The use of nanomaterials (NMs) in orally ingestible products raises concerns about potential hazards. Titanium dioxide (TiO2) particles (of which some are incidentally produced at the nanoscale) are used in cosmetics, biological remediation (photo-catalysis), toothpastes, ingestible pharmaceuticals and food products. The increased surface area to mass ratio of nanoparticles (NPs) potentially makes them more biologically reactive than their coarser (bulk) material counterparts. There is limited data available on the uptake kinetics across the mammalian gastrointestinal tract, and the potential hazard posed to humans. In this study, the uptake and accumulation of TiO2 (nano and bulk) into and across the human intestinal cell line, the isolated perfused rat jejunum and the whole rat were evaluated. Caco-2 monolayers exhibited time-dependent, accumulation, uptake and transport of Ti/TiO2 from TiO2 exposures of 1 mg L-1 over 24 h, which was influenced by the crystal type, irrespective of cell maturity and growth substrate (Chapters 2-3). Electron micrographs of the Caco-2 monolayer showed the presence of particles inside the cells within vesicles and energy dispersive spectroscopy (EDS) confirmed the composition as TiO2. Addition of pharmacological inhibitors altered the Ti concentration in the cells suggesting diffusion is not the primary mechanism of uptake, rather, an active process is responsible (Chapter 2). Whole gut sacs exposures of 1 mg L-1 bulk or nano TiO2 demonstrated the primary regions of the gut associated with accumulation are the small and large intestine, with 70 % or more of the TiO2 accumulating in the mucosa rather than the underlying muscularis. Perfused intestines exposed to 1 mg L-1 bulk or nano TiO2 for 4 h showed a time-dependent accumulation of Ti in the serosal perfusate with the initial rates of Ti flux from the nano exposures being 5 fold higher than the bulk form. Addition of pharmacological inhibitors caused increases in tissue Ti concentration and significantly reduced Ti serosal flux rates for NP exposures. Overall, the data suggests an active absorption mechanism is responsible for Ti uptake from both bulk and nano TiO2 exposures across the perfused rat intestine that is drug sensitive (Chapter 4). In vivo work demonstrated feed status and rat age effected Ti tissue concentrations. Critically, Ti tissue concentrations reduced with increasing age and removal of Ti containing feed caused transient decreases in Ti tissue concentrations in 23 day old rats. Transient decreases in Ti tissue concentration following feed removal were not observed in older rats suggesting young rats may be more sensitive to the uptake hazards presented by titanium (Chapter 5). Overall, the findings presented in this thesis demonstrate Ti/TiO2 from both bulk and nano TiO2 exposures are accumulated and transported across intestinal epithelium and these processes are drug sensitive and affected by crystal structure and particle size. The results in this thesis have contributed to a better understanding of the uptake kinetics and sub-lethal hazards presented by bulk and nano forms of TiO2 exposed to intestinal epithelium which could be used to partially inform policy makers on human dietary risk assessments.

The impact of the intestine in determining the oral bioavailability of drugs has been extensively studied. Its large surface area, metabolic content and positioning at the first site of exposure for orally ingested xenobiotics means its contribution can be significant for certain drugs. However, prediction of the exact metabolic component of the intestine is limited, in part due to limitations in validation of in vitro tools as well as in vitro-in vivo extrapolation scaling factors. Microsomes are a well established in vitro tool for extrapolating hepatic metabolism, however standardised methodologies for preparation in the intestine are limited, in light of complexities in preparation (e.g. presence of multiple non-metabolic cells, proteases and mucus). Therefore, the aims of this study were to establish an optimised method of intestinal microsome preparation via elution in the proximal rat intestine, and to determine microsomal scaling factors by correcting for protein losses during preparation. In addition, to assess species in another preclinical species (dog) and human as well as assessing and regional differences in scaling factors and metabolism. Following optimisation of a reproducible intestinal microsome preparation method in the rat, the importance of heparin in limiting mucosal contamination was established. These microsomes were characterised for total cytochrome P450 (CYP) content, and CYP and uridine 5′-diphosphate glucuronosyltransferase (UGT) activities using maker probes of testosterone and 4-nitrophenol. Loss corrected microsomal scaling factors between two pools of n=9 rats was 9.6±3.5 (recovery 33%). A broad range of compounds (n=25) in terms of metabolic activity and physicochemical properties were screened in rat intestinal microsomes. The prediction accuracy relative to in house generated or literature in vivo estimates of the fraction escaping intestinal metabolism (FG) through in vitro-in vivo extrapolation of observed metabolism and the derived scaling factors and either Caco-2 permeability of physicochemical permeability estimates utilising the Qgut model. In the dog, regional differences in intestinal scaling factors and metabolic activities were explored, as well as relationships between the proximal intestine and liver in matched donors. Positive correlations in both hepatic activity and microsomal scalars were observed. Robust scaling factors were established using the 3 microsomal markers. A total of 24 compounds were screened for hepatic and intestinal metabolism in order to make in vivo estimates of FG, the fraction escaping hepatic metabolism (FH) and oral bioavailability (F). Estimates based on Caco-2 and physicochemical based scaling, as well as utilising a commercial PBPK software platform (ADAM model, Simcyp® v12) were broadly similar with generally reduced prediction accuracy in proximal physicochemical based Qgut scaling, and improved predictions using Caco-2 Qgut or PBPK approaches. Worse predictions were observed for compounds with high protein binding, transporter substrates and/or CYP3A inhibitors. Regional metabolism demonstrated peak metabolism in the proximal intestine, before declining distally. Human intestinal microsomes were prepared for jejunum and ileum tissue. Although samples were limited, regional differences in metabolic activities and scaling factors were also assessed, using correction markers and activity in 23 compounds. In all, 20 compounds overlapped between all three species. Comparison in Fa.FG between rat and human CYP3A substrates showed a modest relationship, however relationships between species and human were generally poor given the observed differing metabolic contributions of testosterone and 4-NP metabolite formation between species limited the observed relationships between species. However, within species, good estimates of oral bioavailability were observed. This is the largest know interspecies comparison of intestinal metabolism and scaling factors with microsomes prepared within the same lab.

