PROTEIN TYROSINE NITRATION UNDER PHYSIOLOGICAL CONDITIONS IN CELLULAR AND ANIMAL MODELS

The significance of NO2Tyr in vivo is highlighted by observations that nitrated proteins are markedly elevated in a broad range of human diseases and clinical disorders. The presence of nitrated tyrosine residues was detected in human fluids and pathological tissues such as atherosclerotic plaques of coronary vessels, amyotrophic lateral sclerosis, and Alzheimer’s lesions, among many others. Although the accumulation of nitrated proteins correlates well with many disease states and is considered a marker of oxidative stress under pathological conditions, substantial evidence has accrued that protein tyrosine nitration is a post-translational modification playing a role in physiological processes, including signal transduction, neuronal differentiation, and embryonic development. On this regard, the aim of the research presented in this PhD thesis is to better understand the significance of protein nitration under normal physiological conditions focusing on differentiation and developmental processes. The PhD thesis is divided in four parts. The first introductory part is dedicated to presenting the biological functions of nitric oxide and its cellular effects in biological system. Particular attention has been devoted to protein tyrosine nitration and its pathological and physiological significance. Two different experimental models (cells and organism) have been used to investigate this issue: (a) a cellular model (PC12 cells) to discuss the effects of micro- and nanoscale topography on neuronal proliferation and differentiation and (b) an animal model (Ciona intestinalis) for studying the role of oxidative stress and NO-derived reactive nitrogen species (RNS) during Ciona development and metamorphosis-related events. In particular, as far as the first model is concerned, my studies were directed to the characterization of PC12 cells behavior on nanostructured TiO2 films in the presence and in the absence of the classical inducer of differentiation NGF. In the second part of the thesis I present the experimental procedures used. Proteomics techniques, including mono- and two-dimensional electrophoresis, electroblotting and immunostaining, and mass spectrometry (MALDI-TOF and LTQ-Orbitrap Velos) have been used to study both experimental models. In the third part the results obtained are reported. Our findings suggest that tyrosine nitration is a physiological event not necessary related to pathological processes and that this NO-mediated post-translational modification of proteins may be regarded as a direct way to NO-signaling transduction. Finally, in the last part I resume the main conclusions and present future perspectives.