Mutations in OPA1 gene have been identified in the majority of patients with Dominant Optic Atrophy (DOA), a blinding disease, and the syndromic form DOA-plus. OPA1 protein is a mitochondrial GTPase involved in various mitochondrial functions, present in humans in eight isoforms, resulting from alternative splicing and proteolytic processing. In this study we have investigated the specific role of each isoform through expression in OPA-/- MEFs, by evaluating their ability to improve the defective mitochondrial phenotypes. All isoforms were able to rescue the energetic efficiency, mitochondrial DNA (mtDNA) content and cristae integrity, but only the presence of both long and short forms could recover the mitochondrial morphology. In order to identify the OPA1 protein domains crucial for its functions, we selected and modified the isoform 1, shown to be one of the most efficient in preserving mitochondrial phenotype, to express three specific OPA1 variants, namely: one with a different N-terminus portion, one unable to generate short form owing to deletion of S1 cleavage site and one with a defective GTPase domain. We demonstrated that the simultaneous presence of the N- and C-terminus of OPA1 was essential for the mtDNA maintenance; a cleavable isoform generating s-forms was necessary to completely rescue the energetic competence and the presence of the C-terminus was sufficient to partially recover the cristae ultrastructure. Lastly, several pathogenic OPA1 mutations were inserted in MEF clones and the biochemical features investigated, to correlate the defective phenotypes with the clinical severity of patients. Our results clearly indicate that this cell model reflects very well the clinical characteristics of the patients, and therefore can be proposed as an useful tool to shed light on the pathomechanism underlying DOA.

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

An increasing number of scientific evidence supports the preventive value of dietary patterns that favor the consumption of plant food. Especially fruit, vegetables, grains and legumes; the correlation between the reduction of risk of chronic disease such as adherence to the Mediterranean diet is the most significant example. The central role of the endothelium in maintaining vascular homeostasis and the correlation between endothelial dysfunction and the development of cardiovascular diseases makes this tissue a primary target for dietary strategies aimed at cardiovascular diseases prevention. Research in the field of nutrition is therefore directed to the identification of food bioactive components with beneficial effects on the endothelium. This study first focused on the evaluation of the potential vascular protective effects of a wheat peptide belonging to the family of non-specific lipid transfer proteins type 2 (nsLTP2). nsLTP2, at physiological concentrations, showed antioxidant and cytoprotective effects in HUVECs undergoing oxidative/inflammatory stimulation and demonstrated modulatory capacity on the expression of adhesion molecules and heme oxygenase-1, both involved in endothelial inflammation. Polyphenols are widely studied antioxidant compounds and research supports the preventive/protective role of a polyphenol-rich diet. Despite experimental evidence of their positive influence on human health, to date there is no clear indication of the compunds responsible for this protective role. In fact, upon ingestion polyphenols are extensively metabolized and the molecule that will act at cellular level will more likely be a metabolite. For this reason the second part of the study focused on the protective effect of polyphenol metabolites belonging to two families: cinnamic acids and anthocyanins. Overall the tested compounds demonstrated antioxidant and cytoprotective activities at endothelial level in oxidative/inflammatory conditions, being also able to affect adhesion molecules expression. These observations may support and characterize biological activities of bioactive peptides and polyphenols metabolites beneficial to vascular health.

Cytochrome b is the only subunit of complex III (CIII) encoded by the mitochondrial DNA. Constituting the central core of the enzyme, the protein is essential for both assembly and catalytic activity of the complex. CIII can associate with complex I (CI) and complex IV to form supercomplexes (SCs). MTCYB mutations can affect CIII only or both CIII and CI, as a consequence of the importance of CIII on the stability of CI. Here, we have investigated the effects of two pathogenic mutations affecting MTCYB: the p.278Y>C missense mutation, causing the substitution of conserved Tyr278 close to the QO site, and the ΔI300-P305 microdeletion, producing the loss of six aminoacids in the sixth transmembrane helix, but leaving the remaining of the MTCYB in frame. We have demonstrated that both MTCYB mutations severely impaired the activity of CIII: the missense mutation produced an oxidative damage of CIII due to increased superoxide production, whereas in cells bearing the ΔI300-P305 microdeletion, CIII was not detected, with consequent derangement also of CI. The detailed analysis of SCs organization revealed in both cases a strong perturbation of the CIII2+IV SC, together with an attempt to preserve the respirasome. These results favor the hypothesis that SCs not only preserve the structure and stability of respiratory complexes, but are essential for attenuating the mitochondrial dysfunction due to pathogenic mutations affecting the respiratory enzymes. Furthermore, the cells bearing ΔI300-P305 deletion showed a marked increase in complex II (CII) redox activity, associated with significant hydrogen peroxide production. It has been suggested that the enhanced CII activity is a compensatory mechanism due to the lacking of CI. Our results instead suggest that it might be a more general phenomenon for cell adaptation to respiratory chain dysfunction, being detected also in CIII-deficient cells where the hydrogen peroxide production is increased.

