This thesis was aimed at investigating the physical-chemical properties and the behaviour in physiological environment of two classes of bioceramics: calcium silicate-based dental cements and alumina-based femoral heads for hip joint prostheses. The material characterization was performed using spectroscopic techniques such as that allow to obtain information on the molecular structure of the species and phases present in the analyzed samples. Raman, infrared and fluorescence spectroscopy was principally used. Calcium silicate cements, such as MTA (Mineral Trioxide Aggregate), are hydraulic materials that can set in presence of water: this characteristic makes them suitable for oral surgery and in particular as root-end filling materials. With the aim to improve the properties of commercial MTA cements, several MTA-based experimental formulations have been tested with regard to bioactivity (i.e. apatite forming ability) upon ageing in simulated body fluids. The formation of a bone-like apatite layer may support the integration in bone tissue and represents an essential requirement for osteoconduction and osteoinduction. The spectroscopic studies demonstrated that the experimental materials under study had a good bioactivity and were able to remineralize demineralized dentin. . Bioceramics thanks to their excellent mechanical properties and chemical resistance, are widely used as alternative to polymer (UHMWPE) and metal alloys (Cr-Co) for hip-joint prostesis. In order to investigate the in vivo wear mechanisms of three different generations of commercial bioceramics femoral heads (Biolox®, Biolox® forte, and Biolox® delta), fluorescence and Raman spectroscopy were used to investigate the surface properties and residual stresses of retrieved implants. Spectroscopic results suggested different wear mechanisms in the three sets of retrievals. Since Biolox® delta is a relatively recent material, the Raman results on its retrievals has been reported for the first time allowing to validate the in vitro ageing protocols proposed in the literature to simulate the effects of the in vivo wear.

Bioinformatics, in the last few decades, has played a fundamental role to give sense to the huge amount of data produced. Obtained the complete sequence of a genome, the major problem of knowing as much as possible of its coding regions, is crucial. Protein sequence annotation is challenging and, due to the size of the problem, only computational approaches can provide a feasible solution. As it has been recently pointed out by the Critical Assessment of Function Annotations (CAFA), most accurate methods are those based on the transfer-by-homology approach and the most incisive contribution is given by cross-genome comparisons. In the present thesis it is described a non-hierarchical sequence clustering method for protein automatic large-scale annotation, called “The Bologna Annotation Resource Plus” (BAR+). The method is based on an all-against-all alignment of more than 13 millions protein sequences characterized by a very stringent metric. BAR+ can safely transfer functional features (Gene Ontology and Pfam terms) inside clusters by means of a statistical validation, even in the case of multi-domain proteins. Within BAR+ clusters it is also possible to transfer the three dimensional structure (when a template is available). This is possible by the way of cluster-specific HMM profiles that can be used to calculate reliable template-to-target alignments even in the case of distantly related proteins (sequence identity < 30%). Other BAR+ based applications have been developed during my doctorate including the prediction of Magnesium binding sites in human proteins, the ABC transporters superfamily classification and the functional prediction (GO terms) of the CAFA targets. Remarkably, in the CAFA assessment, BAR+ placed among the ten most accurate methods. At present, as a web server for the functional and structural protein sequence annotation, BAR+ is freely available at http://bar.biocomp.unibo.it/bar2.0.

Results reported in this Thesis contribute to the comprehension of the complicated world of “redox biology”. ROS regulate signalling pathways both in physiological responses and in pathogenesis and progression of diseases. In cancer cells, the increase in ROS generation from metabolic abnormalities and oncogenic signalling may trigger a redox adaptation response, leading to an up-regulation of antioxidant capacity in order to maintain the ROS level below the toxic threshold. Thus, cancer cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system. Results here reported indicate that the development of new drugs targeting specific Nox isoforms, responsible for intracellular ROS generation, or AQP isoforms, involved in the transport of extracellular H2O2 toward intracellular targets, might be an interesting novel anti-leukaemia strategy. Furthermore, also the use of CSD peptide, which simulate the VEGFR-2 segregation into caveolae in the inactive form, might be a strategy to stop the cellular response to VEGF signalling. As above stated, in the understanding of the redox biology, it is also important to identify and distinguish the molecular effectors that maintain normal biological and physiological responses, such as agents that stimulate our adaptation systems and elevate our endogenous antioxidant defences or other protective systems. Data here reported indicate that the nutraceutical compound sulforaphane and the Klotho protein are able to stimulate the HO-1 and Prx-1 expression, as well as the GSH levels, confirming their antioxidant and protective role. Finally, results here reported demonstrated that Stevia extracts are involved in insulin regulated glucose metabolism, suggesting that the use of these compounds goes beyond their sweetening power and may also offer therapeutic benefits hence improving the quality of life.