Dans le contexte de l'antibiorésistance, l'objet de ce doctorat vise à évaluer l'impact d'antibiotiques sur le microbiote intestinal de porcelets. La colistine et le ceftiofur, pour lesquels les résistances incluent essentiellement et respectivement mutations chromosomiques et gènes plasmidiques, ont été utilisés. La colistine a significativement réduit la population des entérobactéries, mais aucun E. coli résistant n'a été détecté. L'administration de ceftiofur a eu un impact limité sur les populations bactériennes composant l'écosystème digestif mais a conduit à une forte sélection et à la diffusion d'un gène plasmidique codant pour une bêta-lactamase à spectre étendu. Puis, dans le cadre de la réglementation visant à diminuer l'expérimentation animale, un modèle in vitro colique porcin, nommé PigutIVM, a été mis au point afin de simuler l'environnement digestif du porcelet et a permis de confirmer, in vitro, l'effet observé in vivo de la colistine sur le microbiote. Cet outil a ensuite été utilisé pour évaluer l'impact d'un probiotique, Saccharomyces cerevisiae, comme alternative aux antibiotiques. Le modèle PigutIVM devrait se positionner comme un outil de prédiction pertinent dans les domaines d'investigation aussi bien nutritionnels que pharmacologiques. / In the context of antibiotic resistance, the aim of the current PhD work is to assess the impact of antibiotics on intestinal microbiota of piglets. Two antibiotics i.e. colistin and ceftiofur, for which the main resistances include respectively chromosomal mutations and plasmid genes have been used. Colistin significantly reduced the population of Enterobacteriaceae, but there was no selection of resistant E. coli. The administration of ceftiofur had a limited impact on the bacterial populations that make up the digestive ecosystem but it led to strong selection and dissemination of a plasmid gene encoding an extended-spectrum beta-lactamase. Then, in the framework of regulations to reduce animal testing, an in vitro model of colonic pig named PigutIVM was developed in order to simulate the digestive environment of the piglet and then check the effect of colistin on the microbiota simulated in PigutIVM in vitro. Therefore both the approaches i.e. in vivo and in vitro were compared in order to check the effect of colistin on intestinal microbiota of piglets. This tool was then used to evaluate the impact of a probiotic i.e. Saccharomyces cerevisiae, as alternative to antibiotics. Therefore we assume that this PigutIVM model should be positioned as a relevant predictive tool in the fields of nutritional and pharmacological investigations.

