Amino acids are assimilated by membrane-associated transporters into and out of enterocytes either in their free form or in the form of peptides. The peptide transporter, PepT1, is thought to be the major facilitator of peptide transport in the enterocyte. It is unknown if the peptide transporters and free amino acid transporters operate in a compensatory fashion to regulate the amino acid balance within the enterocyte. Therefore, the objective was to examine the regulatory balance between PepT1 and other peptide and free amino acid transporters in enterocytes. The Mouse Small Intestinal Epithelial (MSIE) cells are conditionally immortalized. It was found that MSIE cells express BoAT1, CAT1, CAT2, LAT1, y+LAT1, and y+LAT2, but not PepT1, EAAT3, Bo,+AT, or LAT2, making this model similar to the basolateral membrane of enterocytes. Growing MSIE cells at high temperatures did not affect the nutrient transporter gene expression profile of these cells. Thus, the human colon carcinoma (Caco-2) cell line was used as a small intestinal in vitro model for this study. These cells express PepT1, HPT1, PTR3 EAAT1, EAAT3, rBAT, Bo,+AT CAT1, LAT1, y+LAT1, y+LAT2, ABCC3, ABCC4, which increased from D0 to D21 post confluency, indicating cell maturation. In Caco-2 cells, PepT1 gene silencing was induced in Caco-2 cells. Despite an reduction of PepT1 gene (82%, P < 0.05) protein (96%), no significant difference in any peptide (HPT1, PTR3, ABCC3, ABCC4) or free amino acid transporters (EAAT1, EAAT3, rBAT, Bo,+AT, BoAT1, CAT1, CAT2, LAT1, LAT2, y+LAT1, y+LAT2) between Caco-2 cells treated with PepT1 siRNA and Caco-2 cells treated with Control siRNA was observed. These results suggest no compensation at the gene expression level of these transporters in response to a reduction of PepT1. To account for the limitations of an in vitro and PepT1 kockout mouse model, transgenic chicken models were pursued. Potential cPepT1 overexpressing, cPepT1 shRNA or control shRNA expressing G0 chickens were generated by embryo injection of pseudolentiviral particles followed by ex ovo egg culture. Overall, 9 potential G0 cPepT1 overexpressing chickens, 15 potential G0 cPepT1 shRNA expressing chickens, and 4 potential G0 control shRNA expressing chickens were generated. / Ph. D.

In a number of mammalian species, susceptibility to enteric coronavirus infection has been shown to be age-related. This is thought to be associated with enterocyte maturation and receptor protein expression. One of the factors that can influence differentiation and maturation of enterocytes is the availability of short-chain fatty acids (SCFA) in the intestinal lumen. These compounds are by-products of the bacterial fermentation of dietary fiber and serve as the primary energy source for enterocyte metabolism. The overall objective of this dissertation was to evaluate the effects of dietary fiber and short-chain fatty acids on enterocyte differentiation, maturation, and susceptibility to coronavirus infection in turkeys. Initial work involved the development of an indirect immunoperoxidase assay (IPA) for the identification and localization of turkey coronavirus (TCV) in paraffin-embedded, acid-ethanol fixed tissue. IPA was found to be superior to indirect immunofluorescent antibody test (IFA) for this and other diagnostic purposes. To evaluate cellular differentiation and maturation, an SDS-PAGE/immunoblot technique was developed to determine relative levels of villin expression in turkey embryos. Villin is an actin-bound cytoskeletal protein known to be expressed in increasing quantities at the apical surfaces of maturing enterocytes. Villin expression level was found to increase linearly as a function of embryo age. Villin localization was performed by IPA on paraffin-embedded, acid-alcohol fixed tissue. As enterocytes (embryos) matured, villin was found to concentrate at the apical surfaces and eventually at the basolateral membranes. Experiments were also conducted to see what effect in ovo butyrate administration would have on developing embryonic enterocytes. Butyrate has been shown to enhance differentiation of non-neoplastic and neoplastic cells in culture as well as promote healing of damaged intestinal epithelium in human. Villin expression was significantly enhanced in embryos receiving 0.2 and 0.3 M butyrate 36 hours post-administration. Butyrate appeared to enhance villin expression and therefore enterocyte maturation in a dose-dependent manner. Susceptibility of turkey embryos to TCV infection as a function of age and butyrate treatment was investigated as well as epithelial localization of TCV infection in poults. The level of TCV infection of epithelium was found to increase with embryo age between 17 and 23 days. Poults showed higher levels of infection on the distal 2/3 of villi and no evidence of infection in the intestinal crypts. Butyrate administration in 21-day-old embryos followed by TCV inoculation caused a significant increase of the number of infected cells per villus. This data suggested that butyrate might be used as a means to manipulate enterocyte susceptibility to TCV infection. In the final set of experiments, the effects of fiber-fortified poult diets containing 5% cellulose or 5% guar gum on luminal SCFA levels, enterocyte maturation, and TCV infection were investigated. SCFA levels in cecal contents were determined by gas chromatography. Enterocyte maturation was assessed by the determination of villin expression on immunoblot and the severity of TCV infection was determined by IPA and lesion score. Fiber-fortified diets enhanced SCFA production and villin expression, but contrary to embryo studies, TCV infection appeared to be reduced. In general, poults performed better on the diet containing cellulose. Mechanisms regarding the roles of dietary fiber and SCFA in enterocyte differentiation, maturation, and TCV susceptibility are proposed as well as future directions for research. The in ovo and poults system used in this research may serve as models for further investigation of the influences of host and dietary factors on enteric viral infection and recovery. / Ph. D.