9-hydroxystearic acid (9-HSA) belongs to a class of lipid peroxidation products identified in several human and murine cell lines. These products are greatly diminished in tumors compared to normal tissues and their amount is inversely correlated with the malignancy of the tumor. 9-HSA activity has been tested in cancer cell lines, where it showed to act as a histone deacetylase 1 (HDAC1) inhibitor. In particular, in a colon cancer cell line (HT29), its administration resulted in an inhibition of proliferation together with an induction of differentiation. In this thesis the effect of (R)-9-hydroxystearic acid has been tested in vivo on cell proliferation and differentiation processes, in the early stages of zebrafish development. The final aim of this work was to elucidate the role of (R)-9-HSA in the control of cell differentiation and proliferation during normal development, in order to better understand its molecular control of cancerogenesis. The molecule has been administered via injection in the yolk of zebrafish embryos. The analysis of the histone acetylation pattern showed a hyperacetilation of histone H4 after treatment with the molecule, as detectable in HDAC1 mutants. (R)-9-HSA was also demonstrated to interfere with the signaling pathways that regulate proliferation and differentiation in zebrafish retina and hindbrain. This resulted in a reduction of proliferation in the hindbrain at 24 hours post injection (hpi), and in a hyperproliferation at 48 and 72 hpi in the retina, with a concomitant inhibition of differentiation. Finally, (R)-9-HSA effects were evident on proliferation of stem cell located in the ciliary marginal zone (CMZ) of the retina. The presence of ROS and 4-hydroxynoneal in the CMZ of wild-type embryos supports the hypothesis that oxidative stress could regulate stem cells fate in zebrafish retina.

Diseases due to mutations in mitochondrial DNA probably represent the most common form of metabolic disorders, including cancer, as highlighted in the last years. Approximately 300 mtDNA alterations have been identified as the genetic cause of mitochondrial diseases and one-third of these alterations are located in the coding genes for OXPHOS proteins. Despite progress in identification of their molecular mechanisms, little has been done with regard to the therapy. Recently, a particular gene therapy approach, namely allotopic expression, has been proposed and optimized, although the results obtained are rather controversial. In fact, this approach consists in synthesis of a wild-type version of mutated OXPHOS protein in the cytosolic compartment and in its import into mitochondria, but the available evidence is based only on the partial phenotype rescue and not on the demonstration of effective incorporation of the functional protein into respiratory complexes. In the present study, we took advantage of a previously analyzed cell model bearing the m.3571insC mutation in MTND1 gene for the ND1 subunit of respiratory chain complex I. This frame-shift mutation induces in fact translation of a truncated ND1 protein then degraded, causing complex I disassembly, and for this reason not in competition with that allotopically expressed. We show here that allotopic ND1 protein is correctly imported into mitochondria and incorporated in complex I, promoting its proper assembly and rescue of its function. This result allowed us to further confirm what we have previously demonstrated about the role of complex I in tumorigenesis process. Injection of the allotopic clone in nude mice showed indeed that the rescue of complex I assembly and function increases tumor growth, inducing stabilization of HIF1α, the master regulator of tumoral progression, and consequently its downstream gene expression activation.