O objetivo deste estudo foi investigar a qualidade de vida do paciente com estoma intestinal secundário ao câncer cólon-retal na cidade de Aracaju-Se. Trata-se de estudo descritivo-exploratório, na vertente quantitativa, utilizando-se um instrumento contendo três partes, dados sócio-demográficos e clínicos, itens referentes à Escala de Qualidade de Vida de Flanagan (EQVF), e EORTC-QLQ-C-30. A amostra constou de 18 pacientes atendidos no Centro de Oncologia Dr Oswaldo Leite, localizado na cidade de Aracaju – Sergipe no período de janeiro a dezembro de 2005. Os instrumentos mostraram propriedades psicométricas satisfatórias quanto à consistência interna e validade do construto. Quanto às características sócio-demográficas e clínicas, 50% eram do sexo feminino, e 50% masculino. A média de idade foi de 51,3 anos. Quanto à localização do tumor, 38,9% tumor de reto, seguida de cólon-retal 33,3% e cólon 27,8%. 83,3% apresentavam metástase e 16,7% não apresentavam. Em relação ao tipo de estoma, 72,2% tinham colostomia definitiva e 27,8% colostomia provisória. 77,8% dos pacientes apresentaram menos de um ano de estoma e 22,2% mais de um ano de estoma. A análise de qualidade de vida da amostra mostra para EQVF média de 52,3 (DP=1,40) para EQVF Geral. Para EORTC QLQ-C30, a média estado geral de saúde foi de 35,64 (DP=12,39). Foram estatisticamente significativos os resultados obtidos nas escalas: relações com outras pessoas, atividades sociais e desenvolvimento pessoal e realização. Pacientes com menos de um ano de estoma, apresentam maior atividade social,desenvolvimento pessoal e realização do que aqueles com mais de um ano de estoma. Foram estatisticamente significativas as escalas: função física, dor, estado geral de saúde, dificuldades financeiras, dispnéia, fadiga, náusea e vômito, constipação e função emocional. A função física teve como variável estatisticamente significante o estado civil, com maior média entre pessoas casadas, assim como a escala dor, porém com maior média entre as pessoas viúvas. A função física entre as pessoas casadas foi maior. No sintoma dor, a presença desta, foi maior entre o grupo de pessoas viúvas e separadas. Na escala estado geral de saúde, foram estatisticamente significativos o sexo e complicações do estoma, com médias superiores entre aqueles do sexo masculino e com presença de complicações. Na escala dificuldades financeiras, a procedência e as complicações do estoma foram estatisticamente significantes. Com maiores médias entre aqueles da área rural e com presença de complicações do estoma. No sintoma dispnéia, a variável estatisticamente significante foi a presença ou não de metástase, com maior média entre aqueles com metástases. Já os sintomas fadiga, náusea e vômitos e constipação, apresentou como única variável estatisticamente significante o tempo de estoma. Na escala função emocional, a variável complicações do estoma foi estatisticamente significante, havendo maior média entre aqueles com presença de complicações do estoma. A escala relações com outras pessoas foi influenciada pelas variáveis: sexo, tempo de estoma e estado de origem, conforme cálculo de Regressão Linear Múltipla. As escalas função emocional, dispnéia, dificuldade financeira e estado geral de saúde tiveram influência das variáveis sexo, estado civil, estado de procedência, metástase, tempo de estoma e complicações. / The objective of this study was to investigate the quality of life of the patient with secondary intestinal estoma to the cancer colo-retal in the city of Aracaju-Se. This is an description-exploratory study, in the quantitative source, using an instrument contends three parts, given partner-demographic and clinical, itens referring to Scale of Quality of Life of Flanagan (EQVF), and EORTC QLQ-C30. The sample consisted of 18 patients taken care of in the Center of Oncologia Dr Oswaldo Leite located in the city of Aracaju - Sergipe in the period of January to December of 2005. The instruments had shown to satisfactory psicometrics properties such as the internal consistency and validity of construct. About the partner-demographic and clinical characteristics, 50% were of the feminine sex, and 50% masculine. The age average was 51,3 years. About the localization of tumor, 38,9% tumor of rectum, followed 33,3% colo-rectum and colon 27,8%. Metastase were presented by 83,3% and 16.7% didn’t present. In relation to the type of stoma, 72,2% had definitive colostomia and 27,8% provisory colostomia. 77,8% of the patients had presented less than 1 year of stoma and 22,2% more than 1 year of stoma. The analysis of quality of life of the sample shows for average EQVF of 52,3 (DP=1,40) for general EQVF. For EORTC QLQ-C30, the been average general of health was of 35,64 (DP=12,39). The scales had been statisticaly significant: social relations with other people, activities and personal development and accomplishment. Patients with less than one year of stoma, present greater social activity, personal development and accomplishment of that those with more than one year of stoma. The scales had been statistically significant: physical function, pain, general state of health, financial difficulties, dispneia, fatigue, nausea and vomit, constipation and emotional function. The physical function had as changeable statistically significant the civil state, with average greater between married people, as well as the scale pain, however with average greater between the people widowers. The physical function between the married people was bigger. In the symptom pain, the presence of this, it was bigger enters the group of people separate widowers and. In the scale general of health, the sex and complications of stoma had been statisticaly significant, with superior averages between those of the masculine sex and with presence of complications. In the scale financial difficulties, the origin and the complications of stoma they had been statisticaly significant. With average greaters between those of the agricultural area and with presence of complications of stoma. In the dispneia symptom, the statisticaly significant variable was the presence or not of metastase, with average greater it enters those with metastases. Already the symptoms fatigue, nausea and vomits and constipation, the stoma time presented as only statisticaly significant variable. In the scale emotional function, the variable complications of stoma was statisticaly significant, having bigger average between those with presence of complications of stoma. The scale Relations with other people was influenced by the variable: sex, time of stoma and state of origin, as calculation of Multiple Linear Regression. The scales emotional function, dispneia, financial difficulty and general state of health had had influence of the variable sex, civil state, state of origin, metastase, time of stoma and complications.

