Serogroup B Neisseria meningitidis (MenB) is the cause of the invasive meningococcal disease (IMD). Bexsero is the first genome-derived vaccine against MenB. Neisserial adhesin A (NadA) is one of the three protein antigens included in Bexsero. The main aim of this work was to obtain detailed insights into the structure of vaccine NadA variant 3 (NadAv3) and into the molecular mechanisms governing its transcriptional regulation by NadR (Neisseria adhesin A Regulator). A deep understanding of nadA expression is important for understanding the contribution of NadA to vaccine-induced protection against meningococcal disease. NadA expression is regulated by the ligand-responsive transcriptional repressor NadR. The functional, biochemical and high-resolution structural characterization of NadR is presented in the first part of the thesis (Part One). These studies provide detailed insights into how small molecule ligands, such as hydroxyphenylacetate derivatives, found in relevant host niches, modulate the structure and activity of NadR, by ‘conformational selection’ of inactive forms. In the second part of the thesis (Part Two), strategies involving both protein engineering and crystal manipulation to increase the likelihood of solving the crystal structure of NadAv3 are described. The first approach was the rational design of new constructs of NadAv3, based on the recently solved crystal structure of a close sequence variant (NadAv5). Then, a comprehensive set of biochemical, biophysical and structural techniques were applied to investigate all the generated NadAv3 constructs. The well-characterized trimeric NadAv3 constructs represented a set of high quality reagents which were validated as probes for functional studies and as a platform for continued attempts for protein crystallization. Mutagenesis studies and screenings to identify a new crystal form of NadAv3 were performed to improve crystal quality; structure determination is ongoing. The atomic resolution structure of NadAv3 will help to understand its biological role as both an adhesin and a vaccine antigen.

Marine Protected Areas (MPAs) were initially created to protect the living, non-living, cultural and/or historical values from human activities. The Convention on Biological Diversity (CBD; Earth Summit in Rio de Janeiro on 5 June 1992) has set a target of protecting 10% of the coastal and marine areas by 2020, which has led to a rapid increase in the creation of MPAs worldwide. Within this context, there is a growing concern regarding the number of efficient MPAs. One of the main issues is that biological or ecological features of marine species as well as ecosystem processes are not taken into account in MPA design. Deciding criteria for species management requires considerable information collected from a number of sources, including morphometric data, genetic data and distributional data. Morphometric tools are useful to study species taxonomy, or to provide information about the morphological variability, size and growth of the species, which is essential for MPA monitoring. Genetic tools can be use to resolve species taxonomy or population structure, allowing to estimate genetic diversity and connectivity of populations at different temporal and spatial scales. Both morphometric and genetic data used in combination provide a powerful tool that should be considered in MPA assessment. However, the accurate interpretation and the integration of this information into marine spatial planning is specially challenging. The aim of this PhD thesis was to develop a protocol for monitoring Marine Protected Areas by studying the morphology and genetics of two closely limpet species (Patella rustica and Patella caerulea) across MPAs in the Western Mediterranean sea. Overall, the results of this thesis provides support the inclusion of the morphological and genetic tools into management plans, and in the guidelines for the monitoring to improve and/or maintain MPA health and effectiveness.

Considering the crucial involvement of matrix metalloproteinases’ (MMPs) misregulated activity in the pathogenesis of several degenerative diseases, this class of enzymes has been considered a highly active set of targets for the design of new therapeutic agents. However, the scant success of synthetic MMP inhibitors, largely due to the disappointing results obtained in both clinical and preclinical studies, makes medicinal plants a valuable source of new active compounds able to modulate MMPs activity. In this work, a consistent number of plants, selected on the base of an ethnobotanical research, were tested as inhibitors of collagenase, the founding member of the MMPs family. 1H-NMR-based metabolomic analysis combined with multivariate data treatment (PLS and OPLS) was used to correlate the biological activity to the phytochemical profiles, suggesting tannins as an important class of collagenase inhibitors. Thus, a tannin-removal procedure was developed, which allowed to prove this hypothesis and to identify another class of active metabolites, the glucuronide-conjugated flavonoids (especially quercetin-3-O-β-glucoronide), whose the plant Alchemilla vulgaris was found to be a good source. In another stage of the project, different varieties of tea were investigated as collagenase inhibitors, finding black tea samples particularly potent. Then, an OPLS model was developed with the aim of correlating the biological activity to the UV-Vis spectra of teas, showing that a high activity was related to absorption values in the range 350-440 nm. A subsequent fractionation of the most active tea sample was carried out, and this approach allowed to corroborate the results obtained by the metabolomic analysis. Considering that the absorbance measurement of an extract represents a cheap and simple procedure, the proposed method can be suitable, for instance, to select the best tea variety to be developed as an anti-wrinkles cosmetic or food supplement.

