Made available in DSpace on 2016-12-08T16:24:19Z (GMT). No. of bitstreams: 1 PGCA14MA145.pdf: 1077215 bytes, checksum: 04613ebfde1b73115c405e13c6220fa4 (MD5) Previous issue date: 2014-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / It was conducted a study to evaluate the nutritional value of dual-purpose wheat and the effects of its inclusion over the nitrogen balance, developing, carcass and meat characteristics and economic viability on pig diet in growing and fattening. For digestibility there were used 10 male pigs, castrated (Landrasse x Large White), with 15 kg, 45 kg and 75 kg live weight. The animals were allotted to metabolic cages, type Pekas during seven days for adaptation to the environment, feed management, followed by five days of total collect of faeces and urine. The same conditions were maintained for the nitrogen balance step, where were used 15 animals divided in 5 treatments (3 animals/repetition): 0, 15, 30, 45 e 60 of wheat inclusion on diets, calculated in order to be isonutritive In order to evaluate the development were used pigs with initial weigh of 20.94 ± 2.04 kg and final of 99.27 ± 4.48 kg, subdivided in four steps: initial (20 29 kg), growth I (30 49 kg), growth II (50 69 kg) and finishing (70 100 kg). The animals were distributed in a setting entirely random with five levels of wheat inclusion (0, 15, 30, 45 and 60%) and five repetitions and (one animal/repetition), totalizing 25 experimental units. It was evaluated the increase of daily gain weigh and alimentary conversion for each phase and total period. With the development, it was accompanied the feces microbiota, with periodic collects directly from animals rectum. Based on the development it was determinate the bioeconomic rate to estimate the maximum price of wheat in different diets and phases. With prices of experimental period, the current prices in four market situation and bioindices calculated, it was estimated the maximum cost for wheat to be economically equal to the non-wheat diet. The wheat presented digestible energy of 3.25; 3.56 and 3.59 Mcal/kg and metabolizable energy of 3.19; 3.40 e 3.42 Mcal/kg for the pigs with 15, 45 e 75 kg live weight, respectively. The increase of wheat inclusion decreases the digestibility coefficient in the diet, decreases the efficiency in use of amino acids, but does not influence the other variables in the nitrogen balance. There was reduction on the daily gain weigh and worsen in alimentary conversion of pigs in the phase growth I, according to the increase of wheat inclusion in the diets. The corn, bran and pork values had more influence on the maximum price than the wheat can cost. The use of wheat middling to the wheat analyzed allowed economically the use from 50 kg live weigh of pigs, regardless of the market situation. The wheat that comes from a dual-purpose growing system, mainly when classified as out of the standard (hectoliter weight > 72 kg/hL) can be included until 60% in diets for pigs in growing and finishing process without economic loss / Foi conduzido um estudo para avaliar o valor nutricional do trigo de duplo propósito e os efeitos da sua inclusão sobre o balanço de nitrogênio, desempenho, características de carcaça e carne, microbiota intestinal e a viabilidade econômica das dietas de suínos em crescimento e terminação. Para a digestibilidade foram utilizados 10 suínos machos castrados (Landrasse x Large White), aos 15, 45, e 75 kg de peso vivo (PV). Os animais foram alojados em gaiolas metabólicas do tipo Pekas durante sete dias, para adaptação ao ambiente, rações e manejo alimentar, seguidos por cinco dias de coleta total de fezes e de urina. As mesmas condições foram mantidas para a etapa do balanço de nitrogênio, na qual foram utilizados 15 animais divididos em cinco tratamentos (três animais/repetição): 0, 15, 30, 45 e 60% de inclusão de trigo nas dietas, calculadas de modo a serem isonutritivas. Para avaliar o desempenho foram utilizados suínos com peso inicial de 20,94 ± 2,04 kg e final de 99,27 ± 4,48 kg, subdivido em quatro fases: inicial (20 29 kg), crescimento I (30 49 kg), crescimento II (50 69 kg) e terminação (70 100 kg). Os animais foram distribuídos em um delineamento inteiramente ao acaso com cinco níveis de inclusão de trigo (0, 15, 30, 45 e 60%) e cinco repetições (um animal/repetição), que totalizou 25 unidades experimentais. Foi avaliado o ganho de peso diário (GPD) e a conversão alimentar (CA) para cada fase e período total. Junto ao desempenho, foi acompanhado a microbiota das fezes, com coletas periódicas diretamente do reto dos animais. Com base no desempenho foi determinado índices bioeconômicos para estimar o preço máximo do trigo nas diferentes dietas e fases. Com os preços vigentes no período experimental, em quatro situações de mercado e os bioindices calculados, foi estimado o custo máximo do trigo para as dietas serem economicamente igual às dietas sem trigo. O trigo apresentou valores de energia digestível de 3,25; 3,56 e 3,59 Mcal/kg e energia metabolizável de 3,19; 3,40 e 3,42 Mcal/kg para os suínos aos 