No description available.

L'intestin grêle à un rôle vital, il est impliqué dans de multiples fonctions : absorptive, endocrinienne, lymphoïde, barrière, et dans le métabolisme de l'arginine. L'exploration du grêle est difficile chez les patients graves hospitalisés en réanimation, alors même que ces patients sont à risque d'avoir une dysfonction intestinale aiguë du fait de la sensibilité du grêle à l'ischémie. La découverte et la validation de biomarqueurs entérocytaires, l'un de fonction, la citrullinémie, l'autre de lyse entérocytaire, l'intestinal fatty acid-binding protein (I-FABP), pourrait permettre de mieux évaluer l'intégrité et la fonctionnalité du grêle chez les patients graves. Les objectifs de cette thèse étaient de décrire la prévalence des anomalies entérocytaires chez des patients graves, de comprendre les mécanismes enjeu dans l'atteinte entérocytaire aigiie, d'évaluer la valeur pronostique de ces biomarqueurs, et d'évoquer des perspectives. Entre 2007 et 2013, nous avons mesuré la citrullinémie et/ou l'I-FABP chez des patients hospitalisés au CHU de Besançon ayant un risque théorique de souffrance entérocytaire : patients admis en réanimation, patients nécessitant un pontage aortocoronarien, patients nécessitant une anesthésie générale. Des anomalies de biomarqueurs entérocytaires étaient présentes chez plus de la moitié des patients hospitalisés en réanimation dès leur admission. Il s'agissait d'une citrullinémie basse et/ou d'une concentration plasmatique élevée d'I-FABP. La synthèse des résultats obtenus fait apparaître qu'une cytolyse entérocytaire précoce et courte est fréquente chez le patient admis en réanimation (élévation de la concentration plasmatique d'I-FABP), puis diminue avec la stabilisation clinique des patients. Ce pic initial d'I-FABP semble suivi d'une diminution de la citrullinémie au cours des 48 premières heures d'hospitalisation en réanimation. D'autre part, le sepsis, qu'il soit ou non d'origine digestive, est associé à une diminution de la citrullinémie, ce qui suggère une entéropathie septique indépendante du champ nosologique de la destruction entérocytaire ischémique. Le couple de biomarqueurs, l'un de lyse cellulaire (I-FABP), l'autre de fonction cellulaire (citrulline), semble permettre de caractériser des altérations entérocytaires aiguës du patient grave, en estimant les degrés de destruction ou de dysfonction entérocytaire, et leurs évolutions. Nos études ont mis en évidence une surmortalité chez les patients qui ont une citrullinémie basse et/ou une concentration plasmatique d'I-FABP élevée. La valeur pronostique de l'atteinte entérocytaire nécessite toutefois d'être confirmée par d'autre groupes. Si la compréhension des modifications des valeurs de ces biomarqueurs entérocytaires progresse, avec les limites d'interprétation de leurs variations, et si leur dosage devient plus rapide du fait d'une automatisation de la technique permettant un rendu rapide de résultat, ils pourraient devenir un outil de diagnostic et d'adaptation rapide de la prise en charge thérapeutique des patients graves. / The small bowel is a complex organ involved in numerous vital functions. The small bowel dysfunction is suspected to be the cornerstone of the development of multiple organ dysfunction syndrome in critically ill patients. The development of enterocyte biomarkers is promising for evaluating the small bowel dysfunction and damage in the most severe patients. Plasma citrulline concentration is a validated marker of small bowel function, reflecting the functional enterocyte mass. Plasma citrulline concentration is frequently decreased in critically ill patients, and a decreased plasma citrulline concentration at ICU admission is associated with elevated plasma CRP concentration, and with increased 28-day mortality. Intestinal fatty acid binding protein (I-FABP) is a small cytosolic protein that is released in extracellular space in case of enterocyte destruction. In critically ill patients plasma I-FABP concentration is increased in about one half of patients at ICU admission. Increased plasma I-FABP concentration is associated with shock state, and with elevated catecholamine dose. Both biomarkers are probably complementary and may help the clinician to identify patients presenting enterocyte damage. Further studies are needed to improve the interpretation of plasma citrulline concentration which is probably complex in critically ill patients.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No description available.