In the marine realm, two of the main stressors causing significant changes are ocean warming and acidification. Of particular concern are organisms reliant on the generation of calcium carbonate, such as corals. Reproduction and population structure are strongly related to environmental parameters and may be indicators for stability or decline of coral populations. Part of my research focused on the reproductive cycle of the Mediterranean non-zooxanthellate coral Caryophyllia inornata in relation to seasonal variations of seawater temperature and photoperiod. Moreover, an unusual embryogenesis was found in females, males and inactive individuals during the entire year, indicating a possible agamic origin of embryos. This was a pilot study for a broader investigation along a wide latitudinal gradient of temperature and solar radiation. Reproductive traits of this coral do not vary along the gradient as observed for another non-zooxanthellate species, Leptopsammia pruvoti, indicating that these species could be quite tolerant to environmental changes, probably due to the lack of symbiosis with the zooxanthellae. A study on the spermatogenesis of the zooxanthellate coral Balanophyllia europaea living along a natural pCO2 gradient showed no significant variations with low pH. We hypothesized that high CO2 levels probably enhance zooxanthellae photosynthesis, leading to an increase of the available energy for gonadal development. The ability to reproduce in particular conditions is related to population structure, providing information on coral responses to the environment. I investigated the population structure of the tropical coral Montastraea cavernosa along a depth gradient in Bermuda. Deeper populations were characterized by smaller but more numerous colonies compared to shallow ones, with no variation in the percent cover among depths. Thus, mesophotic populations of M. cavernosa in Bermuda seem quite stable, indicating that these reefs may serve as a source of propagules to maintain shallower reefs and help guide future management and conservation strategies.

Canopy-forming seaweeds are worldwide disappearing due to the combined effects of human activities and climate instabilities. Identifying the type and strength of interactions between multiple anthropogenic and natural stressors can help setting achievable management targets for degraded ecosystems and support ecological resilience through local actions. This thesis aimed to understand how algal forests change from extensive to degraded, and what factors can enhance the ability of forests to withstand or recover from stressors. I contributed to a systematic review to infer potential important synergistic stressors interactions driving the loss of canopy-forming seaweeds at a global level. We found that management of excess nutrient levels would provide the greatest opportunity for preventing the shift from canopy to mat-forming algae, because of the higher prevalence of synergistic interactions between nutrient enrichment with other local and global stressors. Then, I focused my attention on fucoid algae of the genus Cystoseira that are the most typical canopy-forming seaweeds in the Mediterranean Sea. I explored which environmental and anthropogenic factors can explain the current status of the intertidal Cystoseira populations. I found that coastal urbanization and nutrient concentration were the factors most related to the status of Cystoseira. Finally, I carried out a series of manipulative field experiments to explore the effects of nutrient enrichment and heat-wave events on intertidal C. compressa. The results showed that C. compressa is sensitive to heat-wave events and that local biodiversity and thermal history of the alga seem to play a role reducing or increasing respectively the impact of such extreme events. I also characterised the epiphytic bacteria associated to the surface of C. compressa and showed their potential influence on the responses of C. compressa to environmental stressors.