Il core catalitico della DNA polimerasi III, composto dalle tre subunità α, ε e θ, è il complesso minimo responsabile della replicazione del DNA cromosomiale in Escherichia coli. Nell'oloenzima, α ed ε possiedono rispettivamente un'attività 5'-3' polimerasica ed un'attività 3'-5' esonucleasica, mentre θ non ha funzioni enzimatiche. Il presente studio si è concentrato sulle regioni del core che interagiscono direttamente con ε, ovvero θ (interagente all'estremità N-terminale di ε) e il dominio PHP di α (interagente all'estremità C-terminale di ε), delle quali non è stato sinora identificato il ruolo. Al fine di assegnare loro una funzione sono state seguite tre linee di ricerca parallele. Innanzitutto il ruolo di θ è stato studiato utilizzando approcci ex-vivo ed in vivo. I risultati presentati in questo studio mostrano che θ incrementa significativamente la stabilità della subunità ε, intrinsecamente labile. Durante gli esperimenti condotti è stata anche identificata una nuova forma dimerica di ε. Per quanto la funzione del dimero non sia definita, si è dimostrato che esso è attivamente dissociato da θ, che potrebbe quindi fungere da suo regolatore. Inoltre, è stato ritrovato e caratterizzato il primo fenotipo di θ associato alla crescita. Per quanto concerne il dominio PHP, si è dimostrato che esso possiede un'attività pirofosfatasica utilizzando un nuovo saggio, progettato per seguire le cinetiche di reazione catalizzate da enzimi rilascianti fosfato o pirofosfato. L'idrolisi del pirofosfato catalizzata dal PHP è stata dimostrata in grado di sostenere l'attività polimerasica di α in vitro, il che suggerisce il suo possibile ruolo in vivo durante la replicazione del DNA. Infine, è stata messa a punto una nuova procedura per la coespressione e purificazione del complesso α-ε-θ / The DNA polymerase III catalytic core, containing the three subunits α, ε, and θ, is the minimal active polymerase complex responsible of chromosomal DNA replication in Escherichia coli. In the holoenzyme, α displays 5’-3’ polymerase activity and ε features 3’-5’ exonuclease activity, while θ has no enzymatic function. The present study was focused on the core regions directly interacting with ε, i.e. θ (interacting with ε N-terminal domain), and the PHP domain of α (interacting with ε C-terminal domain), for none of which a role has been identified yet. To assign them a function three research lines were engaged. First, the role of θ was investigated using ex-vivo and in vivo approaches. The results presented here show that θ significantly increases the stability of the intrinsically labile ε subunit. During these experiments a new dimeric form of ε was identified. While the dimer function is not clear, it is shown to be actively dissociated by θ, which could then act as its regulator. Furthermore, the first growth phenotype associated to θ is provided. As regard to α PHP domain, a pyrophosphatase activity was determined using a new assay, realized to monitor the kinetics of reactions catalyzed by phosphate- or pyrophosphate-releasing enzymes. PHP-catalyzed pyrophosphate hydrolysis was also shown to be able to sustain α-driven in vitro DNA polymerization, suggesting its possible role in vivo. Finally, a new procedure for α-ε-θ co-expression and purification is presented.

