Spectroscopie électronique de complexes du nickel(II), de lor(I), du ruthénium(II) et de certains lanthanides : caractéristiques inhabituelles de leur structure électronique

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that about 20% of melanomas express major histocompatibility complex (MHC) class II, it is plausible that concomitant antigenic presentation by MHC class I and class II complexes shapes positive (helper T cells) or negative (regulatory T cells) anti-tumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we demonstrated that the gp100 putative amino-terminal signal sequence and the last 70 residues in carboxy-terminus, are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+ endosomal/MIIC. Gp100-targeting sequences were characterized by deleting different sections in the carboxy-terminus (residues 590 to 661). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in carboxy-terminus resulted in decreased MHC class II presentation, without effects on MHC class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100-targeting sequences to mobilize the green fluorescent protein (GFP) to endosomal compartments, and to allow MHC class II and class I presentation of minimal endogenous epitopes. Thus, we concluded that these specific sequences are MIIC targeting motifs. Consequently, these sequences could be included in expression cassettes for endogenously expressed tumor or viral antigens to promote MHC class II and class I presentation and optimize in vivo T cell responses, or as an in vitro tool for characterization of new MHC class II epitopes.

Complexes pinceurs de type diphosphinito (POCOP) de Ni(II) / Ni(III)

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The Independence of CXCR4’s Pathways, Gαi and β-Arrestin2, and Their Modulation by AMD3100 and TC14012

CXCR4, a chemokine receptor involved in metastasis and homing of hematopoietic stem cells, signals through two major pathways: Gαi and β-arrestin2. β-arrestin2 terminates G-protein signaling and targets the receptor to endocytosis. This project proposed to study the effect of a previously described set of CXCR4 mutants on both these signaling pathways, as well as their localization. These mutants were assayed by different Bioluminescence Resonance Energy Transfer (BRET) systems. Using these systems, we confirmed that N119S is a constitutively active mutant (CAM), spontaneously activating Gαi. As well, we found that R134A is a constitutively inactive mutant (CIM), devoided of G-protein signaling, but spontaneously recruiting β-arrestin2. In addition, we studied the dependency of β-arrestin2 recruitment on the Gαi activity. By targeting R134A and N119S with pertussis toxin, an inhibitor of the Gαi activation, we showed efficient blocking of the Gαi pathway, while maintaining the constitutive recruitment of β-arrestin2. This demonstrated that for CXCR4, β-arrestin2 recruitment is independent of the Gαi pathway. Finally, two synthetic ligands of CXCR4, AMD3100 and TC14012 were tested for their ability to recruit β-arrestin2. AMD3100 is a clinically approved drug used for stem cell transplantation, with considerable side effects. We found it to be an antagonist on both Gαi and β-arrestin2 recruitment. On the other hand, TC14012 was found to be an inverse agonist on Gαi and an antagonist on β-arrestin2 recruitment. Based on this finding, it would be preferable to use of TC14012 as it will further reduce any basal Gαi activity, without affecting β-arrestin2 recruitment. These results support the development of TC14012 for stem cell mobilization trials.