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Pentameric ligand-gated ion channel mediate signal transduction at chemical synapses by transiting between resting and open states upon neurotransmitter binding. During my thesis, I investigate the gating transition of the glycine receptor fluorescently labeled at the extracellular domain region and extracellular-transmembrane interface region by voltage-clamp fluorimetry (VCF). The screening of the several pairs of cysteine and tryptophan results in the discovery of two interesting pair: a top extracellular pair and an interface region pair. Fluorescence of the interface pair reports a glycine-elicited conformational transition that precedes pore opening. Low concentrations of glycine, partial agonists or specific mixtures of glycine and strychnine trigger the full fluorescence signal while weakly activating the channel. Molecular dynamic simulations of a partial agonist bound-closed Cryo-EM structure show a highly dynamic personality: a marked structural flexibility at both the extracellular-transmembrane interface and the orthosteric site, generating docking properties that recapitulate VCF data. Data thus illuminate a progressive gating transition towards activation, displaying structural plasticity with novel implication concerning the mechanism of action of allosteric effectors. Fluorescence of the top extracellular pair seems to report a conformational reorganization correlated to the activation and the desensitization of the receptor. Further investigation must be done to elicit what exactly report the pair localized in the extracellular region.; Les récepteurs canaux pentamériques assurent la transduction du signal au niveau des synapses chimiques en transitant entre les états de repos et d'ouverture lors de la liaison des neurotransmetteurs. Dans ma thèse, j'étudie le cycle d'activation du récepteur à la glycine par la méthode de voltage-clamp fluorométrie (VCF) en marquant le domaine extracellulaire et la région d'interface extracellulaire-transmembranaire par des fluorophores. Le criblage de plusieurs paires de cystéine et de tryptophane aboutit à la découverte de deux paires intéressantes : une paire extracellulaire et une paire de régions d'interface. La fluorescence de la paire d'interface met en évidence une transition conformationnelle provoquée par la glycine qui précède l'ouverture du canal. De faibles concentrations de glycine, des agonistes partiels ou des mélanges spécifiques de glycine et de strychnine entraine un variation de fluorescence complète tout en activant faiblement le canal. Les simulations de dynamique moléculaire d'une structure Cryo-EM fermée liée à un agoniste partiel montrent une caractéristique de haute dynamique de la structure: une flexibilité structurelle marquée à la fois à l'interface extracellulaire-transmembranaire et au site orthostérique, générant des propriétés qui récapitulent les données VCF. Les données éclairent ainsi une transition progressive vers l'activation du récepteur, montrant une plasticité structurelle impliquant différents mécanismes d'action des différents effecteurs allostériques. La fluorescence de la paire extracellulaire semble rendre compte d'une réorganisation conformationnelle du récepteur corrélée à l'activation et à la désensibilisation. Une étude plus approfondie est nécessaire pour déterminer ce que rapporte exactement la paire localisée dans la région extracellulaire.

Natural killer cells (NK) are one of the key players in the eradication and control of viral infections. Infections with the hepatitis B virus (HBV) may lead to persistence in a subgroup of patients, and impaired NK cell functions have been observed in these patients. Crosstalk with other immune cells has been shown to modulate the function of NK cells. We studied the functional crosstalk between NK cells and plasmacytoid dendritic cell (pDC) and its modulation by HBV. Healthy human peripheral bloodderived NK cells and pDC were purified and cocultured in the presence or absence of HepG2.2.15-derived HBV under various in vitro conditions. The functionality of NK cells was assessed by evaluation of activation markers, cytokine production and cytotoxicity of carboxyfluorescein succinimidyl ester-labelled K562 target cells by flow cytometry or immunoassays. Additionally, the crosstalk was examined using NK and pDC from patients with chronic HBV. The activation of NK cells in cocultures with pDC, as demonstrated by CD69, CD25 and HLA-DR, was not affected by the presence of HBV. Similarly, when cocultured with pDC, the cytotoxic potential of NK cells was not influenced by HBV. However, HBV significantly inhibited pDC-induced IFN-? production by NK cells both in the presence and in the absence of CpG. As HBV did not affect cytokine-induced IFN-? production by NK cells cultured alone, the suppressive effect of HBV on NK cell function was mediated via interference with pDCNK cell interaction. In contrast to other viruses, HBV does not activate pDCNK cell interaction but inhibits pDC-induced NK cell function. In parallel with NK cells of patients with chronic HBV, which show diminished cytokine production with normal cytotoxicity, HBV specifically suppressed pDC-induced IFN-? production by NK cells without affecting their cytolytic ability. These data demonstrate that HBV modulates pDCNK cell crosstalk, which may contribute to HBV persistence.