Microgravity and immune system: phosphorylative modifications related to the recognition of apoptotic cells by phagocytes in simulated microgravity

This thesis deals with experiments on phosphorylative changes in peripheral blood mononuclear cell cultures grown under simulated microgravity conditions and in normal conditions. Proteomic techniques have been used to evaluate phosphorylative post-translational modifications and to compare the protein pattern of cells grown in a microgravity environment simulated by the Random Positioning Machine. Monodimensional electrophoresis was used to visualize the proteins of interest obtained from peripheral blood mononuclear cells; Blue Coomassie staining was performed to visualize proteins. MALDI-ToF mass spectrometry was also employed to characterize the same proteins. Western Blotting with specific antibodies: antiphosphotyrosine and anti-Syk was prepared to study phosphorylative differences between cells grown in normal conditions (1xg), those grown under simulated microgravity (0xg) and activated by a gravisensitive mitogen such as Concanavalina A.Microgravity seems to influence monocytes with an increase of tyrosine phosphorylations of several proteins; also T lymphocytes and monocytes proteins are extensively modified after Con A, particularly after Random Position Machine exposition. Treatment in the presence of Syk inhibitors decreases the phosphorylation levels in all conditions. Mass spectrometry analysis revealed that some phosphorylated proteins belonged to the class of cytoskeletol proteins. Moreover other identified proteins are HSPs, typically abundant in stress conditions.