Over the past few years the number of deaths caused due to cardiovascular diseases has been increasing and is of major concern. In the United States, 75% of cardiovascular-related deaths have been attributed to atherosclerosis. Western diets containing large quantities of peroxidized lipids are considered atherogenic. Heated oil in the form of fried food brings high levels of peroxidized fat and its decomposition products in the diet. Peroxidized lipids are known to increase the susceptibility of serum lipoproteins to undergo oxidation, thereby contributing to the progression of atherosclerosis. The intestinal cells are responsible for the absorption of dietary fatty acid peroxides (FAOOH) which has been reported to enhance anti-atherosclerotic effects by inducing apolipoprotein A1 (apoA1) gene and protein levels. Therefore, there is a void in the knowledge of when to expect “harmful” or “beneficial” effects of dietary lipid peroxides. The formation of toxic products like aldehydes from the decomposition of FAOOH is well documented. On the other hand, carboxylic acids particularly azelaic acid, formed as an end product of FAOOH decomposition has been reported to have anti-atherosclerotic effects. Hence, we hypothesize that intestinal cells may decompose FAOOH to aldehydes, which might get converted to carboxylic acids that can be transported across the intestine. Linoleic acid is the most abundant polyunsaturated fatty acid (PUFA) present in the diet. So, we will use peroxidized linoleic acid (13- HPODE) and incubate with intestine derived cells or Caco-2 cells as an in-vitro model for determining its decomposition to aldehydes and carboxylic acids. We propose that the decomposition products of FAOOH in the presence of intestinal cells might be iv responsible for causing an increase in apoA1 levels, which might suggest that lipid peroxidation derived products might actually be beneficial for reducing the progression of atherosclerosis as compared to the absorption of intact FAOOH.

Les bactéries lactiques présentent un grand intérêt économique de par leur large utilisation dans l’industrie agroalimentaire. Parmi elles, Streptococcus thermophilus (ST) est d’une importance majeure puisqu’elle est la plus utilisée après Lactococcus lactis, pour la fabrication de produits laitiers fermentés et de fromages. De plus, cette bactérie est le seul streptocoque à bénéficier du statut GRAS (Generally Recognized As Safe). Outre son intérêt en industrie laitière, ST présente des effets bénéfiques sur la santé intestinale de l’Homme. Bien que ces effets soient largement documentés, le statut probiotique de ST reste encore à conforter. C’est pourquoi des études sont actuellement menées afin de sélectionner des souches de ST à fort potentiel probiotique. Parmi les critères importants figurent leur capacité à survivre aux conditions drastiques du tube digestif (TD) et leur capacité à adhérer aux cellules intestinales. Dans cette optique, les objectifs de cette thèse étaient d’étudier, dans un premier temps, la capacité d’adhésion in vitro de la souche ST LMD-9 à différentes lignées cellulaires intestinales d’origine humaine et d’évaluer la survie de cette souche au stress biliaire. Afin de mettre en évidence un rôle potentiel de certaines protéines de surface dans ces deux processus, trois mutants issus de cette souche et inactivés dans les gènes prtS (protéase pariétale), srtA (sortase A) et mucBP (protéine de liaison aux mucines), ont été inclus dans cette étude. Dans un second temps, l’impact de l’adhésion de LMD-9 a été analysé, d’une part sur l’expression de gènes codant certaines mucines dans les cellules eucaryotes, et d’autre part sur l’expression de gènes qui seraient spécifiquement induits durant le processus d’adhésion, ceci en utilisant la technologie R-IVET (Recombinase-based In Vivo Expression Technology). Les résultats obtenus ont permis de montrer que la souche LMD-9 était capable de survivre jusqu’à une concentration de 3 mM en sels biliaires et que les protéines de surface PrtS, SrtA et MucBP seraient impliquées dans la résistance à ce stress. Nos résultats ont également montré que LMD-9 adhérait aux trois différentes lignées cellulaires, suggérant ainsi que la souche pourrait interagir avec les différentes mucines qu’elle peut rencontrer dans le TD. De plus, l’implication de certaines protéines de surface dans l’adhésion de LMD-9 s’est avérée dépendante des caractéristiques de ces lignées, qu’il s’agisse de cellules entérocytaires (Caco-2) ou productrices de mucus (HT29-MTX et HT29-CL.16E). Concernant l’impact de l’adhésion de la souche LMD-9 sur l’expression des gènes MUC2 et MUC5AC, aucun effet sur le taux de transcrits n’a été observé dans nos conditions expérimentales. Par ailleurs, nos résultats ont permis, pour la première fois, d’identifier les gènes spécifiquement induits dans la souche LMD-9 durant l’adhésion aux cellules épithéliales. Nous avons ainsi montré que l’adhésion de la souche LMD-9 ne dépend pas uniquement des protéines de surface, mais d’autres fonctions et voies métaboliques seraient également impliquées. Ce travail de thèse contribue ainsi à apporter de nouvelles connaissances liées (i) au choix du modèle cellulaire dans les études d’adhésion bactérienne in vitro, (ii) à l’aptitude de la souche LMD-9 à survivre au stress biliaire en faisant intervenir certaines protéines de surface et (iii) à la compréhension des mécanismes moléculaires de l’adhésion de LMD-9 aux cellules épithéliales intestinales / Lactic acid bacteria are of great economic interest because of their use in the food industry. Among them, Streptococcus