15, 45 e 75 kg de PV, respectivamente. O aumento da inclusão de trigo reduz o coeficiente de digestibilidade da dieta, reduz a eficiência na utilização de aminoácidos, mas não influencia as demais variáveis do balanço de nitrogênio. Houve redução no GPD e piora na CA dos suínos na fase de crescimento I de acordo com o aumento da inclusão do trigo nas dietas. Houve aumento de coliformes fecais com o aumento do nível de trigo nas dietas. O custo do milho e farelo de soja e valor do quilograma do suíno tiveram maior influência sobre o preço máximo que o trigo pode custar. A utilização do preço do triguilho ao trigo estudado viabilizou economicamente a utilização à partir dos 50 kg de peso vivo dos suínos, independente da situação de mercado. O trigo proveniente de um sistema de cultivo de duplo propósito, principalmente, quando classificado como fora do padrão (peso hectolitro > 72 kg/hL) pode ser incluso até 60% em dietas para suínos em crescimento e terminação sem prejuízos econômicos

O trato gastrointestinal é um sítio de alta exposição antigênica, por isso requer a presença de mecanismos de regulação imunológica mediada por linfócitos T reguladores e T auxiliares produtores de IL-17 (Th17) na mucosa intestinal. Se houvera falha na indução desses mecanismos, pode ocorrer o desequilíbrio das populações de bactérias comensais da microbiota intestinal, denominado de disbiose, geralmente associado à ruptura da barreira intestinal e translocação de bactérias ou LPS para o sangue. Neste sentido, alguns estudos têm evidenciado a importância dos linfócitos Th17 no intestino, já que estas células tem a capacidade de manter a integridade da barreira intestinal e, como conseqüência controlar a colonização e translocação bacteriana. Em adição, em pacientes e animais diabéticos têm sido observada a correlação de altos níveis de LPS circulantes e resistência à insulina. Baseado nessas evidências, nosso objetivo foi avaliar o papel da citocina IL-17 no controle das alterações inflamatórias e metabólicas no modelo de diabetes do tipo 2 (DM2). Para isso, foram utilizados camundongos C57BL/6 selvagens (WT) ou deficientes do receptor da citocina IL-17 (IL-17R-/-) submetidos à dieta controle (DN), composta por 10% de gorduras, 70% de carboidratos e 20% de proteínas ou à dieta hiperlipídica (DH), composta por 60% de gorduras, 20% de carboidratos e 20% de proteínas. Nossos dados demonstraram que a deficiência do receptor de IL-17 protegeu os animais contra a obesidade, mas os mesmos desenvolveram maior hiperglicemia e hiperinsulinemia decorrente da resistência à insulina. Além disso, foi verificada a hiperplasia das ilhotas pancreáticas, anormalidades na arquitetura e intenso infiltrado inflamatório no intestino (íleo) dos animais IL-17R-/- comparados aos WT após DH. Esse fato parece estar correlacionado a um defeito da migração de neutrófilos para a mucosa intestinal, uma vez que foi detectada reduzida expressão gênica da quimiocina CXCL-1 e do receptor CXCR-2 no íleo desses animais. De maneira interessante, as populações de neutrófilos (CD11b+Ly6G+) e de macrófagos anti-inflamatórios (CD11b+CX3CR1+) mostraram-se aumentadas nos linfonodos mesentéricos dos animais IL-17R-/- após DH. Em seguida, foi constatada maior translocação bacteriana no sangue tanto de animais IL-17R-/- submetidos à DN como DH. Entretanto, a análise metagenômica do gene 16S revelou a prevalência de bactérias Bacteroidetes e Proteobacterias, principais representantes de bactérias gram-negativas, somente nas fezes dos animais IL-17R-/- submetidos à DH. Em conjunto, estes dados indicam que o eixo IL-17/IL-17R é importante na manutenção da homeostase intestinal e na regulação das alterações inflamatórias e metabólicas associadas ao DM2 / The gastrointestinal tract is a high antigenic exposure site, so it requires the presence of immune regulation mechanisms mediated by regulatory T lymphocytes and IL-17- producing T helper lymphocytes (Th17) in the intestinal mucosa. If there is a failure in the induction of these mechanisms, may occur the imbalance in the populations of commensal bacteria of the intestinal microbiota, called dysbiosis, generally associated with the break of the intestinal barrier and translocation of bacteria or their products like LPS into the blood. In this regard, some studies have evidenced the importance of Th17 lymphocytes in the intestine, since these cells have the ability to maintain the integrity of the intestinal barrier, and consequently controlling the colonization and bacterial translocation. In addition, in patients and diabetic animals have been observed correlation between high circulating levels of LPS and insulin resistance. Based on this evidence, our objective was to evaluate the role of IL-17 cytokine in the control of inflammatory and metabolic changes in the type 2 diabetes (T2DM). For this reason, were used C57BL/6 wild-type mice (WT) or lacking of IL-17 cytokine receptor (IL-17R-/-) mice undergoing to the control diet (ND) comprising 10% fat, 70% carbohydrate and 20% protein or high fat diet (DH), comprising 