Certaines espèces de phytoplanctons produisent des métabolites secondaires hautement toxiques appelés phycotoxines. Ces phycotoxines peuvent contaminer les fruits et mer et poser un risque sanitaire pour l'homme. Elles sont responsables d'effets néfastes chez l'homme et peuvent provoquer des diarrhées, des nausées et des douleurs abdominales. Pour améliorer l'évaluation du risque sanitaire lié à la présence de phycotoxines dans les coquillages, une meilleure caractérisation du potentiel toxique est indispensable. Aussi, l'objectif de ce doctorat a été d'étudier les effets cytotoxiques des phycotoxines lipophiles seules et en mélange à l'aide de nouvelles approches in vitro en toxicologie. Nos études ont montrées que si les analogues de l'acide okadaïque (AO) induisaient tous l'apoptose, la génotoxicité, l'inflammation cellulaire et la perturbation du cycle cellulaire, sur les lignées intestinales Caco-2 et HT29-MTX, ces effets se produisaient à des concentrations différentes en fonction des toxines. Ensuite, nos recherches sur la lignée hépatique humaine HepaRG ont montrées l'implication des enzymes de phase I dans le processus de détoxication de l'AO et de ses analogues les dinophysistoxines (DTX), et de la pectenotoxin-2 (PTX-2). Pour finir, l'utilisation de la méthode des index de combinaison pour l'étude de la toxicité des mélanges binaires de toxines sur des lignées intestinales a mis en évidence des effets synergiques du mélange Azaspiracide-1/ Yessotoxine. / Some species of phytoplankton produce highly toxic secondary metabolites called phycotoxins. Phycotoxins can contaminate seafood and pose a health risk to humans. They can cause several adverse effects in humans, including diarrhea, nausea and abdominal pain. To improve risk assessment associated with the presence of lipophilic phycotoxins in shellfish, a better characterization of the toxic potential is essential. The aim of this PhD was to study the cytotoxic effects of single and mixed lipophilic shellfish toxins using new approaches in in vitro toxicology. Our studies have shown that, analogs of okadaic acid (OA) induced apoptosis, genotoxicity, cellular inflammation and disturbance of the cell cycle, on intestinal cell lines Caco-2 and HT29-MTX, and that this effects occurred at different concentrations depending on the toxin. Then, our researches on human hepatic lineage HepaRG have shown the involvement of phase I enzymes in the detoxification of the pectenotoxin-2 (PTX-2), and OA and its analogues the dinophysistoxins (DTX). Finally, the use of the combination index method for studying the toxicity of binary mixtures of phycotoxins on intestinal cell lines showed synergistic effects of azaspiracid-1 / yessotoxin mixture.

No description available.

The products of the Cdx genes, Cdx1, Cdx2 and Cdx4, are known to play essential roles in many developmental processes including neural tube closure, axial elongation and patterning the anterior-posterior axis of the developing embryo. Cdx1 and Cdx2 are both expressed in the endoderm of the embryo and persist throughout adulthood in the intestinal epithelium, but their functions and mechanisms of action in this lineage are poorly understood, in part due to the peri-implantation lethality of Cdx2-/- mice. To circumvent this limitation, a conditional loss of function strategy was used to inactivate Cdx2 in the intestinal epithelium. These conditional mutants were also crossed to Cdx1-/- mice, which are viable and fertile, to examine potential functional compensation between these family members. The major findings of this study are that Cdx2 regulates patterning and differentiation of the small intestinal epithelium, while Cdx1 does not appear to make a contribution to either process. Furthermore, Cdx operates upstream of Notch ligand Delta-like 1 (Dll1) in endoderm and mesoderm derivatives, demonstrating that Cdx function is similar in different lineages. Finally, Cdx2 cannot fulfill the requirement for Cdx1 in regulation of its own promoter in the intestine. This is the first in vivo evidence that these two family members have context-dependent functional specificity. Altogether, this study underscores critical roles and mechanisms of action for Cdx members in the developing intestine and mesoderm.

