L’étiologie exacte des maladies inflammatoires chroniques de l’intestin (MICI) reste actuellement méconnue. Mais un déséquilibre de la flore bactérienne, plus connu sous le nom de dysbiose et se traduisant par une augmentation de bactéries potentiellement pathogènes versus une diminution de bactéries bénéfiques, est démontré en permanence. De précédentes études ont permis de mettre en évidence la présence de souches pathogènes d’E. coli chez les patients atteints de la maladie de Crohn (MC). Ces souches appartiennent au pathovar Adherent Invasive E. coli (AIEC) et sont caractérisées par leur capacité à adhérer et à envahir les cellules épithéliales intestinales, à survivre et à se multiplier dans les macrophages en induisant une synthèse intense de TNF. La mise en évidence de ce pathovar a essentiellement été réalisée sur des biopsies de patients présentant une MC. Et bien que les mécanismes de pathogénicité et de virulence de la souche AIEC soient clairement déterminés, il n’existe pas d’études approfondies sur la prévalence des AIEC au niveau des matières fécales chez les patients atteints de MICI en comparaison à des individus sains. Ainsi, ce projet de thèse s’inscrit dans une meilleure compréhension de l’implication de ce pathovar AIEC dans les MICI au niveau luminal. Cette thèse cible différents points: prévalence et détection des AIEC, leur proportion relative par rapport à la flore E. coli totale, leur capacité d'invasion, leur phylogroupe ainsi que leur transmissibilité. A l’issue de ce travail, nous montrons que les AIEC sont retrouvés au niveau luminal chez les patients atteints de la MC mais également chez les patients présentant une rectocolite hémorragique, avec une détection des AIEC chez 33% et 2% respectivement. En outre, ces études ont permis de montrer une prévalence plus forte de ce pathovar dans les matières fécales d’individus sains (51%) en comparaison aux patients atteints de MICI. Et lorsque les AIEC sont présents, que ce soit chez les patients atteints de MICI et chez les témoins, ils représentent en moyenne 20 à 30% de la flore E. coli. Nous avons également pu montrer qu’il n’existe pas de différences significatives des scores d’invasion des isolats AIEC chez les patients atteints de MICI et chez les sujets sains. Certaines souches d’AIEC, isolées chez les patients atteints de MC et chez les sujets sains, ont été caractérisées génétiquement par la technique d’électrophorèse sur gel en champ pulsé. Sur ces souches, différents profils génétiques ont été obtenus attestant de la forte variabilité intra- et interindivuelle des AIEC. En conclusion, les AIEC, au vue de leur forte prévalence chez des sujets en bonne santé, seraient plutôt à reconsidérer comme des pathobionts ce qui définit un symbionte pouvant acquérir des propriétés virulentes chez un hôte prédisposé génétiquement en raison de facteurs environnementaux et/ou diététiques et ainsi favoriser l’inflammation intestinale. / Many studies have reported an imbalance of bacterial flora in patients with inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), defined as a dysbiosis, and resulting in an increase in potentially pathogenic bacteria versus a decrease in beneficial bacteria. Previous studies have highlighted the presence of pathogenic strains of E. coli in patients with CD. These strains belong to the pathovar Adherent Invasive E. coli (AIEC) and are characterized by their ability to adhere and invade intestinal epithelial cells, to survive and to multiply in macrophages by inducing an intense synthesis of TNF. In recent years, many studies established a link between AIEC pathovar and CD. Many of these studies have been performed on biopsies of patients with CD. And although the mechanisms of pathogenicity and virulence of the AIEC strain are clearly determined, there are no in-depth studies on the prevalence of AIEC in feces in IBD patients in comparison to healthy individuals. Thus, the goal of this thesis project is to better understand the involvement of AIEC pathovars in IBD at the luminal level. This thesis is based more precisely on the study of the prevalence of AIEC in feces of patients with IBD in comparison to healthy subjects, targeting different points: prevalence and detection of AIEC, their relative proportion compared to total E. coli flora, their invasion capacity, their phylogroup as well as their transmissibility. AIEC are found at luminal level in patients with CD but also in patients with UC, with detection of AIEC in 33% and 2% respectively. In addition, a higher prevalence of these pathovar is present in the feces of healthy individuals (51%) compared to patients with IBD. And when AIEC are present, both in IBD patients and in controls, they represent on average 20 to 30% of the E. coli flora. We have also been able to show that there are no significant differences in AIEC invasion scores in patients with IBD and in healthy subjects. Some AIEC strains, isolated in patients with CD and in healthy subjects, have been genetically characterized by pulsed-field gel electrophoresis. Different genetic profiles have been obtained attesting the high intra- and interindividual variability of AIEC strains. In conclusion, because of their high prevalence in healthy individuals, AIEC should be reconsidered as pathobionts, which defines a symbiont acquiring virulent properties in a genetically predisposed host due to environmental and / or dietary factors and thus promoting intestinal inflammation.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico-chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico¿chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.