60% fat, 20% carbohydrates and 20% protein. These data demonstrate that IL-17 receptor deficiency protected the animals against obesity, but these mice developed hyperglycemia and hyperinsulinemia due to insulin resistance. Furthermore, we verified a hyperplasia of the pancreatic islets, abnormalities in architecture and intense inflammation in the intestine (ileum) of IL-17R-/- animals undergoing DH compared to WT. This appears to be correlated to a defect in the neutrophil migration to the intestinal mucosa, since was detected reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum of these animals. Interestingly, the populations of neutrophils (CD11b+Ly6G+) and antiinflammatory macrophages (CD11b+CX3CR1+) were shown to be increased in the mesenteric lymph nodes of IL-17R-/- animals after DH. Later, it was found more bacterial translocation in blood, both in IL-17R-/- mice with ND or DH. However, the metagenomic analyzes of the 16S gene revealed increased of Proteobacteria and Bacteroidetes phyla, the main representatives of gram-negative bacteria, only in the faeces of IL-17R-/- mice underwent DH. Together, these data indicate that IL-17/IL-17R axis is important in maintaining intestinal homeostasis and the regulation of inflammatory and metabolic alterations associated to T2DM

Made available in DSpace on 2017-07-10T17:48:27Z (GMT). No. of bitstreams: 1 Marli_ Busanello.pdf: 709375 bytes, checksum: 60f50366eea2e98b3b71af83a9fd0ab6 (MD5) Previous issue date: 2011-07-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective was to evaluate the use oral probiotic of with viable cells and inactivated ("pool" of Lactobacillus sp. and Lactobacillus plantarum of gastrointestinal origin of pigs), on performance, intestinal microbiota and the immune system of piglets during the stages of lactation and creche. Were used, 108 lactating piglets and for nursery phase 72 piglets weaned at 21 days old, divided into an experimental design of randomized blocks with three treatments and six replicates. The treatments were: Treatment A: 1 ml of MRS broth + 1 ml of sterile saline, treatment B: 1 ml of probiotic (8.60 X 107 CFU/ml of a "pool" of Lactobacillus plantarum and Lactobacillus sp) activated MRS broth + 1 ml of saline, treatment C: 1 ml of probiotic inactivated cells in MRS broth + 1 ml of saline. Treatments were administered orally to piglets in the daily morning, from birth to 35 days of age with 1 ml per animal lactating and 2 ml in the nursery phase. The count of acidoláticas bacteria, and coliforms were not affected (P> 0.05) by treatments and sampling times. There was no effect (P>0.05) of treatments for weight gain and weight gain daily of piglets from birth to 21 days old. From 21 to 35 days of age was observed lower consumption and lower daily feed intake (P<0.05) for the control treatment when compared to the probiotic treatments and greater weight gain and weight daily gain (P<0.05) was observed in probiotic treatment with cells inactivated in relation to probiotic treatment with viable cells and control. There was less concentration (P<0.05) globulin in the treatment with probiotics compared to control. There were no significant differences (P>0.05) for the variables serum protein, albumin, glucose, hemoglobin, leukocytes, hematocrit, red blood cells, eosinophils, sticks, segments, lymphocytes, monocytes, platelets and serum levels of IgA between treatments. It was concluded that oral use of probiotics with viable cells and inactivated did not alter microbial counts, the values of serum protein, albumin, glucose, blood count and IgA and use of probiotics with viable and inactivated cells diminished serum globulin / O objetivo foi avaliar o uso oral de probiótico com células viáveis e inativadas ( pool de Lactobacillus sp. e Lactobacillus plantarum de origem gastrointestinal de suínos), no desempenho, na microbiota intestinal e no sistema imune de leitões durante as fases de aleitamento e creche. Foram utilizados, na fase de aleitamento, 108 leitões e para a fase de creche 72 leitões desmamados aos 21 dias de idade, distribuídos em um delineamento experimental de blocos ao acaso, com três tratamentos e seis repetições. Os tratamentos foram: tratamento A: 1 ml de caldo MRS + 1 ml de solução salina estéril; tratamento B: 1 ml de probiótico (8,60 X 107 UFC/ml de um pool de Lactobacillus sp e Lactobacillus plantarum) ativadas no caldo MRS + 1 ml de solução salina; tratamento C: 1 ml de probiótico contendo células inativadas no caldo MRS + 1 ml de solução salina. Os tratamentos foram administrados via oral aos leitões diariamente no período da manhã, do nascimento aos 35 dias de idade sendo 1 ml por animal na fase de aleitamento e 2 ml na fase de creche. As contagens de bactérias acidoláticas e coliformes não foram influenciadas (P>0,05) pelos tratamentos e tempos de coleta. Não foi observado efeito (P>0,05) dos tratamentos para ganho de peso e ganho diário de peso dos leitões do nascimento aos 21 dias de idade. Dos 21 aos 35 dias de idade observou-se para o tratamento controle menor consumo e menor