Sox9 est un facteur de transcription exprimé au cours du développement de l'intestin et son expression est maintenue à l'âge adulte dans trois populations cellulaires : les cellules souches, les cellules de Paneth, et les cellules tuft. L'inactivation de Sox9 dans l'épithélium intestinal embryonnaire entraîne, chez l'adulte, une hyperplasie des cryptes ainsi que l'absence de cellules de Paneth. Ce projet de thèse vise à déterminer le rôle de Sox9 dans les cellules de Paneth (dont la fonction est altérée chez les patients atteints de la maladie de Crohn), à identifier les mécanismes par lesquels Sox9 régule la prolifération et à proposer des cibles de Sox9 dans les cellules tuft. À l'aide de modèles murins d'inactivation de Sox9 au niveau de l'épithélium intestinal adulte, nous avons montré que la perte de ce facteur conduit à une augmentation de la prolifération dans les cryptes, confirmant ainsi que Sox9 régule négativement ce processus. Nos résultats indiquent que Sox9 est essentiel au maintien de l'identité des cellules de Paneth et nous proposons qu'il assure cette fonction en réprimant des gènes requis pour la différenciation des cellules de Goblet : Muc2 et Klf4. La perte de Sox9 dans les cellules de Paneth s'accompagne d'une réduction importante des molécules antimicrobiennes, ce qui entraîne une dysbiose intestinale. Dans un environnement spécifique (en présence du « mouse norovirus »), les souris déficientes en Sox9 présentent une perméabilité intestinale augmentée et une susceptibilité à l'inflammation accrue. Les dysfonctionnements des réponses antimicrobiennes et immunitaires dans notre modèle sont comparables à ceux observés chez les patients atteints de la maladie de Crohn, suggérant une implication potentielle de Sox9 dans cette pathologie. De plus, ces altérations pourraient expliquer l'augmentation de l'apparition des tumeurs observée chez les souris dont l'épithélium intestinal est déficient en Sox9, dans le contexte d'une mutation du gène suppresseur de tumeur Apc. Enfin, nous avons identifié des gènes potentiellement régulés par Sox9 qui pourraient expliquer son rôle dans le contrôle de la prolifération. Ces découvertes seront importantes pour mieux comprendre le processus du renouvellement de l'épithélium intestinal et identifier précisément le rôle de Sox9 dans le maintien de l'homéostasie et au cours du processus de la tumorigenèse intestinale. / Sox9 is a transcription factor expressed during the intestinal development and its expression is maintained throughout adult age in at least three populations of cells: stem cells, Paneth cells and tuft cells. Sox9 inactivation in the embryonic intestinal epithelium leads to crypts hyperplasia and to the loss of the Paneth cell lineage. The aim of this project is to determine Sox9 function in the adult intestinal epithelium, especially its role in Paneth cells (which function is altered in patients affected by inflammatory diseases such as Crohn disease), to identify how Sox9 controls proliferation and to propose molecular targets of Sox9 in tuft cells. Using mice models to inactivate Sox9 in adult intestinal epithelium, we could show that Sox9 is required to limit proliferation in the crypts, thus validating the hypothesis that Sox9 regulates negatively proliferation. Our results indicate that Sox9 is essential to maintain Paneth cells identity and we proposed that it ensures this function by repressing genes specific for Goblet cells differentiation: Muc2 and Klf4. Loss of Sox9 in Paneth cells is associated with a reduction of antimicrobial molecules which causes intestinal dysbiosis. In a specific environment (in presence of the « mouse norovirus »), Sox9-deficient mice have a defective intestinal permeability and are more susceptible to inflammation. The dysfunctions of the mucosal defences and of immunity responses in our model resemble those observed in Crohn patients, thus suggesting a potential implication of Sox9 in this pathology. In addition, these alterations could be responsible for the increased susceptibility of our deficient model to develop tumors in the context of a mutation of the tumor suppressor gene Apc. We started to unravel potential molecular targets of Sox9 that are involved in the control of proliferation, that will be important to better understand Sox9 function in the intestinal epithelium self-renewal and to identify precisely Sox9 function to maintain homeostasis and during intestinal tumorigenesis.

