritonavir. Nausea, vomiting and cliarrhea were present in 80% of the individuais at the second part of the study. Overweight was noticed in 70% and malnutrition anel eutrophia were noticed in 10% of the individuais without alterations during the study. AST, ALT, Gamma-GT and triglycerides and LDL-cholesterol enzymes were increased at the second part and normalized 30 days after the end of medication. Significant reduction of the three genera of anaerobic bacteria - Lactobacil/us, Bifidobacterium and Bacteroides - evaluated was observed in reiation to the controi group at the three moments. The use of antiretrovirals caused significant impact in the intestinal microbiota of the normal individuais, without recovery 30 days after the end of medication. / Mestre

A microbiota gastrointestinal humana normal é um ecossistema complexo constituído por microrganismos anaeróbios que desempenham papel fundamental na manutenção da saúde e de funções fisiológicas do hospedeiro. O presente estudo objetivou caracterizar a microbiota intestinal de pacientes portadores de constipação funcional e de pacientes com espinha bífida e intestino neurogênico e compará-las com pacientes saudáveis. Estudo transversal inclui 25 crianças com constipação funcional, 25 pacientes saudáveis e 14 pacientes com intestino neurogênico e espinha bífida. A metodologia molecular foi utilizada para determinação do perfil da microbiota intestinal. O DNA total das amostras de fezes foi extraído com o kit QIAamp DNA Stool®-QIAGEN e realizado sequenciamento do gene 16S rRNA (MiSeq (Illumina). As sequências obtidas foram processados no QUIIME. A análise dos dados obtidos foi realizada por meio de estatística descritiva. A diversidade e riqueza da microbiota intestinal foi avaliada pelos índices Shannon, Simpson e Chao e a contribuição de outras variáveis (sexo, tempo de amamentação exclusiva, uso de medicação nos pacientes constipados, tipo de parto e presença de sintomas no momento da coleta da amostra de fezes) foi obtida por análises multivariada. Firmicutes foi o filo mais predominante seguido do filo Bacteroidetes nos três grupos de estudo. Bifidobacterium foi mais abundante nos constipados funcionais do que nos saudáveis. O filo Tenericutes foi mais abundante em pacientes que nasceram por parto cesárea se comparados com parto vaginal independente do grupo de estudo. Participantes sintomáticos no momento da coleta, apresentaram maior abundância do gênero Ruminoclostridium em relação aos indivíduos assintomático, independente do grupo de estudo. No grupo dos pacientes saudáveis, os participantes sintomáticos no momento da coleta possuíam nível maior do gênero Phascolarctobacterium. Quanto ao sexo, tempo de amamentação exclusiva e medicações utilizadas para tratamento da doença, não foram encontradas nenhuma diferença na microbiota entre os grupos. Concluímos, portanto, que pacientes com constipação intestinal funcional apresentaram maior abundância de Bifidobacterium, em relação ao grupo controle, tal achado não foi observado no grupo de intestino neurogênico / The human gastrointestinal microbiota is a complex ecosystem consisting of anaerobic microorganisms that play a key role in maintaining the health and physiological functions of the host. The present study aimed to characterize the intestinal microbiota of patients with functional constipation and patients with spina bifida and neurogenic gut and to compare them with healthy patients. A cross-sectional study included 25 children with functional constipation, 25 healthy patients and 14 patients with neurogenic gut and spina bifida. The molecular methodology was used to determine the profile of the intestinal microbiota. Total DNA from the faeces samples was extracted with the QIAamp DNA Stool®-QIAGEN kit and sequenced the 16S rRNA gene (MiSeq (Illumina)). The sequences obtained were processed in QUIIME. Data analysis was performed using descriptive statistics. The diversity and richness of the intestinal microbiota was evaluated by the Shannon, Simpson and Chao indices and the contribution of other variables (sex, exclusive breastfeeding time, use of medication to treat constipation, type of delivery and presence of symptoms in the sampling time) was obtained by multivariate analysis. Firmicutes was the most predominant phylum followed by the phylum Bacteroidetes in the three study groups. Bifidobacterium was more abundant in patients with functional constipation than in healthy ones. The phylum Tenericutes was more abundant in patients who were born by cesarean section compared to vaginal delivery independent of the study group. Symptomatic participants at the time of collection had greater abundance of the genus Ruminoclostridium in relation to the asymptomatic individuals, independent of the study group. In the group of healthy patients, the symptomatic participants at the time of collection had a higher level of the genus Phascolarctobacterium. Regarding sex, exclusive breastfeeding time and medications used to treat the disease, no difference was found in the microbiota between the groups. We conclude, therefore, that patients with functional intestinal constipation had a greater abundance of Bifidobacterium, in relation to the control group, such finding was not observed in the group of neurogenic intestine.