consumo diário de ração (P<0,05) quando comparado aos tratamentos probióticos e maior ganho de peso e ganho diário de peso (P<0,05) foi observado no tratamento probiótico com células inativadas em relação ao tratamento probiótico com células viáveis e o controle. Observou-se menor concentração (P<0,05) de globulina nos tratamentos com probióticos em relação ao controle. Não foram observadas diferenças significativas (P>0,05) para as variáveis proteínas séricas totais, albumina, glicose, hemoglobina, leucócitos, hematócrito, hemácias, eosinófilos, bastões, segmentados, linfócitos, monócitos, plaquetas e para os níveis séricos de IgA entre os tratamentos. Concluiu-se que o uso oral de probiótico com células viáveis e inativadas não alterou as contagens microbiológicas, os valores das proteínas séricas totais, albumina, glicose, hemograma e IgA e uso de probióticos com células viáveis e inativadas diminuiu os níveis séricos de globulina

De todas as influências exógenas que possam alterar a microbiota intestinal, os antimicrobianos são capazes de causar as mais rápidas e drásticas mudanças. O impacto da exposição aos antimicrobianos na microbiota intestinal causa diminuição no número de microrganismos ou mesmo supressão, dependendo do antimicrobiano utilizado, da dose e do tempo de exposição. Assim, o objetivo deste estudo foi analisar de forma comparativa alguns microrganismos que compõem a microbiota fecal de crianças com e sem antibioticoterapia em idade escolar; bem como avaliar a susceptibilidade aos antimicrobianos e os genes de resistência envolvidos. Foram coletadas amostras fecais não diarreicas de 30 crianças sem antibiótico (controle) e 31 de crianças com antibioticoterapia. Na análise quantitativa foi observada redução no número de cópias por g/fezes de: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii e do filo Firmicutes nas amostras das crianças com antibióticos em relação ao grupo controle, exceto para Lactobacillus spp. e P. distasonis que apresentaram quantificação maior no grupo antibióticos quando comparados com o controle. E. coli foi isolada em 26 (86,7%) crianças controles e em 23 (74,2%) tratadas com antibióticos. A resistência foi verificada para diversas drogas no grupo controle exceto para ciprofloxacina, meropenem e tigeciclina; entretanto o grupo com antibioticoterapia apresentou elevada resistência para todas as drogas avaliadas, caracterizando os isolados desse estudo como MDR. Todos os isolados do grupo controle e antibióticos albergaram diversos genes de resistência, entretanto o gene blaKPC foi o único não detectado nos isolados do grupo controle. Desta forma, nossos dados demonstram que a antibioticoteria causa alterações qualitativas e quantitativas na microbiota intestinal; além disso, a elevada resistência as diversas classes de antimicrobianos das cepas de E. coli, bem como a presença de diversos genes de resistência ressalta a importância de cepas comensais serem MDR e albergarem esses genes. / Of all the exogenous influences that may alter the intestinal microbiota, antimicrobial agents are able to cause the more rapid and dramatic changes. The impact of exposure to antimicrobial agents on intestinal microbiota causes a decrease in the number of certain genera and species, depending on the antimicrobial agent used, dose and duration of exposure. Thus, the aim of this study was to analyze comparatively some microorganisms that composing the fecal microbiota of children with and without antibiotic therapy in school age; and evaluates the antimicrobial susceptibility and resistance genes involved. Stool samples (not diarrhea) were collected of 30 children without antibiotic (control) and 31 children with antibiotic therapy. In quantitative analysis was observed decrease in the number of copies per g/feces: Bifidobacterium spp., B. fragilis, C. perfringens, E. coli, M. smithii and the phylum Firmicutes in samples of children with antibiotic therapy in relation to control group, except Lactobacillus spp. and P. distasonis that showed a higher quantification in the antibiotics group when compared with control group. E. coli was isolated in 26 (86.7 %) children controls and in 23 (74.2 %) children treated with antibiotics. The resistance was verified for several drugs in the control group except for ciprofloxacin, meropenem and tigecycline; however the group with antibiotic therapy showed high resistance to all drugs evaluated, characterizing isolates of this study as MDR. All isolates from control group and antibiotics harbored several resistance genes, however blaKPC gene was the only one not detected in isolates from the control group. Thus, our data demonstrate that the antibiotic therapy cause qualitative or quantitative changes in intestinal microbiota leading to a decrease in the diversity and the elimination of microorganisms; in addition, the high resistance the various classes of antimicrobial of the strains of E. coli, as well as the presence of several genes of resistance highlights the importance of commensal strains are MDR and harboring these genes.