INTRODUCTION: Small intestinal bacterial overgrowth refers to the proliferation of abnormal numbers and types of microorganisms in the lumen of the proximal bowel. Bacterial overgrowth has been implicated as a possible factor in prolonging some episodes of infantile gastroenteritis. This thesis examines 2 different aspects of the duodenal flora of infants with gastroenteritis, and has therefore been divided into 2 separate studies. CARBOHYDRATE STUDY: Objective: To test the hypothesis that during a diarrhoeal episode the presence of malabsorbed carbohydrate in the duodenal lumen acts as a factor promoting bacterial proliferation. Patients and methods: Infants admitted to the rehydration ward with acute gastroenteritis were selected for study if they fulfilled various criteria in terms of age, nutritional status, previous diarrhoeal episodes and antibiotic administration. They were admitted to the research ward. Weights were measured and if they had severe diarrhoea (≥ 30g/kg) were included in the study. Twenty patients were entered into the study. On admission into the trial the first duodenal intubation was done to measure the duodenal flora quantitatively and qualitatively. Thereafter the patients were assigned on an alternate basis to one of 2 groups. One group (carbohydrate-containing group) received a soy-based infant formula containing carbohydrates (Isomil, Ross). The other group (carbohydrate-free group) received an identical milk but from which all carbohydrate had been omitted (Ross CHO-free). To these infants carbohydrate was given intravenously. Stool output was measured daily. After 3 days of the respective diets the duodenal flora was re-examined. Results: Longitudinal analysis of the duodenal flora of the carbohydrate-containing group showed a small decrease in the number of bacterial isolates and in their magnitude. The duodenal flora of the carbohydrate-free group was virtually unchanged. Comparing the duodenal bacteriology of the groups the only significant difference was that the number of isolates and the magnitude of Haemophilus was greater in the carbohydrate-free group- (p < 0.05). The diarrhoea resolved in 5 patients: 2 in the carbohydrate-containing and 3 in the carbohydrate-free group. Conclusions: The lack of difference in the response of the duodenal flora between the two groups studied suggests that the presence of carbohydrates in the lumen is not important in encouraging the growth of bacteria in that site. The possible causes for an increase in Haemophilus numbers in the carbohydrate-free group are discussed. BOWEL COCKTAIL STUDY: Objective: Small intestinal bacterial overgrowth has been proposed as a cause of progression of acute diarrhoeal episodes to persistence. The "bowel cocktail", a combination of oral gentamicin and cholestyramine, has been shown to be effective in terminating episodes of persistent diarrhoea. It has been postulated to work by eradicating small intestinal bacterial overgrowth, but its mode of action is not known. The objective of this study was to examine the changes in the duodenal flora associated with administration of the bowel cocktail in order to elucidate its possible mechanism or mechanisms of action. Patients and methods: The study group comprised 15 patients. Fourteen were infants from the carbohydrate study who had ongoing diarrhoea. The remaining infant (the "late entry") was selected from the rehydration ward. Severe diarrhoea, as defined by a stool output equal to or greater than 30g/kg/day, was a pre-requisite for entry into the study. The investigation involved 2 duodenal intubations for microbiological analysis of the duodenal fluid. After the first intubation (which was the second intubation for the 14 infants who had been in the carbohydrate study) the bowel cocktail was administered. This comprised a 3-day course of oral gentamicin and 5 days of oral cholestyramine. Forty-eight hours after the start of therapy the duodenal bacteriology was repeated. The patient management was the same as during the carbohydrate study and the feeding regimen of the infants was not altered. The study ended immediately after completion of the bowel cocktail course. Results: Administration of the bowel cocktail was associated with a decreased stool output in all patients. Bacteriological analysis of the duodenal flora after this treatment showed a statistically significant decrease in the total microbial count, the aerobic microbial fraction and the Enterobacteriaceal fraction. On analysis of the bacterial genera a significant decrease was noted in Neisseria, Haemophilus, and aerobic lactobacilli. Analysis of individual patients' duodenal fluid bacteriology in conjunction with the stool bacteriology results before administration of the bowel cocktail often provided an explanation as to the possible aetiology of the diarrhoea and its resolution by therapy. Conclusions: Small intestinal bacterial overgrowth, in the accepted sense of a luxuriant flora teeming with faecal organisms, did not appear to be a feature of the patients in this study. The total bacterial count was only slightly above the accepted upper limit of normal. Although the decrease in the number of Enterobacteriaceae could possibly be interpreted in the context of bacterial overgrowth, a study of the individual patients' duodenal flora shows that these microorganisms were more likely to be acting as specific enteric pathogens. It is concluded that small intestinal bacterial overgrowth, as currently defined, is not an important cause of persistent diarrhoea. The efficacy of the bowel cocktail is more likely to reside in its ability to eradicate specific enteric pathogens. The author ends by questioning the validity of the whole concept of small intestinal bacterial overgrowth.

