Biochemical, computer, and spectroscopic techniques applied to the study of prions and of combinations of antineoplastic drugs

This thesis collects the work I have done during the three-year PhD Course. The results obtained are divided according to the research topics addressed: · Drug discovery of anticancer agents and development of synergistic associations (Part I); · Studies on the prion structure and the pathogenesis of prion diseases (Part II). Studies referring to the Part I have been carried out at the University of Cagliari and were focalized on the evaluation and experimental validation of the method known as Artificial Neural Network (ANN), which allows to determine, and also to predict, types and degree of interaction of two or more drugs in combination with one another(s). I have successfully applied the ANN approach to combinations of two cytotoxic compounds, i.e. cis-platinum (CDDP), a potent antineoplastic used in the therapy of some types of carcinoma, with Cu(II) complexes that were previously shown to be endowed with potent cytotoxic activity in vitro (Pivetta et al., J Inorg Biochem. 2011 and 2012). Binary combinations comprising CDDP and Cu(II) complexes revealed (Part I, Chapter I) a strong synergistic cytotoxic effect against leukemia cell lines (Pivetta et al., Talanta 2013). The synergistic effect was confirmed in CDDP-resistant leukemia and ovarian cancer cell lines (Part I, Chapter II). Investigations on molecular bases of the CDDP – Cu(II) complexes synergism are in progress. As for the Part II of my research, I firstly investigated the mechanisms underlying the process of generation of pathogenic prion protein (PrPSc). These studies were carried out in part at the University of Cagliari, and in part at the Rocky Mountains Laboratories (RML) of the NIAID/NIH in USA (Hamilton, Montana), during my 14-month research internship as supplemental visiting fellow in a graduate partnership program. The studies carried out in Cagliari have investigated 1H NMR modifications of brain metabolite profiles in sheep from a Sardinian farm hit by natural Scrapie with the aim to discriminate infected vs. Uninfected, and early vs. late phases of the prion infection (Part II, Chapter I). The overall results, obtained by different chemometric tools, were able to describe a metabolite profile of scrapie-infected sheep brain, with and without clinical sign, different to that of healthy ones, and to suggest Ala as a biomarker of PrPSc deposition (Scano et al., J Molecular Biosystem, 2015) Studies conducted at the RML of NIAID/NIH have addressed the structural features of PrPSc in order to identify regions of the protein involved in the process of misfolding (Part II, Chapter II). So far, data suggest that i) the N-terminal portion of the prion protein may have a role in the modulation of the protein aggregation process, typical of prion diseases, while ii) the central portion seems capable to undergo aggregation into fibrils even in the absence of other regions of the protein. However, further studies are needed to confirm the proposed role in promoting/hindering the misfolding and subsequent aggregation process of the various protein regions.