Orientador: Mário Roberto Maróstica Junior / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T07:02:00Z (GMT). No. of bitstreams: 1 Cazarin_CinthiaBauBetim_D.pdf: 7421314 bytes, checksum: 3937f33ea2104648545ff81374161389 (MD5) Previous issue date: 2014 / Resumo: A doença inflamatória intestinal (DII) édoença crônica recidivante que atinge milhões de pessoas no mundo, englobando a Doença de Crohn (DC) e a Retocolite Ulcerativa (RCU). A diferença entre as duas éa sua localização, sendo a RCU específica da região do cólon e reto. O tratamento atual para estas patologias é realizado a base de corticosteróides, imunomoduladores ou anti -TNF-a, conhecido como terapia biol ógica, os quais apresentam diversos efeitos colaterais ao paciente. A patogênese desta doença está relacionada com fatores gen éticos, imunol ógicos e ambientais. Acredita-se que o desequilíbrio da microbiota, assim como a ruptura na barreira natural exercida pela mucosa intestinal seja o primeiro passo para o desencadeamento da resposta inflamatória. As fibras alimentares apresentam função sobre a modulação da microbiota, sendo utilizada como substrato para a formação de ácidos graxos de cadeia curta (AGCC), gerados por meio do processo de fermentação. Em adição, os compostos fenólicos presentes nos alimentos apresentam atividade antioxidante e anti -inflamatória que podem atuar na modulação do processo inflamatório. Sendo assim, o objetivo deste trabalho foi avaliar a utilização de um subproduto da indústria de alimentos, a casca do maracujá, como fonte de fibras e compostos fenólicos, na alimentação de ratos com colite induzida por TNBS e sua influência no processo inflamatório. Ratos Wistar foram alimentados com dieta AIN-93, sendo substituídos 50% da celulose da dieta padrão (AIN) por fibras da casca do maracujá (PFF), em dois ensaios biológicos: prevenção e tratamento. O dano causado àmucosa foi avaliado macro e microscopicamente, assim como a expressão de marcadores inflamatórios. Avaliação da microbiota e formação de AGCC foram realizadas no conteúdo cecal. Embora a avaliação macroscópica da mucosa tenha apresentado um escore maior para o grupo PFF no ensaio prevenção, a avaliação microscópica em ambos os ensaios não mostrou diferença no dano àmucosa entre os grupos. O ensaio tratamento mostrou diminuição da peroxidação lipídica do cólon, diminuição na contagem de enterobactérias e aeróbios totais, assim como aumento de ácido acético e butírico nas fezes do grupo PFF. Jáno ensaio prevenção foi observada modulação dos lactobacilos e bifidobactérias. Com relação aos marcadores inflamatórios, foram observadas modulações significativas da expressão de IKK?, COX-2 e iNOS nos animais alimentados com a dieta PFF. Estes resultados sugerem que a casca do maracujá Passiflora edulis pode modular a microbiota aumentando a produção de AGCC, assim como a expressão de marcadores inflamatórios observados na colite induzida por TNBS. Desta forma, a casca do maracujá poderia ser utilizada como coadjuvante na terapêutica da DII como fonte de fibras e polifenóis / Abstract: Inflammatory bowel disease (IBD) is a chronic relapsing disease that affects millions of people worldwide, encompassing Crohn's disease (CD) and ulcerative colitis (UC). UC is an inflammation specific to the region of the colon and rectum. Current treatments for these diseases are based on the use of corticosteroids, immunomodulators or biological therapy, which have various side effects to the patient. The pathogenesis of IBD is related to genetic, immunological and environmental factors. It is believed that the microbial imbalance as well as natural break in the barrier exerted by the intestinal mucosa is the first step in triggering the inflammatory response. Food dietary fiber presents capacity to modulate the microbiota and improve short chain fatty acids (SCFA) formation, by fermentation process. In addition, the phenolic compounds present in the food have antioxidant and anti-inflammatory activities that can modulate the inflammatory process. Thus, this study aimed to evaluate the use of a byproduct of the food industry, the passion fruit peel, as a source of fiber and phenolic compounds in the diet of rats with TNBS-induced colitis and its influence on the inflammatory process. Wistar rats were fed a modified AIN-93 (50% of cellulose was replaced by passion fruit peel PFF) to evaluate prevention and treatment of colitis induced by TNBS. The damage to the mucosa was evaluated macroscopically and microscopically, as well as the expression of inflammatory markers. Evaluation of the microbiota and formation of SCFA in cecal contents were performed. The macroscopic appearance of the mucosa damage in the group PFF was higher than AIN in prevention trial. However, the microscopic evaluation in both trials showed no difference in mucosal damage amongst the groups. Treatment trial showed that PFF could promote a decrease in lipid peroxidation of the colon, decrease