Abstract This study utilized normal and defective epithelial cell cultures and epidermal skin samples to examine intra- and intercellular calcium signaling. The main interests of this thesis were Hailey-Hailey disease (HHD), Darier disease (DD), neurofibromatosis 1 (NF1) and transitional cell carcinoma (TCC). HHD and DD diseases are rare autosomal dominant skin disorders characterized by dissociation of epidermal keratinocytes (acantholysis) at the suprabasal layer of the epidermis. HHD and DD diseases are caused by mutations in the genes encoding the calcium pumps in the Golgi apparatus (hSPCA1) and endoplasmic reticulum (SERCA2b), respectively. Due to these mutations calcium uptake into the Golgi apparatus or ER is diminished, which is believed to cause abnormal cell junction protein processing and dissociation of keratinocytes. This study utilized electron probe microanalysis (EPMA) and demonstrated for the first time that lesional areas of HHD and DD and non-lesional areas of DD epidermis display abnormally low calcium content in the basal cell layer. Furthermore, ATP mediated calcium signaling was impaired in cultured HHD and DD keratinocytes and epidermal ATP receptor localization was disrupted. In conclusion, these results suggest that the low calcium content in the basal cell layer is the reason for suprabasal ruptures in HHD but not necessarily in DD lesions, and that abnormal ATP receptor localization contributes to the calcium signaling defects. NF1 deficient keratinocytes display abnormally low resting cytosolic calcium levels and it has been suggested that the calcium concentration in the lumen of the endoplasmic reticulum is decreased. This study demonstrated that NF1 keratinocytes rely mostly on ATP mediated calcium signaling while normal keratinocytes rely mostly on gap junctional intercellular communication (GJIC). Studies with TCC cells have demonstrated that gap junctions participate in intercellular calcium wave propagation. This thesis demonstrated that the ATP mediated pathway was also operational in calcium wave propagation in normal uroepithelial and TCC cell cultures. Furthermore, impaired calcium wave propagation in the TCC cell culture could be improved through PKC α/βI –isoenzyme inhibition with Gö6976. Gö6976 treatment increased connexin 26 clustering at plasma membrane but did not alter expression level of the protein. This thesis contains a wide repertoire of calcium detection techniques including a new cutting-edge technology for elemental calcium detection of epidermal samples. These techniques can be used for molecular specific analysis of calcium signaling in epithelial cells.

La mesure des effets des éléments en traces métalliques (ETM) sur les microorganismes, acteurs majeurs du fonctionnement des sols, constitue une approche complémentaire des analyses physico-chimiques dans l'évaluation de leur impact éco-toxicologique. La démarche scientifique présentée ici visait à déterminer dans quelle mesure la diversité, l'abondance et l'expression de gènes bactériens de résistance aux métaux et métalloïdes pourraient constituer des biomarqueurs pertinents des concentrations biodisponibles en ETM dans les sols contaminés et de leur impact sur les communautés bactériennes. Dans cette étude, nous nous sommes focalisés sur les gènes de résistance arsB/ACR3(1) codant les pompes d'efflux de l'arsénite, largement répandues chez les procaryotes, et aioA codant une arsénite-oxydase. Une technique de PCR en temps réel, utilisant des amorces dégénérées conçues au cours de travaux de thèse précédents (Achour et al., 2007 ; Quéméneur et al., 2008), a été développée pour quantifier ces gènes cibles et leurs transcrits dans des systèmes d'étude de plus en plus complexes, de la culture liquide aux microcosmes de sols. Cette recherche d'interprétations physiologiques et écologiques des réponses bactériennes à une exposition à l'arsenic a été intégrée dans le projet ANR Multipolsite (ANR CES 2008). Il a dès lors été possible d'étudier, dans un contexte d'atténuation naturelle et de phytoremédiation, l'abondance des gènes arsB, ACR3(1) et aioA in situ et sur le long terme en fonction de différents traitements telles que la présence de végétation et la désorption-thermique de ce sol multi-contaminé aux HAP et aux ETM. Ces travaux ont également abouti à la conception d'amorces dégénérées ciblant les gènes de résistance aux cadmium, zinc et plomb, gènes codant les ATPases de type PIB étant des pompes d'efflux spécifiques de ces ETM / Arsenic is a widespread toxic metalloid which is a major issue of public health. Its presence in the environment is naturally due to the geochemical background, i.e. the weathering of parent material and volcanic eruptions, but the main contamination sources are anthropogenic activities such as mining and metalworking industry. We describe a real-time PCR assay for the quantitative detection of arsB and ACR3(1) arsenite transporter gene families, two ubiquitous and key determinants of arsenic resistance in prokaryotes. The aioA gene encoding the large subunit of arsenite-oxidase was monitored in parallel. This study aimed to determine whether diversity, abundance and expression of these arsenite efflux pumps could serve as suitable biomarkers of metalloid stress and provide means to assess the impact of contamination on soil bacterial communities. The assay was applied in batch growth experiments using a wasteland soil bacterial community as an inoculum to investigate the effect of increasing arsenic concentrations on genes and transcripts abundances. To confirm previous results, further studies on the abundance and expression of arsB and ACR3(1) in indigenous soil bacterial communities exposed to different levels of arsenic over various time periods have helped to gain a better understanding of how these genes contribute to the adaptation of the communities to arsenic stress and their role in shaping the community structure and diversity. On the other hand, metal transporting PIB-type ATPases are critical components of bacterial resistance to cadmium, zinc and lead. We therefore designed degenerate PCR primers targeting PIB-type ATPases and tested their specificity on reference strains, metal-resistant soil isolates and soil metagenomic DNA

Major histocompatibility complex molecules (MHCII) are cell surface glycoproteins that present extracellular antigens to CD4+ T lymphocytes and initiate adaptive immune responses. Apart from their protective role, overexpression of MHCII contributes to autoimmune disorders where the immune system attacks our own tissues. Autoimmune diseases are characterized by self-reactive responses to autoantigens, promoting tissue damage, inflammation mediated by proinflammatory cytokines, autoreactive lymphocytes, and autoantibodies. MHCII molecules are tightly regulated at the level of transcription by Class II transactivator (CIITA). CIITA associates with an enhanceosome complex at MHCII promoters and regulates the expression of MHCII. It is thus crucial to understand the regulation of CIITA expression in order to regulate MHCII in autoimmune diseases. Our lab has shown that the 19S ATPases of the 26S proteasome associate with MHCII and CIITA promoters and play important roles in gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear. Here, we define novel roles of the 19S ATPases Sug1, S7, and S6a in expression of CIITApIV genes. These ATPases are recruited to CIITApIV promoters and coding regions, interact with the elongation factor PTEFb, and with Ser5 phosphorylated RNA Pol II. Both the generation of CIITApIV transcripts and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by knockdown of 19S ATPases. Alternatively, inflammation is also suppressed via the Regulator of G-protein signaling 10 (RGS10) in microglial cells which express high levels of RGS10 and promote homeostasis in the central nervous system. However, chronic activation of microglial cells leads to release of cytokines which cause neuroinflammation. Our investigation of roles played by RGS10 in chronically activated microglial cells indicates that RGS10 binds to promoters of IL-1β, and TNF-α and regulates these genes, while the molecular mechanism remains to be investigated. Together, our observations indicate roles for the UPS in modulating gene expression and for RGS10 in regulating proinflammatory cytokines in microglial cells, each of which provides novel therapeutic targets to combat inflammation in autoimmune and neurodegenerative diseases.

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