Effects of Fe and Se status on GSHPx activity and lipid peroxidation in liver and intestinal mucosa were studied. Rats were Se and Fe supplemented ( +Se+Fe), Sedeficient and Fe-supplemented (-Se+Fe), Se supplemented and Fe overloaded (+Se++Fe) by intramuscular injection, or Se deficient and Fe overloaded (-Se++Fe) for 20d. Fe overloaded tissues had more Fe, but hemoglobin was unaffected. Liver and mucosal GSHPx activity was low in Se-deficient rats. Thiobarbituric acid reactive substances (TBARS) were higher in Fe overloaded and -Se++Fe vs +Se++Fe tissues. Mucosal TBARS was higher in -Se++Fe rats gavaged with CBrCl3. In experiment 2, Fe overload was induced by a 2% carbonyl iron, low-Se diet fed for 2mo, the +Se++Fe and -Se++Fe groups. Low Se-, low Fe-diet was fed to rats supplemented with Fe or Fe and Se in the water, the -Se+Fe and +Se+Fe controls. Iron overloaded tissues had more Fe. Liver and mucosal GSHPx activity was lower in Se-deficient and +Se++Fe vs +Se+Fe rats. TBARS was higher in Fe overloaded, -Se++Fe vs +Se++Fe, and CBrCl3 tissues. Hemoglobin and serum Fe were lower in the -Se++Fe group. In experiment 3, low-Se, low-Fe diet was fed for 20d, the -Se-Fe, +Se-Fe, -Se+Fe and +Se+Fe groups. Mucosal Fe was lower in the Fe-deficient rats. Cytochrome P-450 and GSHPx activities were lower and TBARS was higher in Se-deficient tissues. In experiment 4, the +Se+Fe, +SeFe, -Se+Fe, -Se-Fe, +Se++Fe, and -Se++Fe groups treated as in experiment 3. Iron overloaded tissues had more Fe and TBARS, but hemoglobin and serum Fe were unaffected. GSHPx and Cytochrome P-450 activities were lower in Se-deficient and in +Se++Fe vs +Se+Fe rats. CBrCl3 did not affect TBARS. High TBARS occurred in liver and mucosa of Fe overloaded rats. Chronic Fe overload was required to reduce liver and mucosal GSHPx activity. The combination of Se deficiency and Fe overload caused very high TBARS. Low oral CBrCl3 doses elevated mucosal TBARS, a first report of extrahepatic action of CBrCl3 in vivo. Iron deficiency did not affect GSHPx activity or CBrCl3 induced lipid peroxidation.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19021号 / 農博第2099号 / 新制||農||1030(附属図書館) / 学位論文||H27||N4903(農学部図書室) / 31972 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 久米 新一, 教授 祝前 博明, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20233号 / 医博第4192号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 斎藤 通紀, 教授 松田 文彦, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The primary role of 1,25(OH)₂D and the vitamin D receptor (VDR) during growth is to mediate intestinal calcium (Ca) absorption by regulating the expression of genes (e.g., Trpv6, S100g) that control Ca fluxes through enterocyte. In contrast to the well-defined role during rapid growth, the understanding of 1,25(OH)₂D signaling in post-growth, mature adult or the elderly is poor. Some observational studies suggest intestinal 1,25(OH)₂D signaling is not important to Ca absorption and bone for mature adult. In the elderly, intestine develops resistance to 1,25(OH)₂D action which might be due to age-related reductions in intestinal VDR level. In addition, there is lack of evidence that directly tests the role of 1,25(OH)₂D action in the distal intestine post-growth. My dissertation research focuses on discovering the importance of 1,25(OH)₂D signaling from both the temporal and spatial perspectives. By using 4-month old, whole intestine or large intestine VDR knockout mice, we found that for mature mice, 1,25(OH)₂D signaling plays a minimal role in regulating Ca absorption and protecting bone mass when dietary Ca intake is adequate. In contrast, 1,25(OH)₂D signaling in the whole intestine, and to a lesser extent the proximal colon, is critical to upregulate Ca absorption and protect bone when dietary Ca intake is low. Next, we proved that the Ca absorptive machinery in the proximal colon can be locally stimulated to enhance the expression of Trpv6 by 1,25(OH)₂D released from glycoside and glucuronide forms of calcitriol. Furthermore, our transcriptomic analysis on 1,25(OH)₂D-regulated genes in the duodenum of mature (4-month old), middle-aged (11.5-month old), and old (20.5-month old) mice suggest although aging did reduce the induction of some genes by 1,25(OH)₂D, this effect is not universally present across the genome and it is not related to intestinal Vdr expression. The findings from my dissertation research serve as a foundation for future research to identify a) the potential of specifically targeting proximal colon to increase intestinal Ca absorption and protect bone in adult; b) the molecular mechanisms that contribute to the aging-associated, non-universal resistance to 1,25(OH)₂D action.