in enterobacteria and total aerobics counts, as well as increase in acetic and butyric acid in the stool. On the other hand, the prevention trial showed that the ingredient could exert modulation on lactobacilli and bifidobacteria. The inflammatory markers showed significant modulation, mainly IKK?, COX-2 and iNOS in animals fed with PFF diet. These results suggest that the passion fruit peel, Passiflora edulis, can modulate the microbiota, increase the production of SCFA, and modulate the expression of inflammatory markers observed in TNBS-induced colitis. Passion fruit peel could be used in the treatment of IBD as a source of fiber and polyphenols / Doutorado / Nutrição Experimental e de Alimentos / Doutora em Alimentos e Nutrição

Le développement des méthodes de séquençage de nouvelle génération a permis d’approfondir les connaissances sur le rôle des communautés bactériennes commensales pour la santé de leur hôte, et l’impact négatif de la perturbation de leur équilibre. Les antibiotiques sont les principaux perturbateurs de cet équilibre, mais leur impact n’a pas été quantifié précisément.Nous avons quantifié la relation entre les concentrations fécales d’antibiotiques et la perturbation de la diversité bactérienne au sein du microbiote intestinal, et modélisé le lien entre la perte de diversité bactérienne et la probabilité de décès dans un modèle animal de colite à Clostridium difficile induite par les antibiotiques. Nous avons montré que l’indice de diversité de Shannon et la distance UniFac non pondérée étaient les indices de diversité qui étaient le plus prédictif du décès dans ce modèle d’infection.Chez des volontaires sains, nous avons développé un modèle mathématique semimécanistique de l’évolution de la diversité au sein du microbiote, mesurée par deux indices de diversité, après perturbation antibiotique, et quantifié la relation entre l’exposition individuelle plasmatique et fécale à un antibiotique, et son effet sur la perturbation de la diversité bactérienne au cours du temps. Nous avons également analysé le rôle de la voie d’élimination des antibiotiques pour la limitation de l’impact d’un antibiotique sur le microbiote. Ces travaux nous ont permis de montrer que le microbiote intestinal présente une grande sensibilité aux antibiotiques, et que la voie d’élimination ne semble de ce fait pas jouer un rôle prépondérant dans la perspective de limiter l’impact des antibiotiques sur le microbiote intestinal. / The development of next generation sequencing broadened our knowledge on the role of commensal bacterial communities on their host’s health, and the negative impact of their disruption. Antibiotics are the main disrupting factor, but their impact has not been precisely quantified.We quantified the relationship between antibiotic fecal concentrations and the loss of bacterial diversity in the intestinal microbiota, and modelled the link between the loss of diversity and mortality in a hamster model of antibiotic-induced Clostridium difficile infection. We showed that the Shannon diversity index and the unweighted UniFrac distance are the 2 indices that best predict mortality in this model. In healthy volunteers, we developed a semi-mechanistic model of the evolution over time of bacterial diversity – measured by two indices – after an antibiotic perturbation, and quantified the relationship between antibiotic concentrations in plasma and feces and the loss of bacterial diversity in the intestinal microbiota. We also analyzed the role of the antibiotic elimination pathway in the reduction of their impact on the microbiota. In this work, we showed that the intestinal microbiota is highly susceptible to antibiotics, and that the elimination route doesn’t have a major role, in the perspective of limiting antibiotics’ impact on the intestinal microbiota.

No description available.

La dépression représente l’affection neuropsychiatrique la plus répandue dans le monde. Son impact socio-économique est important et la prise en charge des patients est souvent confrontée aux limites d’efficacité des traitements actuels. Les mécanismes sous-jacents responsables de cette affection sont en partie inconnus. Néanmoins, un nombre grandissant de données désignent aujourd’hui le microbiote intestinal comme un acteur potentiel de la physiopathologie de la dépression. En particulier, des déséquilibres dans la nature et la quantité des métabolites bactériens qu’il produit pourraient être impliqués. L’indole est un métabolite du tryptophane produit par le microbiote intestinal. Il joue un rôle (i) dans la physiologie bactérienne et les relations bactérie-bactérie au sein du microbiote, (ii) dans le fonctionnement des cellules intestinales, et (iii) certains de ses dérivés sont connus pour être neuro-actifs. L’objectif de la thèse est de mieux comprendre comment un excès de production de ce métabolite bactérien peut influencer le cerveau et le comportement dans le contexte de la dépression