Le vieillissement est un processus physiologique au cours duquel l’ensemble de l’organisme voit son fonctionnement modifié. Il s’agit d’un remodelage génotypique et phénotypique, en lien avec le stress oxydatif. Les molécules antioxydantes comme les composés phénoliques (CP) ont pris une place importante dans la diète humaine, à titre de compléments alimentaires et/ou à titre thérapeutique. Toutefois, les conséquences d’un usage à long terme de ces molécules visant à reverser les effets du vieillissement sur les fonctions organiques, restent encore mal élucidées. Dans ce contexte, et compte tenu de notre intérêt pour le cœur, le rein et le microbiote intestinal, l’objectif de cette thèse est d’évaluer, sur de jeunes rats mâles, les effets des CP administrés à différentes concentrations, pendant quatorze mois. Les groupes traités par les CP ont montré une préservation, avec l’âge, de la performance cardiaque par rapport aux témoins non traités. De plus, les myocardes de rats âgés traités ont présenté de moindres signes d’inflammation, de fibrose et d’apoptose que les témoins. Ces modifications sont soutenues par un remodelage du niveau d’expression protéique des marqueurs de l’hypertrophie et du stress oxydatif, et des résultats préliminaires suggèrent une activation du courant potassique KATP en présence des CP sur les myofibroblastes. Le tissu rénal conserve son architecture normale avec l’âge chez tous les groupes. Enfin, les métabolites issus des CP ont montré une modulation sélective du microbiote intestinal vers un phénotype sain. Nos travaux montrent dans un modèle murin qu’une consommation régulière de CP permet de préserver le cœur, les reins et le microbiote du remodelage lié au vieillissement. / Aging is a physiological process in which the entire body sees its normal functional capacities modified. It is associated with a genotypic and phenotypic remodeling, related to oxidative stress. Antioxidant molecules such as phenolic compounds (PC), have taken an important place in the human diet, as food supplements and/or as therapeutics. However, the consequences of long-term use of these molecules to reverse the effects of aging on organic functions, remain poorly understood. In this context, and considering our interest for the heart, the kidney and the intestinal microbiota, the aim of this thesis is to evaluate, in young male rats, the effects of PC administered at different concentrations for a period of fourteen months. PC treated groups showed a dose-dependent preservation of cardiac morphology and performance compared to control untreated ones. Additionally, myocardia from treated aged rats exhibited less inflammation, fibrosis and cardiomyocyte apoptosis than controls. These modifications were supported by a remodeling of the proteins level expression of the markers of hypertrophy and oxidative stress, and preliminary data suggest a concomitant activation of potassium current KATP on myofibroblasts in the presence of PC. Renal tissues retained their normal architecture with age in all groups. Finally, derived-metabolites from PC showed a selective modulation of intestinal microbiota towards a healthy phenotype. Our work shows that regular consumption of PC may preserve the heart, kidneys and microbiota of age-related remodeling.