Não consta resumo na publicação / Biological and immunological properties of phytohemagglutins from two brazilian varieties of beans (Phaseolus vulgaris) were studied. Through immunoprecipitation in agar gel, immuoenzymatic assays (ELISA) and eletrophoretic behavior after affinity chromatography purification it was shown that the lectins are similar. Both lectins when given to rats are toxic. They reduce growth and intestinal dissacharidase activity. They also able to smooth the \"microvillous\" of the enterocyte and to induce the appearance of autophagosomes in the enterocyte citoplasm. All this modification result in a reduced rate of carbohydrates absorption either in \"vitro\" or \"in vivo\".

O objetivo do presente estudo foi avaliar o desempenho, saúde intestinal e metabolizabilidade de dietas para frangos de corte alimentados com o uso contínuo ou mudança brusca de diferentes rações à base de milho e sorgo moído ou inteiro. No Exp. 1 foi estudada a mudança no tipo de grão com as dietas: M100% (ração à base de milho); S100% (ração à base de sorgo); M:S50% (ração à base de 50% milho e 50% sorgo); PS-M (ração à base de sorgo na fase pré-inicial e milho nas demais fases); PM-S (ração à base de milho na fase pré-inicial e sorgo nas demais fases). No Exp. 2 foi estudada a mudança na forma do grão de sorgo: Sm100% (ração à base de sorgo moído); Si100% (ação à base de sorgo inteiro); PSm-Si (ração à base de sorgo moído na fase pré-inicial e inteiro nas demais fases) e PSi- Sm (ração à base de sorgo inteiro na fase pré-inicial e moído nas demais fases). Os experimentos foram conduzidos em um delineamento em blocos casualizados (blocos no tempo) para avaliar o desempenho e epitélio jejunal (r=8), microbiota intestinal (r=4) e metabolizabilidade das dietas (r=10). As mudanças entre milho e sorgo não alteraram o desempenho, epitélio jejunal nem a metabolizabilidade das rações, mas influenciaram a porcentagem de Clostridium, Weissella, Bacillus e Alkaliphilus no intestino delgado e Lactobacillus e Desulfotomaculum nos cecos. O uso de sorgo inteiro piorou o desempenho aos sete dias. Aos 40 dias, Sm100% e PSm-Si apresentaram desempenhos semelhantes, PSi-Sm apresentou menor ganho de peso, mas melhor conversão alimentar e Si100% apresentou o pior desempenho. Si100% e PSm-Si apresentaram aumento no peso relativo da moela e na metabolizabilidade das rações, assim como diminuição de Clostridium e aumento de bactérias das famílias Actinomycetales e Bacillales no intestino delgado, Si100% resultou em menor porcentagem de Alkaliphilus e Enterococcus que Sm100% nos cecos. Conclui-se que a melhor estratégia de uso do sorgo é a substituição total de milho por sorgo moído ao alojamento com posterior mudança para sorgo inteiro, pois não afeta o desempenho e epitélio jejunal das aves, melhora a metabolizabilidade das rações e potencializa a redução de Clostridium no intestino delgado de frangos de corte. / The aim of this study was to evaluate performance, intestinal health and metabolizability of diets in broilers fed different corn- or sorghum (ground or whole)- based diets, continuously or with abrupt change between the diets. Exp. 1 - Change in the type of grain: C100% (corn-based diet); S100% (sorghum-based diet); C:S50% (50% corn and 50% sorghum-based diet); PC-S (corn-based diet in pre-starter phase and sorghum-based diet in other phases); PS-C (sorghum-based diet in pre-starter phase and corn-based diet in other phases). Exp. 2 - Change in the form of sorghum grain: Gs100% (ground sorghum-based diet); Ws100% (whole sorghum-based diet); PGs-Ws (ground sorghum-based diet in pre-starter phase and whole sorghum-based diet in other phases); PWs-Gs (whole sorghum-based diet in pre-starter phase and ground sorghum-based diet in other phases). Experiments were conducted in a randomized block design for to evaluate performance and jejunal epithelium (r=8), intestinal microbiota (r=4) and metabolizabilty of diets (r=10). The changes between corn and sorghum did not affect performance, jejunal epithelium or metabolizability of the diets, but influenced the genera Clostridium, Weissella, Bacillus and Alkaliphilus in the small intestine, and Lactobacillus and Desulfotomaculum in the caecum. Whole sorghum resulted in decreased performance at seven days of age. At 40 days, Gs100% and PGs-Ws showed similar performance, PWs-Gs showed lower weight gain and the best feed conversion rate, and Ws100% showed the worst performance. Ws100% and PGs-Ws resulted in the biggest gizzard relative weight and the highest diet metabolizability values, as well as the lowest level of Clostridium and highest level of Actinomycetales and Bacillales in the small intestine. Ws100% showed lower level of Alkaliphilus and Enterococcus than Gs100% in the caecum. The best strategy to use sorghum in broilers diets is replacing 100% of corn for ground sorghum since the first day followed by change to whole sorghum, because this diet did not affect performance or jejunal epithelium, improved diet metabolizability values, and reduced Clostridium in the small intestine of broilers.