et de troubles mentaux qui lui sont souvent associés, les troubles anxieux. Mon travail de thèse comporte 3 parties.La première a pour but de tester si une dysbiose du microbiote intestinal induisant une surproduction d’indole est un facteur de vulnérabilité aux troubles anxieux et dépressifs, et d’étudier les modifications biochimiques et moléculaires associées. Une étude comportementale chez des souris gnotoxéniques produisant de l’indole en excès ou n’en produisant pas montre qu’une surproduction intestinale d’indole exacerbe les comportements de type anxieux et dépressif induits par l’exposition à un stress chronique modéré. L’étude de l’expression de gènes des glandes surrénales impliqués dans la synthèse de la corticostérone et de l’adrénaline montre que les souris surproductrices d’indole et soumises au stress chronique surexpriment un gène impliqué dans la synthèse de l’adrénaline. Des dosages de neurotransmetteurs cérébraux et des analyses d’expression de gènes dans le cerveau et la muqueuse intestinale ont aussi été conduits. La seconde partie de la thèse porte sur l’identification des circuits neuronaux cérébraux activés par l’indole. Pour ce faire, des souris conventionelles ont été gavées avec de l’indole et la protéine c-Fos marquée par immunohistochimie dans toutes les régions du cerveau, du tronc cérébral au cortex préfrontal. La troisième partie de la thèse consiste à moduler la disponibilité du tryptophane alimentaire dans le tube digestif de souris conventionnelles, et à en étudier l’impact sur la composition bactérienne du microbiote intestinal et sa capacité à produire de l’indole. La composition du microbiote fécal des souris a été déterminée par séquençage de l’ADN codant l’ARNr 16S et les concentrations fécales de tryptophane et d’indole ont été déterminées par analyse HPLC.En conclusion, ce projet de thèse aura contribué à une meilleure compréhension du rôle de l’indole dans les réponses comportementales et neuro-endocrines au stress. Il aura également permis d’initier l’étude des circuits neuronaux activés par l’indole, et de tester comment la modulation de la digestibilité de protéines riches en tryptophane peut influencer l’équilibre du microbiote intestinal et ses capacités à produire de l’indole. / Depression is the most spread neuropsychiatric disorder worldwide. It is a socio-economical burden and efficacy of the treatments is very limited. Mechanisms underlying this disorder are mainly unknown. However, a growing number of data has highlighted the potential role of gut microbiota dysbioses in the pathophysiology of depression. Particularly, an unbalance in the diversity and abundance of metabolites produced by the gut microbiota might be implicated. Indole is a tryptophan derivative produced by the gut microbiota. It is known to influence (i) the bacterial physiology and quorum sensing within the gut microbial ecosystem, (ii) the intestinal cells functioning, and (iii) some of its derivatives are known to affect the brain. The aim of this work is to investigate how an overproduction of indole by the gut microbiota can modulate the brain and behaviour in the context of depression and its main co-morbidity, anxiety. This thesis work contains 3 sections.In the first one, we investigated whether an intestinal microbiota dysbiosis leading to an overproduction of indole could confer vulnerability toward anxiety and depression. We also looked for potentially associated biochemical and molecular changes. A behavioural study in gnotobiotic mice overproducing or non producing indole showed the overproduction of indole exacerbated the anxiety-like and depressive-like behaviours induced by a chronic mild stress. Gene expression analysis in the adrenal glands showed chronically stressed mice overproducing indole up-regulated the expression of one gene implicated in adrenaline synthesis. Brain neurotransmitters quantification and gene expression in the brain and intestinal mucosa were also carried out. The second part of the thesis work focused on the brain neurocircuitry of indole. Conventional mice were force-fed with indole and the c-Fos protein was labelled by immunohistochemistry in all brain areas from brainstem to prefrontal cortex. In the third and last part, we modulated dietary tryptophan availability in the gastro-intestinal tract of mice, to study how this modulation could affect the composition and the indole production ability of the gut microbiota. The mice fecal microbiota composition was determined by 16S rRNA sequencing, and fecal tryptophan and indole concentrations were measured by HPLC.In summary, this work improves the understanding of the role of indole in the behavioural and neuro-endocrine responses to stress. This study also initiated the deciphering of brain circuits activated by indole. Finally, it brings some evidence about how modulating food digestibility can impact the gut microbiota composition and its indole production capacity.