La consommation excessive d’alcool est la première cause de cirrhose en France. L’atteinte hépatique débute par une stéatose (accumulation de triglycérides dans les hépatocytes) qui peut évoluer vers un état inflammatoire (hépatite alcoolique) lors d’une consommation chronique d’alcool. La maladie peut ensuite évoluer vers la fibrose, la cirrhose et jusqu'à l’hépatocarcinome. La mortalité des formes aiguës de l’hépatite alcoolique sévère est comprise entre 50 et 75%. La corticothérapie est le seul traitement qui peut améliorer le pronostic à court terme. D’autres facteurs que la seule consommation excessive d’alcool interviennent dans la genèse des lésions hépatiques. Ainsi, parmi les sujets ayant une forte consommation d’alcool à long terme, la majorité des patients développent une stéatose mais seulement 10 à 35% développeront une hépatite et 8 à 20% évolueront vers la cirrhose. La recherche de facteurs qui relient la consommation d’alcool et la nature et progression des lésions hépatiques est donc essentielle pour trouver de nouvelles cibles thérapeutiques améliorant la prise en charge de ces formes graves. Afin de rechercher quels sont ces facteurs, nous avons utilisé un modèle murin d’alcoolisation. Nous avons utilisé un régime Lieber de Carli (LDC) enrichi en graisses, additionné d’alcool ou non. Nous avons orienté notre projet vers un axe microbiote-inflammation hépatique en analysant l’évolution des populations bactériennes intestinales au cours de la surconsommation d’alcool chez la souris. Des résultats nous ont montré que les Bacteroides variaient grandement d’une animalerie à l’autre. L’objet de nos travaux était d’étudier l’effet de la modulation des Bacteroides sur l’apparition des lésions hépatiques lors de la maladie alcoolique du foie. / Excessive alcohol consumption in the first cause of liver cirrhosis in France. Liver damages begin with steatosis (accumulation of fat in hepatocytes), which can progress to an inflammatory state (alcoholic hepatitis) during a long-term chronic alcohol consumption. Then, the disease can degenerate in liver fibrosis, cirrhosis and reach the hepatocarcinoma state. The mortality rate of acute alcoholic hepatitis ranges from 50% to 75%. Treatment based on corticoids is the only available and efficient treatment to improve the short-term prognosis. Other factors than only excessive alcohol consumption play a role in the onset of hepatic damages. Among patients having a long-term alcohol excessive consumption, most of them will develop a steatosis, whereas only 10% to 35% will develop alcoholic hepatitis and only 8 to 20% a liver cirrhosis. Researching these factors linking alcohol consumption and the nature and onset of liver injuries is of the utmost importance in order to find new therapeutic targets improving the patient life expectancy.In order to research these actors, we used a mice alcoholization model. We used a Lieber DeCarli diet, enriched in fat, added with ethanol or not. We studied the link between intestinal microbiota composition and the onset of liver injuries, by analyzing bacterial populations during an excessive alcohol consumption in mice. We discovered that one of the major bacterial population composing the intestinal microbiota, Bacteroides, underwent differential modifications from one animal housing facility to the other. The aim of our work was to modulate the Bacteroides population and study its effects on liver injuries.

Este estudo teve o objetivo de avaliar a eficiência do probiótico em manter o equilíbrio da microbiota intestinal de aves submetidas à antibioticoterapia e as associações com o desempenho. Os tratamentos dietéticos consistiram de uma dieta basal única, à base de milho e farelo de soja, à qual foi acrescido ou não o probiótico Bacillus subtillis (100 g/ton de ração), na concentração de 10? UFC/g. Por 3 dias consecutivos a partir de 21 dias de idade, as aves foram submetidas à antibioticoterapia via água de bebida consistindo de 200 ppm de bacitracina metileno dissacilato (efeito em bactérias Gram-positivas ) e 1000 ppm de sulfato de neomicina (efeito em bactérias Gram-negativas). O experimento foi conduzido com frangos de corte no período de 1 a 42 dias, sendo que de 1 a 21 dias as aves receberam somente o tratamento dietético, e, a partir de 21 dias, as aves receberam os tratamentos dietético e terapêutico. Aos 2, 4 e 6 dias após a antibioticoterapia, 3 aves de cada unidade experimental foram sacrificadas para coleta do conteúdo do intestino delgado e do ceco e obtidos \"pools\" dos conteúdos intestinais em cada unidade experimental para constituir uma repetição. O experimento foi realizado com 4 tratamentos, obedecendo a esquema fatorial 2×2 para as variáveis de desempenho (com e sem probiótico × com e sem antibioticoterapia) com 9 