Mitochondria have a central role in energy supply in cells, ROS production and apoptosis and have been implicated in several human disease and mitochondrial dysfunctions in hypoxia have been related with disorders like Type II Diabetes, Alzheimer Disease, inflammation, cancer and ischemia/reperfusion in heart. When oxygen availability becomes limiting in cells, mitochondrial functions are modulated to allow biologic adaptation. Cells exposed to a reduced oxygen concentration readily respond by adaptive mechanisms to maintain the physiological ATP/ADP ratio, essential for their functions and survival. In the beginning, the AMP-activated protein kinase (AMPK) pathway is activated, but the responsiveness to prolonged hypoxia requires the stimulation of hypoxia-inducible factors (HIFs). In this work we report a study of the mitochondrial bioenergetics of primary cells exposed to a prolonged hypoxic period . To shine light on this issue we examined the bioenergetics of fibroblast mitochondria cultured in hypoxic atmospheres (1% O2) for 72 hours. Here we report on the mitochondrial organization in cells and on their contribution to the cellular energy state. Our results indicate that prolonged hypoxia cause a significant reduction of mitochondrial mass and of the quantity of the oxidative phosphorylation complexes. Hypoxia is also responsible to damage mitochondrial complexes as shown after normalization versus citrate synthase activity. HIF-1α plays a pivotal role in wound healing, and its expression in the multistage process of normal wound healing has been well characterized, it is necessary for cell motility, expression of angiogenic growth factor and recruitment of endothelial progenitor cells. We studied hypoxia in the pathological status of diabetes and complications of diabetes and we evaluated the combined effect of hyperglycemia and hypoxia on human dermal fibroblasts (HDFs) and human dermal micro-vascular endothelial cells (HDMECs) that were grown in high glucose, low glucose concentrations and mannitol as control for the osmotic challenge. / I mitocondri hanno un ruolo fondamentale nella produzione di energia nella cellula, ma sono coinvolti anche in altri processi tra cui la produzione di ROS e l’apoptosi. Disfunzioni del metabolismo mitocondriale sono state associate a diversi disordini, tra cui: diabete di tipo II, malattia si Alzheimer, infiammazione, cancro ed ischemia cardiaca. Quando i livelli di ossigeno nella cellula diventano limitanti, la funzione mitocondriale viene modulata per consentire l’adattamento biologico. La via dell’AMP- activated protein kinase (AMPK) ha il compito di monitorare lo stato energetico della cellula mantenendo i livelli fisioligici di ATP/ADP. In seguito all’esposizione prolungata in ambiente ipossico, l’attivazione di HIF-1 e’ in grado di upregolare diversi geni coinvolti nella sopravvivenza cellulare a basse concentrazioni di ossigeno. In questo lavoro, e’ stata valutata la bioenergetica mitocondriale in fibroblasti primari coltivati a basse concentrazioni di ossigeno (1 % O2) per 72 ore; in particolare, abbiamo preso in considerazione l’organizzazione mitocondriale nella cellula e il loro contributo nel mantenere lo stato energetico cellulare. I nostri risultati indicano che l’esposizione prolungata all’ipossia causa una significativa riduzione della massa mitocondriale e della quantita’ dei complessi della fosforilazione ossidativa, nonostante le cellule siano in grado di mantenere i livelli intracellulari di ATP. Inoltre abbiamo studiato l’ipossia nel contesto patologico del diabete ed in particolare delle complicanze del diabete. E’ noto che l’iperglicemia e l’ipossia, dovuta ad ischemia a danni vascolari, hanno un ruolo importante nell’insorgenza delle complicanze del diabete. HIF-1α rappresenta uno stimolo nella rigenerazione delle ferite, in quanto stimola la vascolarizzazione e la migrazione dei cheranociti ed e’ stato ipotizzato che le cellule perdano la capacita’ di adattarsi e rispondere all’ipossia quando sono coltivate in presenza di elevate concentrazioni di glucosio (>25 mM). Abbiamo valutato il ruolo della destabilizzazione di HIF-1α nella produzione di ROS, considerati i principali responsabili della progressione del diabete.