Le rôle crucial du tractus gastrointestinal dans la pathogenèse et la pathophysiologie des troubles cardiométaboliques (TCM) et du syndrome métabolique (SM) est actuellement bien établi. Plusieurs facteurs, incluant le stress oxydatif (SOx), l'inflammation et la résistance à l'insuline (RI), perturbent l'homéostasie intestinale et causent des TCM. Les polyphénols (PP) ont des effets biologiques bénéfiques dans la prévention de pathologies métaboliques. Cependant, leurs mécanismes d'actions, surtout au niveau de l'axe intestin-foie, ne sont pas bien compris. Par ailleurs, malgré les nombreuses études sur les effets biologiques et la biodisponibilité des PP, il existe encore des zones d’ombres concernant les interactions entre le microbiote intestinal et les PP et les conséquences subséquentes sur la santé intestinale et métabolique. Dans ce travail de recherche, nous favorisons l’axiome selon lequel les PP, notamment ceux de grande taille moléculaire tels que les proanthocyanidines (PACs), pourraient être utile pour combattre les maladies métaboliques grâce à leurs actions antioxydante et anti-inflammatoire. Toutefois, ces actions précitées des PACs dépendraient d’une régulation en amont du microbiote intestinal. L’objectif central consiste à démontrer les effets bénéfiques des PACs dans la prévention des dérèglements métaboliques dans deux modèles distincts, l’un cellulaire et l’autre animal et d’en étudier les mécanismes. Les effets des PACs sur la RI, les dérangements métaboliques intestinaux grâce à la production de métabolites ont été étudiés. Dans une première étape, nous avons étudié les mécanismes d’actions des PACs et de l’un de leurs métabolites majeurs, le 4,5-dihydroxyphenyl valerolactone (DHPVL), dans la prévention des maladies métaboliques et dans le maintien de l’homéostasie intestinale en utilisant la lignée cellulaire intestinale Caco-2/15. Ces cellules constituent un outil de choix pour l’investigation du SOx, la défense antioxydante et l’inflammation en relation directe avec nos objectifs. Les résultats suggèrent que la capacité des PACs à augmenter la défense antioxydante et anti-inflammatoire et à améliorer l’homéostasie intestinale passeraient en partie probablement par leurs métabolites microbiens. Dans une deuxième étape, en utilisant le modèle murin C57BL6, nous avons déterminé l’impact des PACs sur l’homéostasie métabolique intestinale et hépatique, via l’atténuation du SOx et l’inflammation, le maintien de l’intégrité de la barrière intestinale, la prévention de l’endotoxémie métabolique et les modifications du profil lipidique et de la fonction du microbiote intestinal. Cette partie a évalué les aspects préventifs et thérapeutiques des PACs en spécifiant leurs bénéfices biologiques et voies mécanistiques dans des organes métaboliques clés. Pour étudier ces mécanismes et les comprendre, nous avons utilisé le modèle dysmétabolique de souris C57BL6 soumises à une diète riche en lipides et en sucrose (HFHS), servant à développer le SM et les complications cardio-métaboliques afin d’examiner l’action des PACs. Le développement de l’obésité, de la RI ainsi que la survenue d’autres altérations métaboliques ont été prévenus par l’administration de PACs. Les résultats de cette thèse permettent une meilleure compréhension des mécanismes d’actions qui sous-tendent les effets préventifs et thérapeutiques des PACs dans les désordres métaboliques, en particulier dans l’axe intestin-foie. / The crucial role of the gastrointestinal tract in the pathogenesis and pathophysiology of cardiometabolic disorders (CMD) and metabolic syndrome (MetS) is currently recognized. Several factors, including oxidative stress (OxS), inflammation and insulin resistance (IR), disrupt intestinal homeostasis and cause CMD. Polyphenols (PP) have beneficial biological effects in the prevention of metabolic pathologies. However, their mechanisms of action, especially in the gut-liver axis, are not well understood. Moreover, despite numerous studies on the biological effects and bioavailability of PP, there are still grey areas concerning the interactions between the intestinal microbiota and PP and the subsequent consequences for intestinal and metabolic health. In this research work, we promote the axiom that PP, particularly those of large molecular size such as proanthocyanidins (PACs), could be useful in combating metabolic diseases thanks to their antioxidant and anti-inflammatory actions. However, the aforementioned actions of PACs would depend on upstream regulation of the intestinal microbiota. The central objective is to demonstrate the beneficial effects of PACs in preventing metabolic disorders in two distinct models, one cellular and the other animal, and to study the mechanisms involved. The effects of PACs on IR and intestinal metabolic disturbances through metabolite production were studied. In a first step, we investigated the mechanisms of action of PACs and one of their major metabolites, 4,5-dihydroxyphenyl valerolactone (DHPVL), in the prevention of metabolic diseases and in the maintenance of intestinal homeostasis using the Caco-2/15 intestinal cell line. These cells are a tool of choice for investigating OxS, antioxidant defense and inflammation in direct relation to our objectives. The results suggest that the ability of PACs to enhance antioxidant and anti-inflammatory defense and improve intestinal homeostasis is probably partly mediated by their microbial metabolites. In a second step, using the C57BL6 mouse model, we determined the impact of PACs on intestinal and hepatic metabolic homeostasis, via attenuation of OxS and inflammation, maintenance of intestinal barrier integrity, prevention of metabolic endotoxemia and changes in lipid profile and gut microbiota function. This section assessed the preventive and therapeutic aspects of PACs, specifying their biological benefits and mechanistic pathways in key metabolic organs. To investigate and understand these mechanisms, we used the dysmetabolic model of C57BL6 mice subjected to a high-fat, high-sucrose diet (HFHS), used to develop MetS and cardio-metabolic complications to examine the action of PACs. The development of obesity, IR and other metabolic alterations was prevented by the administration of PACs. The results of this thesis provide a better understanding of the mechanisms of action underlying the preventive and therapeutic effects of PACs in metabolic disorders, particularly in the intestine-liver axis.

yes / Abstract Objective Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). Design A randomised, open-label, cross-over trial of 8 weeks’ treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week ‘washout’ period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. Results Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or β diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. Conclusion Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. / NIHR/EME Yorkshire Cancer Research (YCR)