repetições e 2×2×3 para as análises da microbiota intestinal (com e sem probiótico na dieta × com e sem antibioticoterapia × 2, 4 e 6 dias após a antibioticoterapia) com 3 repetições. O DNA total foi extraído dos conteúdos do intestino delgado e ceco para o isolamento da região 16S rRNA e análise das comunidades bacterianas. As técnicas moleculares utilizadas foram a de fingerprinting Terminal Restriction Length Polymorphism (T-RFLP), PCR em tempo real (qPCR) e o sequenciamento, sendo que, para todas as técnicas, a região alvo foi o gene 16S rRNA. Os fatores dietético e terapêutico modularam a microbiota intestinal de forma independente. Houve efeito dos fatores probiótico e antibioticoterapia nos grupos predominantes do conteúdo do intestino, desde a classificação taxonômica filo até a classificação gênero. Alguns grupos filogenéticos foram igualmente afetados pelos 2 fatores em estudo, enquanto que outros grupos foram alterados de forma específica em função do probiótico ou antibioticoterapia. A antibioticoterapia, assim como o probiótico dietético, reduziu o número de unidades taxonômicas operacionais (OTUs) no conteúdo do ceco. O melhor desempenho observado nas aves alimentadas com dietas com probiótico provavelmente está relacionado às alterações observadas na estrutura das comunidades intestinais e grupos filogenéticos, como o acréscimo de Lactobacillus e redução de Clostridiales. A antibioticoterapia modificou a estrutura da comunidade bacteriana, entretanto não provocou queda no desempenho das aves. A comunidade bacteriana do intestino das aves medicadas e suplementadas com probiótico apresentou alta similaridade com a comunidade das aves que receberam apenas probiótico dietético, indicando a possível recuperação da microbiota intestinal aos 6 dias após a antibioticoterapia. / The purpose of this study was to verify the ability of a probiotic in the feed to maintain the stability of gut microbiota in chickens after antibiotic therapy and associations with the performance. The dietary treatments consisted of a cornsoybean meal basal diet that was supplemented or not with a probiotic (Bacillus subtilis) in the concentration of 3×109 cfu/kg of feed. Starting on day 21, the birds were submitted to the antibiotic therapy consisting of 200 ppm of bacitracin methylene disalicylate (for Gram-positive bacteria) and 1,000 ppm of neomycin sulfate (for Gram-negative bacteria) in the drinking water, during 3 days. The trial was conducted with broiler chickens from 1 to 42 days of age, however, from 1 to 21 days, the chickens received only the dietary treatment, and after of the 21 days, the birds received the dietary and therapeutic treatments. At 2, 4 and 6 days after the antibiotic therapy, three chickens from each experimental unit were euthanized and the contents of the small intestine and ceca were collected and pooled by pen. The trial was conducted in a completely randomized design with 4 treatments and 9 replicates in a 2×2 factorial arrangement for performance characteristics (with and without probiotic × with and without antibiotic therapy), and in a 2×2×3 factorial arrangement for gut microbiota (with and without probiotic × with and without antibiotic therapy × 2, 4 and 6 days after of the antibiotic therapy) with 3 replicates per treatment. The DNA was extracted from the contents of the small intestine and ceca to isolate the 16S r RNA and study of the bacterial communities. The molecular techniques used were the terminal restriction fragment length polymorphism (TRFLP), quantitative PCR (qPCR) and sequencing, considering the 16S rRNA -genetargeted. The dietary and therapeutic factors modulated the gut microbiota independently. The probiotic and antibiotic therapy affected the main groups within of the gut content from the phylogenetic classification at the phylum level until the phylogenetic classification at the genera level. Some phylogenetic groups were equally affected by the two factors while other groups were changed in a distinct form depending on of the probiotic or antibiotic therapy. The antibiotic therapy and dietary probiotic decreased the number of taxonomic operational unit (OTUs) in cecal content. The improved performance observed in birds supplemented with probiotic was probably related to changes in the structure of intestinal bacterial communities and phylogenetic groups such as higher Lactobacillus and decreased Clostridiales. Antibiotic therapy modified the bacterial community structure; however it did not cause loss of broiler performance. The gut bacterial community in birds medicated and supplemented with probiotic had high similarity with the gut community of birds that received dietary probiotic only, indicating the possible recovery of the gut bacterial community 6 days after the antibiotic therapy.