thermophilus (ST) is of major interest since it is the most used after Lactococcus lactis, for the manufacture of fermented dairy products and cheese. In addition, this bacterium is the only streptococcus to benefit from GRAS status (Generally Recognized As Safe). Beside its interest in the dairy industry, ST has beneficial effects on human intestinal health. Although these effects are widely documented, the probiotic status of ST remains to be consolidated. Therefore, studies are currently being conducted in order to select strains of ST with a high probiotic potential. Among criteria that are important to select ST strains include their ability to survive stress the drastic conditions of the digestive tract (DT) and their ability to adhere to intestinal cells. In this context, the aim of this thesis was to investigate firstly the in vitro adhesion capacity of the ST LMD-9 strain to different human intestinal cell lines and to evaluate the survival of this strain to bile salt stress. In order to highlight the potential role of some surface proteins in these two processes, three mutants derived from this strain and inactivated in the genes prtS (parietal protease), srtA (sortase A) and mucBP (Mucin Binding-protein) were also included in this study. Secondly, the impact of LMD-9 adhesion was analyzed, on one hand on the expression of some mucin-encoding genes in eukaryotic cells, and on the other hand on the expression of genes that would be specifically induced during adhesion process using the R-IVET (Recombinase-based In Vivo Expression Technology) approach. The results obtained demonstrated the ability of LMD-9 to survive up to the concentration of 3 mM of bile salts and that the PrtS, SrtA and MucBP surface proteins would be involved in the resistance to this stress. Our results also showed that the LMD-9 strain was capable of adhering to three cell lines used suggesting that this strain could interact with different mucins that may encounter in the DT. Moreover, the involvement of some surface proteins in the adhesion of LMD-9 has been found to be dependent on the surface characteristics of these cell lines, whether they are enterocytic (Caco-2) or mucus-secreting cells (HT29-MTX and HT29-CL.16E). Regarding the impact of LMD-9 adhesion on MUC2 and MUC5AC gene expression, no effect has been observed on the transcript level under our experimental conditions. Furthermore, for the first time, our results allowed us to identify genes specifically induced in the LMD-9 strain during adhesion process to epithelial cells. We have thus shown that the LMD-9 adhesion does not depend solely on surface proteins, but other functions and metabolic pathways are also involved. This thesis work contributes thus to new knowledge related to (i) the choice of the cellular model in in vitro bacterial adhesion studies, (ii) the ability of LMD-9 to survive bile salt stress by involving some surface proteins and (iii) understanding the molecular mechanisms of LMD-9 adhesion to epithelial cells

1,25-dihydroxyvitamin D3 [1,25(0H)2D3] has been shown to promote phosphate transport rapidly in the perfused duodenal loop, relative to controls, reaching treated/av basal at T = 40 min = 1.82 ± 0.42 and 1.11 ± 0.21, respectively. By using isolated chick enterocytes, studies were undertaken to determine whether 1,25(0H)2D3 has a direct effect on isolated intestinal cells that is manifested by either enhanced uptake or extrusion of phosphate. In time course studies, with 4- to 8-wk-old chicks, 32P uptake in enterocytes at 10 min after addition of test substance was 0%, 130%, 151%, and 123% of controls for 10 pM, 50 pM, 130 pM, and 300 pM 1,25(0H)2D3, respectively. The metabolite 24,25- dihydroxyvitamin D3 [24,25(0H)2D3] exerted an inhibitory effect on phosphate uptake by 1,25(0H)2D3 at a concentration of 130 pM. This result was in agreement with perfusion studies and supports the physiological relevance of isolated cell studies. For signal transduction studies, isolated enterocytes were incubated with 20 µM forskolin (an activator of protein kinase A), 100 nM phorbol ester (an activator of protein kinase C), or 2 µM BAY K 8644 (a calcium channel activator). Enhanced 32P levels relative to controls were found for phorbol ester (126% of controls at T = 7 min, P < 0.05) and BAY K 8644 (150% of controls at T = 7 min, P < 0.05) but not for forskolin, suggesting involvement of protein kinase C and calcium channel signal transduction pathways in uptake. These results paralleled those observed for the perfused duodenal loop. For aging studies, white leghorn roosters were raised for 7, 14, and 28 wk prior to experiments. These studies showed a 1,25(0H)2D3-mediated increase in 32P uptake in isolated cells at 7 wk, but not at 14 or 28 wk. Further analysis of isolated basal lateral membrane (BLM) on SDS-PAGE followed by Western analysis with a well characterized antibody (Ab099) showed a decreased expression of the putative membrane receptor for 1,25(0H)2D3 with increasing age, paralleling the results obtained for 32P uptake in isolated intestinal cell studies. Analyses of 1,25(0H)2D3 effect on protein kinase C activity likewise revealed hormone-mediated stimulation in cells from 7-wk- old chicks, with decreasing responsiveness at a later age. The combined results indicate a physiologically important role for 1,25(0H)2D3 membrane-initiated phosphate uptake in enterocytes of young, rapidly growing animals. Furthermore, these studies validate the use of isolated intestinal cells for further studies on ribozyme-mediated ablation of the 1,25(0H)2D3 membrane receptor function.