Several studies have reported beneficial effects of autophagy in preventing chondrocyte death, OA-like changes in gene expression and cartilage degeneration. Many microRNAs have been identified as key modulators of autophagy pathway. So far, to our knowledge no relationship has been revealed between nutraceuticals and microRNA network in OA. First aim of this thesis is to evaluate molecular mechanisms of action of hydroxytyrosol (HT) a promising compound already tested for protective efficacy in OA chondrocytes. HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires SIRT-1, which accumulated in the nucleus following HT treatment. Second aim consists in identifying a microRNA implicated in HT-mediated protective response to oxidative stress and examining the effects after modulation of miR levels by approach of transient transfection. After in silico analysis we identify miR-9 as a speculative candidate able to target SIRT-1 and confirm this hypothesis by means of luciferase gene-reporter assay. Moreover miR-9 mediates cell death induced by H2O2 and the protective effect of HT, as observed in human primary chondrocytes and C/28-I2 cell line. Third aim is to investigate the potential role of miR-155, found to be one of the most highly upregulated microRNAs in human OA knee cartilage, in autophagic pathway. Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy. The identification of a single microRNA, involved simultaneously in several disease-related pathways, discloses a potent therapeutic target. Indeed the unveiling of bioactive compounds, exerting a beneficial effect through induction of epigenetic changes, may open a new topic of research not yet well explored.

DNA elongation is performed by Pol III α subunit in E. coli, stimulated by the association with ε and θ subunits. These three subunits define the DNA Pol III catalytic core. There is controversy about the DNA Pol III assembly for the simultaneous control of lagging and leading strands replication, since some Authors propose a dimeric model with two cores, whereas others have assembled in vitro a trimeric DNA Pol III with a third catalytic core, which increases the efficiency of DNA replication. Moreover, the function of the PHP domain, located at the N-terminus of α subunit, is still unknown. Previous studies hypothesized a possible pyrophosphatase activity, not confirmed yet. The present Thesis highlights by the first time the production in vivo of a trimeric E. coli DNA Pol III by co-expressing α, τ, ε and θ subunits. This trimeric complex has been enzymatically characterized and a molecular model has been proposed, with 2 α subunits sustaining the lagging-strand replication whereas the third core replicates the leading strand. In addition, the pyrophosphatase activity of the PHP domain has been confirmed. This activity involves, at least, the H12 and the D19 residues, whereas the D201 regulates phosphate release. On the other hand, an artificial polymerase (HoLaMa), designed by deleting the exonuclease domain of Klenow Fragment, has been expressed, purified and characterized for a better understanding of bacterial polymerases mechanism. The absence of exonuclease domain impaired enzyme processivity, since this domain is involved in DNA binding. Finally, Klenow enzyme, HoLaMa, α subunit and DNA Pol III αεθ have been characterized at the single-molecule level by FRET analysis, combining ALEX and TIRF microscopy. Fluorescently-labeled DNA molecules were immobilized, and changes in FRET efficiency enabled us to study polymerase binding and DNA polymerization.