La continua immissione in ambiente dei farmaci ad uso umano, e la loro incompleta rimozione nei depuratori rende questi composti pseudo-persistenti e potenzialmente pericolosi per gli organismi acquatici. La ricerca ha inteso valutare gli effetti sub-letali di farmaci di rilevanza ambientale su due organismi d’interesse commerciale: il mitilo mediterraneo e l’anguilla europea, tramite lo sviluppo e l’applicazione di indicatori biologici, in laboratorio e in campo. Gli studi sull’anguilla hanno permesso di identificare nella glicogenolisi, valutata in termini di glucosio rilasciato dagli epatociti, il parametro ideale per valutare alterazioni sul metabolismo glucidico. Con l’utilizzo di un metodo in vitro dinamico e sensibile, la perifusione di epatociti isolati in colonna, sono stati valutati gli effetti dei β-bloccanti propranololo ed atenololo sul metabolismo glucidico. I risultati hanno mostrato la maggior potenza del propranololo nell’alterare la glicogenolisi e la maggior sensibilità dei pesci quando esposti al farmaco. Questa metodologia potrebbe permettere di rilevare la presenza di composti adrenergici, noti e non, nelle matrici ambientali. La consolidata batteria di biomarker applicata sui mitili in condizioni controllate, ha permesso di valutare gli effetti specifici della caffeina dopo esposizione di 7 giorni a concentrazioni ambientali. I risultati hanno evidenziato una moderata induzione della sindrome da stress, confermando la bassa pericolosità della caffeina per gli organismi acquatici, rispetto ad altri farmaci. L’applicazione degli stessi biomarker in un sistema naturale soggetto ad effetti antropici (la laguna Piallassa Piombone), ha permesso di valutare la qualità biologica della laguna, ma non ha potuto imputare gli effetti osservati ai residui farmaceutici, risultati minoritari rispetto ad altri inquinanti. In conseguenza di effetti osservati a basse dosi, concludiamo che è necessario aumentare le conoscenze sugli effetti che questi contaminanti hanno sulle specie non target, e di sviluppare nuove e sensibili metodologie applicabili nella valutazione di rischio ambientale derivante da farmaci. / The continuous release and incomplete removal of human pharmaceuticals in the environment poses a risk for aquatic wildlife. The research aimed at evaluating sub-lethal effects of pharmaceuticals of environmental concern on two commercial aquatic species: the Mediterranean mussel and the European eel. The effects were assessed through the development and application of biological indicators in experiments under controlled conditions and in the field. Studies on eel allowed to identify that glycogenolysis, assessed in term of glucose released from the hepatocytes, is the best parameter to evaluate the alterations on hepatic glucose metabolism. The development of a sensitive in vitro dynamic method (perifusion of isolated hepatocytes on columns), permitted to assess the effects of two β-blockers (propranolol and atenolol) on fish glucose metabolism. The results showed that propranolol is more potent than atenolol in modifying the glycogenolysis. Moreover, fish resulted more sensitive when exposed to therapeutic concentrations of the pharmaceutical. The use of this technique could allow the search for unknown adrenergic compounds in environmental matrices. The well-established set of biomarkers used in controlled conditions, allowed the evaluation of the specific effects of caffeine on mussels health status, after 7-days exposure to environmentally relevant concentrations. Results showed a moderate induction of the stress syndrome, confirming the low risk posed by caffeine for aquatic species, in comparison with other pharmaceuticals. The application of the same set of biomarkers in a natural impacted ecosystem (the Piallassa Piombone lagoon), allowed to assess the biological quality of the lagoon, but could not attribute the observed effects to pharmaceuticals amongst other pollutants. Considering the effects observed at low pharmaceutical doses, we highlight the need for a wider knowledge regarding the effects that these contaminants on non-target species, and for implementing new and sensitive methodologies to be used in the environmental risk assessment posed by pharmaceuticals.

Much progress has been made in the past decade in elucidating the physiological, pathophysiological and pharmacological role of Hydrogen Sulphide (H2S). Recently a function of H2S virtually in every tissue of the human organism has emerged. However, the H2S-mediated regulation of bone homeostasis has been scarcely investigated. Despite a recent increased interest in the field, many fundamental issues remain indeterminate. The main objective of this study was to increase the basic knowledge on the role of H2S in bone through in vitro and in vivo studies and develop novel therapeutic strategies for bone diseases. Ex vivo experiments revealed that H2S-generating enzymes (Cystathionine-β-synthase, CBS; Cystathionine-γ-lyase, CSE) are expressed in human bone tissues and human bone-derived cells. In vitro experiments evidenced that CBS and CSE expression is a distinctive feature of the transition of mesenchymal stromal cells (h-MSCs) toward mature osteoblast. Furthermore, loss of function experiments on CBS and CSE during osteogenic differentiation of h-MSCs revealed an impaired mineralization ability. In vivo experiments in mice highlighted the role of CBS, CSE and H2S in the maintenance of bone homeostasis and CBS, CSE and H2S were found to be depleted in post-menopausal osteoporosis. Furthermore, our in vitro and in vivo data validated the use of H2S-donors as novel potential candidates for the treatment of bone pathologies. In particular H2S administration prevented and reversed ovariectomy-induced bone loss in mice. Based on these evidences, we firstly developed an H2S-releasing hybrid drug (DM-22) by modifying a clinically relevant anti-resorptive drug in order to improve the therapy of bone loss. DM-22 displayed improved biological properties compared to the parent drug; in particular, it increased the osteogenic differentiation ability of h-MSCs. Secondly, we developed an H2S-releasing scaffold to improve bone regeneration which was permissive for h-MSCs colonization and supported their osteogenic differentiation.