Este estudo teve por objetivos avaliar diferentes níveis de suplementação de ?-ácidos do lúpulo como aditivos de rações para frangos de corte sobre o desempenho e a manutenção do equilíbrio da microbiota intestinal após desafio com Eimeria. No experimento 1, foram alojados 1440 pintos de corte da linhagem Cobb 500, no período de 1 a 42 dias de idade, com 6 tratamentos e 6 repetições. Os tratamentos utilizados foram: controle negativo, ração sem antimicrobiano; controle positivo, ração com 30 mg/kg de bacitracina de zinco e rações com 30, 60, 120 ou 240 mg/kg de ?-ácidos do lúpulo, compondo 4 tratamentos adicionais. Os ?-ácidos foram microencapsulados com 30%de extrato. As rações foram formuladas à base de milho e farelo de soja, com inclusão de 5% de farelo de trigo e 5% de farinha de penas e vísceras. Aos 7 dias de idade todas as aves foram vacinadas contra coccidiose. As aves e as rações foram pesadas semanalmente para cálculo de desempenho. No experimento 2, foram alojados 1440 pintos de corte da linhagem Ross 308, no período de 1 a 42 dias de idade, com 6 tratamentos e 6 repetições. Os tratamentos foram: controle negativo, dieta basal; controle positivo, ração com 30 mg/kg de bacitracina de zinco; controle negativo mais desafio; controle positivo mais desafio; controle negativo suplementado com 30 mg/kg de ?-ácidos mais desafio; controle negativo suplementado com 240 mg/kg de ?-ácidos mais desafio. As rações foram formuladas à base de milho e farelo de soja com inclusão de 5% de farinha de penas e vísceras. Aos 14 dias de idade, as aves dos tratamentos 3, 4, 5 e 6 foram desafiadas, via oral, com 2x105 e 5x104 oocistos de Eimeria acervulina e E. tenella, respectivamente. As aves e as rações foram pesadas semanalmente para obtenção dos dados de desempenho. Aos 21 e 35 dias de idade das aves, coletou-se o conteúdo do intestino delgado e cecos das aves para análise da microbiota intestinal, com auxílio de técnicas moleculares. No experimento 1, aos 21 dias de idade as aves os tratamentos com 30 ou 60 mg/kg de ?-ácidos apresentaram desempenho semelhante às do controle positivo. Aos 42 dias houve melhora na conversão alimentar das aves recebendo 30 e 240 mg/kg de ?-ácidos ou antimicrobiano. No segundo experimento, a coccidiose causou significativa redução no desempenho das aves e nenhum dos aditivos utilizados foi capaz de reverter esta situação. Houve aumento de bactérias do gênero Clostridium no intestino delgado aos 21 dias, em consequência do desafio, entretanto, o maior nível de ?-ácidos reduziu esta população. Aos 35 dias de idade a infecção de coccidiose não alterou a comunidade bacteriana do intestino delgado, mas nos cecos houve aumento do gênero Bacteroides.Os ?-ácidos possuem potencial para serem utilizados nas dietas de frangos de corte em situações de baixo desafio, e podem auxiliar no controle da proliferação de Clostridium, embora ineficazes contra a Eimeria. / The objective was to evaluate increasing level of hops ?-acids in the feed on performance of broiler chickens. A pen trial using 1440 one-day old chickens, from 1 to 42 days, with 6 treatments and 6 replicates was conducted (Experiment 1). The experimental treatments were: negative control, basal diet; positive control, basal diet supplemented with zinc bacitracin, 30 mg/kg; and basal diet supplemented with 30, 60, 120 or 240 mg/kg of hops ?-acids, for 4 additional treatments. The corn soybean meal basal diet was formulated with inclusion of 5% poultry by-product meal and 5% wheat bran. At 7 days of age all birds were vaccinated against coccidiosis. The chickens and the feed were weighted weekly to calculate the performance. In the second experiment, the objective was to evaluate the supplementation of hops ?-acids on performance and the balance of intestinal microbiota of broilers, following challenge with Eimeria acervulina and E. tenella. A pen trial using 1440 one-day-old chickens, from 1 to 42 days, with 6 treatments and 6 replicates was conducted. The experimental treatments were: negative control, basal diet; positive control, basal diet supplemented witn zinc bacitracin, 30 mg/kg; negative controle + challenge; Positive control + challenge; negative control supplemented with 30 mg/kg of hops ?-acids + challenge; negative control supplemented with 240 mg/kg of hops ?-acids + challenge. The corn soybean meal basal diet was formulated with inclusion of 5% poultry byproduct meal. At 14 days of age, the birds in treatments 3, 4, 5 and 6 were challenged with 2x105 and 5x104 oocists of Eimeria acervulina and E. tenella, respectively. The chickens and the feed were weighted weekly to calculate the performance. At 21 and 35 days of age, the small intestine and ceca content was collected to analyze the intestinal microbiota, using molecular techniques. In the first experiment, at 21 days of age the treatment with 30 or 60 mg/kg of ?-acids had the same performance of chickens in positive control. At 42 days, the treatments containing 30 or 240 mg/kg of ?-acids and positive control had improved feed conversion. In the second experiment, there was worse performance in broilers chickens challenged with coccidiosis and the additives were not able to oppose this situation. There was increase in the Clostridium population in the small intestine at 21 days, due to challenge, however, the highest level of ?-acids decreasead this genus. At 35 days, the coccidiosis did not alter the bacterial community in the small intestine, although the ceca had higher level of Bacteroides. The hops ?-acids have the potential to be used in the diets of broiler chickens, under low level of challenge, and to proliferation of Clostridium, although ineffective against Eimeria.