Iodide transport is necessary for the synthesis of thyroid hormones following accumulation in the follicular lumen out of thyroid cells, via channels unknown with the exception of pendrin. According to our hypothesis, TMEM16A could be the main molecular identity of the channel mediating iodide efflux in the thyroid gland. TMEM16A is the prior candidate for calcium-activated chloride conductance (CaCC). TMEM16A belongs to the TMEM16/anoctamin family comprising ten members (TMEM16A-K). Higher affinity of TMEM16A for iodide and predicted expression in the thyroid gland suggest its mediation of iodide efflux. The aim of this project was to identify the role of TMEM16A in iodide transport in the thyroid gland, by characterizing its molecular expression and functional properties. We demonstrated that TMEM16F, H, K transcripts are expressed in FRTL-5 thyroid cells, as well as TMEM16A, which is TSH-independent. Tumor tissue from human thyroid maintains TMEM16A expression. Functional in vivo experiments in FRTL-5, stably expressing YFP-H148Q/I152L fluorescent protein as a biosensor, showed that iodide efflux is stimulated by agonists of purinergic receptors with an order of potency of ATP>UTP>ADP (compatible with an involvement of P2Y purinergic receptors), and by agonists of adrenergic receptors (epinephrine, norepinephrine and phenylephrine). Iodide efflux was blocked by α-receptor antagonists prazosin and phentolamine, consistent with a role of α1 adrenergic receptors. Iodide efflux was specifically dependent on calcium mobilized from intracellular compartments and induced by the calcium ionophore ionomycin. CaCC blockers suppressed ionomycin-/ATP-/epinephrine-stimulated iodide efflux. Heterologous expression of TMEM16A in CHO K1 cells induced calcium-activated iodide fluxes. All these results support the hypothesis of the involvement of TMEM16A in calcium-dependent iodide efflux induced by receptor agonists in thyroid cells. TMEM16A may represent a new pharmacological target for thyroid cancer therapy, since its blockade may enhance the retention of radioiodide by tumour cells enhancing the efficacy of radioablative therapy.

Il progresso tecnologico nel campo della biologia molecolare, pone la comunità scientifica di fronte all’esigenza di dare un’interpretazione all’enormità di sequenze biologiche che a mano a mano vanno a costituire le banche dati, siano esse proteine o acidi nucleici. In questo contesto la bioinformatica gioca un ruolo di primaria importanza. Un nuovo livello di possibilità conoscitive è stato introdotto con le tecnologie di Next Generation Sequencing (NGS), per mezzo delle quali è possibile ottenere interi genomi o trascrittomi in poco tempo e con bassi costi. Tra le applicazioni del NGS più rilevanti ci sono senza dubbio quelle oncologiche che prevedono la caratterizzazione genomica di tessuti tumorali e lo sviluppo di nuovi approcci diagnostici e terapeutici per il trattamento del cancro. Con l’analisi NGS è possibile individuare il set completo di variazioni che esistono nel genoma tumorale come varianti a singolo nucleotide, riarrangiamenti cromosomici, inserzioni e delezioni. Va però sottolineato che le variazioni trovate nei geni vanno in ultima battuta osservate dal punto di vista degli effetti a livello delle proteine in quanto esse sono le responsabili più dirette dei fenotipi alterati riscontrabili nella cellula tumorale. L’expertise bioinformatica va quindi collocata sia a livello dell’analisi del dato prodotto per mezzo di NGS ma anche nelle fasi successive ove è necessario effettuare l’annotazione dei geni contenuti nel genoma sequenziato e delle relative strutture proteiche che da esso sono espresse, o, come nel caso dello studio mutazionale, la valutazione dell’effetto della variazione genomica. È in questo contesto che si colloca il lavoro presentato: da un lato lo sviluppo di metodologie computazionali per l’annotazione di sequenze proteiche e dall’altro la messa a punto di una pipeline di analisi di dati prodotti con tecnologie NGS in applicazioni oncologiche avente come scopo finale quello della individuazione e caratterizzazione delle mutazioni genetiche tumorali a livello proteico. / The technological advancement in the molecular biology field is strongly dependent from the availability of bioinformatics support in order to manage and to interpret the very large amount of biological sequences produced. A new level of knowledge is derivable from the Next Generation Sequencing technology (NGS), that allows to obtain whole genomes and transcriptomes in few days and with low costs. Cancer research is one of the most relevant applications of NGS technology. With the NGS analysis is possible to perform the genomic characterization of tumors highlighting all the detectable mutations such as single nucleotide variants, chromosomal rearrangements, insertions and deletions, in order to develop new diagnostic, prognostic and therapeutic approaches in the cancer treatment. However, the genetic variation found in the samples must be analyzed at protein level since they directly alter the tumor cell phenotype. The bioinformatic expertise is essential to implement the NGS data analysis and also to perform the annotation of the genes emerging from the sequenced genome and of the protein expressed by it. In this work of thesis two computational methods for protein annotation have been developed (prediction of targeting and signal peptides); moreover we will introduce a bioinformatic pipeline to analyze NGS data and to annotate the genetic variants in the cancer genomic research.

