Short term training abroad on lymphocytic Choriomeningitis virus

• Grant number: 13/16550-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: October 01, 2013
• End date: November 30, 2013
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: João Gustavo Pessini Amarante Mendes
• Grantee: Narciso Junior Vieira
• Supervisor: Pamela S. Ohashi
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Toronto (U of T), Canada
• Associated to the scholarship: 11/20352-3 - Role of rab2a, rab5a, rab17 and rab18 in effector function of cytotoxic cells, BP.DD

Abstract

At this point of my PhD project, we have already characterized the presence of RAB members in cytotoxic granules by confocal as well as electron microscopy. Consequently, we want to carry out functional assays in wild-type and RAB-deficient cytotoxic cells. To obtain RAB-deficient cells, we will use modern techniques of molecular and cellular biology such as iRNA, to construct shRNA vectors and develop stable natural killer (NK) and/or CD8 T lymphocytes (CTL) lines. To address the importance of our proteins in cytotoxic killing, we will compare vector and shRNA-transfected cells in in vitro and, if possible, in in vivo cytotoxic assays. To accomplish these tasks, we will have the support of Prof. Pamela Ohashi and her group. Prof. Ohashi agreed to receive me at Ontario Cancer Institute in Toronto, Canada, where I can learn their well-established model of infection with lymphocytic choriomeningitis virus (LCMV) and related techniques and, therefore, develop part of my PhD project. The LCMV, a natural pathogen in mice, is an excellent tool widely used in studies of antiviral immune responses, particularly mediated by CTLs. Dominant and subdominant epitopes have been characterized and the frequency of epitope-specific CTL can be easily evaluated using the technology of MHC multimers, which are oligomeric forms of MHC molecules loaded with specific desired antigenic peptide. Based on these characteristics, this infection model provides an incredible platform for biological studies of cytotoxic cells. Among the academic gains that this short term training project will provide, we can highlight the learning of a new methodology/experimental system to evaluate the behavior of cytotoxic cells in vivo and in vitro by using LCMV that we will subsequently transfer to our lab, expanding the potential of the research developed by our group. Part of the training includes proper handling of the virus in order to ensure student safety and to ensure optimal conditions of culture, in addition to growth and titration of microorganism. Moreover, I will be trained to conduct in vitro cytotoxicity assays and flow cytometry staining with using the tetramer labeling to evaluate antigen-specific T cell responses. Finally, I will have the opportunity to participate in scientific meetings held by Prof. Ohashi's group and the Institute's seminars, as well as to discuss with Prof. Ohashi the different aspects of my PhD project. This collaboration will therefore provide specific tools and expertise that will directly influence the development of my PhD project and the experience to work as part of such an important international research group will allow an intellectual and personal development. (AU)