La maladie alcoolique du foie (MAF) regroupe l’ensemble des lésions qui apparaissent suite à une consommation excessive et chronique d’alcool. A consommation d’alcool égale, les patients n’évolueront pas tous vers les formes sévères de la maladie. Le microbiote intestinal est un cofacteur déterminant dans la sévérité de la MAF. Parmi les métabolites fécaux entre des souris recevant le microbiote intestinal de patients avec des lésions sévères ou non, les acides biliaires ont été identifiés comme les plus discriminants. Le récepteur aux acides biliaires TGR5, exprimé sur les cellules de Kupffer, favorise leur profil anti-inflammatoire.Nous avons évalué l’impact du récepteur TGR5 dans la MAF chez des souris déficientes pour ce récepteur. La déficience en TGR5 aggrave la MAF, sans passer par une modulation de la cellule de Kupffer. C’est en fait le microbiote intestinal qui est impacté chez les souris déficientes pour TGR5, et qui médie cette aggravation.Par ailleurs, afin de moduler le microbiote intestinal au cours de la MAF, nous avons évalué le rôle de la pectine, qui favorise la croissance de certaines bactéries et peut chélater les acides biliaires. Malgré ses propriétés chélatantes, ce sont bien les modifications du microbiote intestinal induites par la pectine qui jouent un rôle protecteur et curatif dans la MAF.Ces différentes études devraient permettre d’identifier des cibles thérapeutiques potentiellement applicables chez des patients alcooliques et basées sur la modulation du microbiote intestinal. / Alcoholic liver disease (ALD) includes all the liver injuries occurring as a result of excessive and chronic alcohol consumption. Nevertheless, among heavy drinker, only a subset of individuals will develop severe liver injury. Intestinal microbiota was identified as a major player in the mechanisms involved in ALD. Moreover, bile acids were the most discriminant faecal metabolites between mice with or without liver injury. The bile acids receptor TGR5, which is expressed on Kupffer cells, promotes their anti-inflammatory profile.We assessed the role of bile acids receptor TGR5 in ALD using TGR5-deficient mice. TGR5-deficiency worsens ALD, but without modulating the Kupffer cells profile. However, intestinal microbiota is impaired in TGR5-deficient mice, and this is responsible for ALD worsening.Furthermore, in order to modulate the intestinal microbiota during ALD, we assessed the role of pectin, which is known to promote the growth of certain bacteria and that is a bile acids sequestrant. Despite its sequestrant properties, pectin-induced changes in intestinal microbiota play a protective and curative role in ALD.These studies will allow the identification of new therapeutic targets that could be used for alcoholic patients, using intestinal microbiota modulation.