Le alterazioni della funzionalità mitocondriale detengono un ruolo cruciale nella patogenesi della malattia di Alzheimer (AD), sostenendo il processo neurodegenerativo attraverso meccanismi quali la riduzione della disponibilità energetica e la iperproduzione di ROS. Alle numerose ipotesi di patogenesi dell’AD, si è recentemente affiancata la cosiddetta ipotesi vascolare. Nei soggetti AD è stata riscontrata una significativa riduzione della disponibilità di ossigeno a livello neuronale (ipossia neuronale). Da numerosi studi è poi emerso che l’ipossia gioca un ruolo fondamentale nello sviluppo dell’AD contribuendo a più vie patogenetiche contemporaneamente. Tuttavia, non sono stati ancora chiariti tutti i meccanismi attraverso cui l’ipossia esplica la sua azione di danno. Lo scopo di questo studio è stato quello di contribuire a chiarire il ruolo patologico dell’ipossia nell’AD, analizzando principalmente le alterazioni della funzionalità mitocondriale indotte dalla riduzione della disponibilità di ossigeno. Nella prima fase dello studio cellule PC12 sono state coltivate in presenza di β-amiloide e ipossia. In questo modello abbiamo osservato un potenziamento dei fenomeni di deplezione dell’ATP e di generazione delle ROS indotti dalla Aβ quando anche l’ipossia era presente come fonte di danno cellulare, ipotizzando per i due fattori un effetto congiunto di tipo additivo. Nella seconda fase abbiamo esposto all’ipossia fibroblasti prelevati da pazienti AD portatori di mutazioni a carico dei geni APP e PSEN. La presenza di mutazioni predisponenti ad un fenotipo AD era in grado di determinare un danno bioenergetico e ossidativo. Le alterazioni bioenergetiche riscontrate in normossia risultavano ulteriormente potenziate quando i fibroblasti erano coltivati in ipossia, mentre lo stato di stress ossidativo veniva evidenziato solo in condizioni ipossiche. Sulla base dei risultati finora conseguiti si può ipotizzare che uno dei meccanismi attraverso cui l’ipossia esplica la sua azione di danno nella AD, possa essere dovuto alla capacità di potenziare ulteriormente le alterazioni della funzionalità mitocondriale. / Mitochondrial dysfunctions play a crucial role in the pathogenesis of Alzheimer’s disease (AD), sustaining the neurodegenerative process through mechanisms such as energy deprivation and ROS overproduction. The etiopathogenesis of AD is still obscure, although several hypothesis have been formulated. Recently the vascular hypothesis has been proposed. In AD patients, the cerebral blood flow is significantly decreased, with a consequent reduction of oxygen tension (neural hypoxia). Several studies have shown that hypoxia has a central role in AD pathogenesis, since it is involved in different pathogenic pathways. However, the role of hypoxia in AD is not fully understood. The aim of this study was further to elucidate the pathological role of hypoxia in AD, mainly analyzing the mitochondrial dysfunction induced by reduction of oxygen tension. In the first part of the study, PC12 cells were cultured in the presence of amyloid-β peptide and hypoxia. In this model, hypoxia and amyloid-β showed a synergistic effect, strongly contributing to ATP depletion and ROS overproduction. In the second part of the study we used, as cellular model, AD patient skin fibroblasts exposed to hypoxia. These fibroblasts are characterized by mutations of APP and PSEN genes that increase the production of amyloid-β peptide. The AD fibroblasts showed an alteration of energy metabolism and a condition of oxidative stress. The bioenergetic alterations found under normoxia were further increased by hypoxia, while the oxidative stress was observed only under hypoxic conditions. In the light of the results, we suggest that one of the pathological consequences of hypoxia in AD is the inhibition of mitochondrial function, which may contribute to the neuronal dysfunction and the impairment of cognitive capacities.