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The research is focused on the relationship between some Mg2+-dependent ATPase activities of plasma- and mitochondrial membranes from tissues of cultured marine bivalve molluscs and potentially stressful environmental conditions, such as the exposure to contaminants both of natural origin (ammonia nitrogen, the main contaminant of aquaculture plants) and of anthropic source (alkyltins). The two filter-feeding bivalve species selected colonize different habitats: the common mussel Mytilus galloprovincialis binds to hard substrates and the Philippine clam Tapes philippinarum burrows into sea bottom sandy beds. The choice of typical species of coastal waters, extremely suitable for environmental studies due to their features of poor motility, resistance to transport and great filtering efficiency, may constitute a model to evaluate responses to contaminants of membrane-bound enzyme activities involved in key biochemical mechanisms, namely cell ionic regulation and mitochondrial energy production. In vitro and in vitro approaches have been pursued. In vitro assays were carried out by adding the contaminants (NH4Cl and alkyltins) directly to the ATPase reaction media. In vivo experiments were carried out by exposing mussels to various tributyl tin (TBT) concentrations under controlled conditions in aquaria. ATPase activities were determined spectrophotometrically according to the principles of the method of Fiske and Subbarow (1925). The main results obtained are detailed below. In Tapes philippinarum the interaction of NH4 +, the main form of ammonia nitrogen at physiological and seawater pHs, with the Na,K-ATPase and the ouabaininsensitive Na-ATPase was investigated in vitro on gill and mantle microsomal membranes. The proven replacement by NH4 +of K+ in the activation of the Na,KATPase and of Na+ in the activation of the ouabain-insensitive ATPase displayed similar enzyme affinity for the substituted cation. on the one hand this finding may represent one of the possible mechanisms of ammonia toxicity and, on the other, it supports the hypothesis that NH4 + can be transported across the plasma membrane through the two ATPases. In this case both microsomal ATPases may be involved and co-operate, at least under peculiar circumstances, to nitrogen excretion and ammonia detoxification mechanisms in bivalve molluscs. The two ATPase activities stimulated by NH4 + maintained their typical response to the glycoside ouabain, specific inhibitor of the Na,K-ATPase, being the Na++ NH4 +-activated ATPase even more susceptive to the inhibitor and the ouabain-insensitive ATPase activity activated indifferently by Na+ or NH4 + unaffected by up to 10-2 M ouabain. In vitro assays were carried out to evaluate the response of the two Na-dependent ATPases to organotins in clams and mussels and to investigate the interaction of TBT with mussel mitochondrial oligomycin-sensitive Mg-ATPase. Since no literature data were available, the optimal assay conditions and oligomycin sensitivity of mussel mitochondrial MgATPase were determined. In T. philippinarum the ouabain-insensitive Na-ATPase was found to be refractory to TBT both in the gills and in the mantle, whereas the Na,K-ATPase was progressively inhibited by increasing TBT doses; the enzyme inhibition was more pronounced in the gills than in the mantle. In both tissues of M. galloprovincialis the Na,K-ATPase inhibition by alkyltins decreased in the order TBT>DBT(dibutyltin)>>MBT(monobutyltin)=TeET(tetraethyltin) (no effect). Mussel Na-ATPase confirmed its refractorimess to TBT and derivatives both in the gills and in the mantle. These results indicate that the Na,K-ATPase inhibition decreases as the number of alkyl chains bound to tin decreases; however a certain polarity of the organotin molecule is required to yield Na,K-ATPase inhibition, since no enzyme inhibition occurred in the presence of tetraalkyl-substituted derivatives such as TeET . Assays carried out in the presence of the dithioerythritol (DTE) pointed out that the sulphhydrylic agent is capable to prevent the Na,K-ATPase inhibition by TBT, thus suggesting that the inhibitor may link to -SH groups of the enzyme complex.. Finally, the different effect of alkyltins on the two Na-dependent ATPases may constitute a further tool to differentiate between the two enzyme activities. These results add to the wealth of literature data describing different responses of the two enzyme activities to endogenous and exogenous modulators . Mussel mitochondrial Mg-ATPase was also found to be in vitro inhibited by TBT both in the gills and in the mantle: the enzyme inhibition followed non competitive kinetics. The failed effect of DTE pointed out that in this case the interaction of TBT with the enzyme complex is probably different from that with the Na,K-ATPase. The results are consistent with literature data showing that alkyltin may interact with enzyme structures with different mechanisms. Mussel exposure to different TBT sublethal doses in aquaria was carried out for 120 hours. Two samplings (after 24 and 120 hrs) were performed in order to evaluate a short-term response of gill and mantle Na,K-ATPase, ouabain-insensitive Na-ATPase and Mg-ATPase activities. The in vivo response to the contaminants of the enzyme activities under study was shown to be partially different from that pointed out in the in vitro assays. Mitochondrial Mg-ATPase activity appeared to be activated in TBTexposed mussels with respect to control ones, thus confirming the complexity of evaluating in vivo responses of the enzyme activities to contaminants, due to possible interactions of toxicants with molluscan metabolism. Concluding, the whole of data point out that microsomal and mitochondrial ATPase activities of bivalve molluscs are generally responsive to environmental contaminants and suggest that in some cases membrane-bound enzyme activities may represent the molecular target of their toxicity. Since the Na,K-ATPase, the Na-ATPase and the Mg-ATPase activities are poorly studied in marine bivalves, this research may contribute to enlarge knowledge in this quite unexplored field.