Studio della trasduzione del Segnale Nucleare Inositide-Dipendente: identificazione di eEF1A2 come nuovo Fosfosubstrato di PKC βI

Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3). PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic differentiation. Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear localization and relative distribution of eEF1A2. Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS. Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2). We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized at the nucleolar level. eEF1A2 could be phosphorylated in many sites among which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the serine residue of the motif recognized by the antibody that is specifically phosphorylated by PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during myoblasts differentiation.

Aging of nuclear power plant cables: in search of non-destructive diagnostic quantities

The safety systems of nuclear power plants rely on low-voltage power, instrumentation and control cables. Inside the containment area, cables operate in harsh environments, characterized by relatively high temperature and gamma-irradiation. As these cables are related to fundamental safety systems, they must be able to withstand unexpected accident conditions and, therefore, their condition assessment is of utmost importance as plants age and lifetime extensions are required. Nowadays, the integrity and functionality of these cables are monitored mainly through destructive test which requires specific laboratory. The investigation of electrical aging markers which can provide information about the state of the cable by non-destructive testing methods would improve significantly the present diagnostic techniques. This work has been made within the framework of the ADVANCE (Aging Diagnostic and Prognostics of Low-Voltage I\&C Cables) project, a FP7 European program. This Ph.D. thesis aims at studying the impact of aging on cable electrical parameters, in order to understand the evolution of the electrical properties associated with cable degradation. The identification of suitable aging markers requires the comparison of the electrical property variation with the physical/chemical degradation mechanisms of polymers for different insulating materials and compositions. The feasibility of non-destructive electrical condition monitoring techniques as potential substitutes for destructive methods will be finally discussed studying the correlation between electrical and mechanical properties. In this work, the electrical properties of cable insulators are monitored and characterized mainly by dielectric spectroscopy, polarization/depolarization current analysis and space charge distribution. Among these techniques, dielectric spectroscopy showed the most promising results; by means of dielectric spectroscopy it is possible to identify the frequency range where the properties are more sensitive to aging. In particular, the imaginary part of permittivity at high frequency, which is related to oxidation, has been identified as the most suitable aging marker based on electrical quantities.