Les douleurs abdominales chroniques, souvent associées à une hypersensibilité viscérale d’origine colique (HSVC), sont l’un des symptômes majeurs constatés lors du syndrome de l’intestin irritable (SII). Le SII est une colopathie chronique fonctionnelle touchant environ 11% de la population mondiale et altérant significativement la qualité de vie des patients. L’étiologie multifactorielle de cette pathologie rend la physiopathologie complexe. Les gastroentérites infectieuses ont été décrites comme l’un des facteurs de risque dans le développement du SII-post-infectieux (SII-PI). Le SII-PI survient en effet dans 4 à 31% des cas suite à une gastroentérite aigüe d’origine bactérienne, virale ou parasitaire. Ces infections peuvent avoir de nombreuses répercussions et en particulier sur l’intégrité de la barrière épithéliale intestinale, le système immunitaire ou encore sur le microbiote intestinal. Par ailleurs, suite à une infection parasitaire, le risque de développer un SII est de 40% contre seulement 14% suite à une infection bactérienne.Blastocystis est le parasite intestinal le plus fréquemment retrouvé chez l’Homme. Néanmoins, malgré de récentes études épidémiologiques rapportant une plus forte prévalence de ce parasite chez les patients atteints de SII, son rôle en santé humaine reste débattu. De plus, d’autres études rapportent que les individus porteurs de ce parasite présentent des douleurs abdominales et sont atteints d’une dysbiose intestinale. Actuellement, l’absence d’un modèle animal d’infection par Blastocystis reproductible ne permet pas d’étudier les mécanismes physiopathologiques liés à l’infection et donc d’explorer la contribution éventuelle de ce parasite dans le SII.Les objectifs de ce travail de thèse étaient tout d’abord de mettre en place un modèle murin d’infection expérimentale par Blastocystis pour dans un deuxième temps évaluer si ce parasite est capable d’induire une HSVC et une dysbiose intestinale avec l’objectif d’établir un nouveau modèle de SII d’origine infectieuse chez le rat. Pour répondre au premier objectif, le pouvoir infectieux de différents sous-types et différentes formes du parasite (formes vacuolaires ou kystes), isolés de cultures axéniques ou purifiés à partir de selles de patients et d’animaux a été évalué chez des animaux de laboratoire (rats et souris). Nous avons ainsi réussi à établir un modèle reproductible d’infection chronique par Blastocystis chez le rat de laboratoire à l’aide de kystes purifiés à partir de selles humaines.L’utilisation de ce modèle in vivo, nous a permis de mettre en évidence que le sous-type 4 (ST4) de Blastocystis induit une HSVC sans origine inflammatoire chez les rats expérimentalement infectés. De plus, les animaux développent un comportement type anxio-dépressif corrélé à l’HSVC. La dysbiose intestinale associée à l’infection se caractérise par une augmentation de la richesse bactérienne et une diminution du ratio Firmicutes/Bacteroidetes. Par ailleurs, nous avons corrélé l’HSVC à l’augmentation de l’abondance relative du genre Bacteroides et la diminution de l’abondance relative de la famille des Clostridiaceae, bactéries productrices d’acides gras à chaine courte (AGCC). Une diminution des taux d’AGCC fécaux a d’ailleurs été constatée chez les rats infectés. De plus, nous avons mis en évidence une augmentation de l’activité de protéases à sérine dans les fèces des animaux infectés pouvant expliquer l’HSVC.Ces données suggèrent qu’une infection gastro-intestinale par Blastocystis serait associée à une hypersensibilité viscérale d’origine colique (HSVC) et à un déséquilibre de la flore intestinale (dysbiose). Ainsi, ce nouveau modèle d’infection pourrait être un bon modèle de SII d’origine infectieuse et pourrait donc contribuer à un meilleur diagnostic et au développement de nouvelles stratégies thérapeutiques pour des pathologies chroniques de l’intestin chez certains individus. / Chronic abdominal pain often associated with colonic hypersensitivity (CHS) is one of the major symptoms of irritable bowel syndrome (IBS). IBS is a functional chronic disorder affecting ~11% of worldwide population and disturbing patients’ quality of life. Etiology is multifactorial and thus pathophysiology is complex and remains poorly understood. Infectious gastroenteritis has been described as one of the risk factors for development of post-infectious IBS (PI-IBS). PI-IBS can occur in 4-31% of patients following acute gastroenteritis of bacterial, viral or parasitic origin. Numerous studies support a role for pathogen-mediated modifications in the resident intestinal microbiota, epithelial barrier integrity and immune activation in PI-IBS. Interestingly, the risk of IBS is highest with protozoal enteritis, with ~40% of individuals developing IBS against ~14% following bacterial infection. Blastocystis is the most common intestinal parasite found in human intestinal tract. Nevertheless, clinical relevance remains controversial, despite recent epidemiological studies showing a higher prevalence of this parasite in IBS patients. Interestingly, studies report that individuals carrying Blastocystis display abdominal pain and intestinal dysbiosis. Currently, the lack of reproducible animal model of Blastocystis infection does not allow to study the pathological mechanisms related to infection and thus to explore the potential contribution of this parasite in IBS.The aims of this study were first to develop a murine model of Blastocystis infection and then to investigate whether this parasite could lead to the development of intestinal dysbiosis associated CHS with the aim of developing a new PI-IBS rat model.The first aim was to evaluate the infectivity of different parasitic subtypes and stages (vacuolar and cystic forms) isolated from axenic cultures or purified from human or animal feces, into laboratory animals (rats and mice). Interestingly, we succeeded in the development of a reproducible model of chronic infection by Blastocystis in laboratory rats using cysts purified from human stool.Using this animal model, we found that Blastocystis ST4 induced non inflammatory CHS in infected rats. In addition infected rats developed anxiety- and depressive-like behavior correlated with CHS. Infection associated intestinal dysbiosis was characterized by increased bacterial richness and decreased Firmicutes/Bacteroidetes ratio. Interestingly, we correlated CHS with the increase in the relative abundance of genus Bacteroides and the decrease in the relative abundance of the family Clostridiaceae, some bacteria producing Short Chain Fatty Acids (SCFAs). Indeed, fecal SCFAs levels were decreased in infected rats. These decreases were correlated with the relative abundance of genus Oscillospira which was also described increased in Blastocystis individual carriers. In addition, we have demonstrated an increase in fecal serine protease activity in infected animals that may explain development of CHS.These data suggest that a gastrointestinal infection with Blastocystis may be associated with the establishment of intestinal dysbiosis associated CHS. Thus, this new infectious model could be a good model of PI-IBS and could therefore contribute to a better diagnosis and development of new therapeutic strategies for chronic bowel diseases.