No description available.

Intestinally derived chylomicron remnants (CM-r) may contribute to atherogenic dyslipidemia during the Metabolic Syndrome (Mets). However, the combined effects of ezetimibe (EZ) and simvastatin (SV) on post-prandial (PP) dyslipidemia during MetS remains unclear, nor is it known whether the combination has a synergistic anti-atherogenic effect on CM-r arterial retention. The first objective was to delineate the effects of EZ+SV therapy on intestinal cholesterol flux and CM PP metabolism in the JCR:LA-cp rat, a model of MetS. The second objective was to quantify the impact of EZ+SV therapy on arterial retention of CM-r and subsequent myocardial lesion development in the JCR:LA-cp rat. EZ+SV therapy decreased net intestinal cholesterol absorption in MetS rats. Furthermore, EZ+SV therapy reduced arterial retention of CM-r and frequency of myocardial lesions in MetS rats. In conclusion, EZ+SV therapy reduces arterial retention of CM-r and myocardial lesion development possibly through its beneficial effects on cholesterol transport and PP-metabolism. / Nutrition and Metabolism

Overproduction of intestinal chylomicrons (CM) has been proposed to contribute to fasting and post-prandial (PP) dyslipidemia and may accelerate the development of cardiovascular disease (CVD) during obesity, insulin resistance (IR) and diabetes. However, the impact of morphological changes in intestinal mucosa structure have not been investigated during IR and intestinal dyslipidemia. The first objective of this thesis was to characterize intestinal villi morphology and to determine whether a morphological relationship exists with enterocytic apoB48 (a marker of CM), and intestinal lymph secretion of apoB48 in the obese and IR JCR:LA-cp rat. The second objective was to assess the impact of n-3 PUFA supplementation on PP dyslipidemia in the JCR:LA-cp rat. Intestinal hypertrophy was observed in IR rats, corresponding to an increase in intestinal and lymphatic apoB48 expression. Further, a dietary intervention of n-3 PUFA showed lower PP plasma concentrations of apoB48 and PP plasma inflammatory markers. We conclude that intestinal hypertrophy may contribute to intestinal CM overproduction during obesity and IR. Additionally, dietary n-3 PUFA improves PP lipemia and the associated PP inflammatory response in the JCR:LA-cp rat model. / Nutrition and Metabolism

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).