Background and Aim: Bacterial infections are a well described complication of cirrhosis and occur in 37% of hospitalized patients. Culture positive infections in addition to the presence of bacterial products and DNA lead to loss of liver function and decompensation in cirrhosis. The mechanisms and molecular pathways associated with Bacterial Translocation (BT) are unknown. The aims of this study were to determine: i. macrophage phenotype and molecular pathways associated with bacterial translocation ii. if intestinal macrophages in liver cirrhosis are capable of modulating intestinal permeability.iii. structural integrity of the epithelial barrier. Methods: Duodenal biopsies and serum samples were collected from 29 patients with decompensated cirrhosis, 15 patients with compensated and 19 controls. Duodenal macrophages were characterized by means of flow cytometry and IHC. Gene expression analysis was performed to determine molecular pathways involved in BT. Inflammatory cytokine determination was done in serum and culture supernatant by means of customized cytometric bead arrays. Results: Patients with decompensated cirrhosis demonstrated: increased frequency of CD33+/CD14+/TREM-1+ and iNOS+ macrophages in their duodenum, elevated mRNA levels of nitric oxide synthase 2 (NOS2), chemokine ligand 2 (CCL2), chemokine ligand 13 (CCL13) and interleukin 8 (IL8) and increased serum levels of interleukin 6 (IL6), IL8 and lipopolysaccharides (LPS). Additionally, patients with decompensated cirrhosis showed an increase in NO, IL6, IL8 and CCL2 levels in culture supernatant after short term duodenal biopsy culture. Although the epithelial barrier on EM seemed intact, significantly increased expression of the “pore” forming tight junction claudin 2 was observed. Conclusion: This study showed the presence of activated CD14+Trem- 1+iNOS+ intestinal macrophages and increased levels of NO, IL-6 and claudin-2 levels in the duodenum of patients with decompensated liver cirrhosis, suggesting that these factors enhance intestinal permeability to bacterial products. / Afrikaans: Inleiding: Bakteriele infeksie is ‘n beskryfde komplikasie van lewersirrose wat in 37% van gehospitaliseerde pasiente voorkom. Kultuur positiewe infeksies asook die teenwoordigheid van bakteriele produkte en DNA lei tot verlies van lewerfunksie en dekompensasie. Die molekulere meganismes wat verband hou met bakteriele translokasie is nog onbekend. Die doel van hierdie studie was om: i. Makrofaag fenotipe en molekulere meganismes geassosieerd met bakteriele translokasie te beskryf, ii. te bepaal of intestinale makrofage dermdeurlaatbaarheid beinvloed, asook iii. om die struktruele integriteit van die dermwand te bepaal. Methods: Serum en dunderm biopsies was verkry van 29 pasiente met gedekompenseerde lewer sirrose, 15 pasiente met gekompenseerde sirrose en 19 kontroles. Dunderm makrofage was gekarakteriseer met behulp van vloeisitometrie en immunohistochemie. Molekulere meganisms belangrik tydens bakteriele translokasie was bepaal met behulp van geneekspressie. Serum en selkultuur supernatant sitokien bepalings was met Bioplex assays gedoen. Resultate: Pasiente met gedekompenseerde sirrose demonstreer: ‘n verhoogde frekwensie van CD33+/CD14+/TREM-1+ en iNOS+ makrofage in hul dunderm, verhoogde mRNA vlakke van NOS2, CCL2, CCL13 en IL8 asook verhoogde serum vlakke van IL6, IL8, LPS. Addisioneel het pasiente met gedekompenseerde sirrose vehoogde supernatant vlakke van NO, IL6, IL8 and CCL2 na kort termyn dunderm biopsie kulture. Alhoewel elekronmikroskopie gewys het dat die dundermwand intak is, was daar statisties-beduidend verhoogde ekspressie van die “porie” vormende vasteaansluitings- proteien, claudin 2 sigbaar. Gevolgtrekking: Gesamentlik het die studie gewys dat geaktiveerde CD14+/Trem-1+/iNOS+ intestinale makrofage asook verhoogde vlakke van NO, IL-6 en claudin-2 teenwoordig is in die dunderm van pasiente met gedekompenseerde sirrose. Dit dui daarop dat diè faktore derm deurlaatbaarheid vir bakteriele produkte kan verhoog. / Dissertation (MSc)--University of Pretoria, 2011. / Immunology / MSc / Unrestricted