REST (RE-1 silencing transcription factor, or NRSF –neuron-restrictive silencing factor) binds to a conserved RE-1 motif present in the promoter region of regulated genes and represses their transcription in neuronal and non-neuronal cells (Bruce et al., 2004). REST recruits corepressors (CoREST, mSin3a) and multiple chromatin modifying enzymes (HDAC1/2, demethylase LSD1 and methyltransferase G9a), causing chromatin compaction and altering gene expression by changing epigenetic tagets (Ballas et al., 2005). REST contributes to orchestrate the epigenetic regulation of target genes through several miRNAs including miR-9/9*, miR-29a, miR-124a, miR-218 and others (Wu and Xie, 2006). My thesis has ascertained an anti-tumor properties of transcription factor REST on a model of cervical adenocarcinoma where class I histone deacetylase (HDAC) inhibitor: valproic acid (VPA) down-regulates REST and its corepressors CoREST and HDAC1 at mRNA and protein level. These effects are related to a potent effect on cell apoptosis, possibly mediated by miR-9 overexpression as consequence of REST and CoREST down-regulation. I report the presence of a double-negative feedback loop between REST and miR-9 in HeLa cell line: in absence of REST, miR-9 levels substantially increase while miR-9 overexpression promotes REST down-regulation. Interestingly, I have observed that REST is sufficient to induce a noteworthy chromatin remodeling in HeLa cells. HeLa cell apoptosis induced by these events, involves mitochondrial control of apoptosis signaling pathways, particularly Bcl-2 family gene BAX. In conclusion, the present study aims to contribute to a more accurate comprehension of the processes responsible for REST activity in a model of epithelial cervical adenocarcinoma, and relevant for a detailed knowledge of important events causing oncogenesis. Moreover, considering the crucial role of epigenetic regulation of gene transcription in the etiology of many pathological conditions, any further knowledge in this field could find important and innovative pharmacological applications.

L’importanza del microambiente nella progressione metastatica iniziò a delinearsi sin dalla fine del XIX secolo attraverso la teoria del “seed and soil”, proposta dal chirurgo inglese Stephen Paget (1855-1926), secondo la quale determinati tumori sono in grado di formare metastasi in organi specifici, proprio come un seme trova il terreno giusto per poter attecchire. In termini molecolari, Paget intuì come la cellula tumorale potesse esprimere molecole riconosciute solo in determinati tessuti. Negli stessi anni, il patologo americano James Ewing (1866-1943) elaborò una teoria complementaria, la “anatomical-mechanical hypothesis”, suggerendo l’idea che i sistemi linfatico e vascolare svolgessero un ruolo preponderante nella diffusione passiva delle cellule verso un determinato sito anatomicamente accessibile. Nel 1982, Bissell delineò la teoria moderna secondo cui, alla base dello sviluppo del tumore, sia il microambiente sia le mutazioni genetiche svolgono un ruolo fondamentale. Negli ultimi decenni, infatti, accanto alla definizione di cancro come malattia genetica, si è delineata l’importanza fondamentale delle interrelazioni che intercorrono tra l'epitelio tumorale e il microambiente tissutale nel processo di tumorigenesi. Il tumore è da tempo considerato un tessuto complesso, le cui cellule mutate non agiscono da sole durante la progressione del cancro, ma reclutano le normali cellule circostanti come collaboratori attivi per instaurare un fenotipo neoplastico. Se da un lato l’instabilità genomica dei tumori garantisce loro un vantaggio evolutivo continuo, permettendo a cellule sempre più resistenti e aggressive di sopravvivere alle terapie, dall’altro la loro natura malleabile ha evidenziato la necessità di attuare strategie multiple: molti studi pre-clinici e clinici hanno suggerito l’efficacia di terapie combinate che abbiano come target non solo le cellule tumorali, ma anche le componenti del microambiente e, in particolare, quelle dello stroma. / The relevance of the microenvironment in metastatic progression began to take shape since the late nineteenth century through the theory of the "seed and soil", given by the English surgeon Stephen Paget (1855-1926), according to which certain cancers are able to form metastases in specific organs, just like a seed finds the right soil to take root. In molecular terms, Paget understood as cancer cell could express molecules recognized only in certain tissues. In the same years, the American pathologist James Ewing (1866-1943) developed a complementary theory, "anatomical-mechanical hypothesis ", suggesting that the lymphatic and vascular systems could play a predominant role in the passive diffusion of cells toward a particular site anatomically accessible. In 1982, Bissell delineated the modern theory that, at the basis of tumor growth, both the microenvironment and the genetic mutations play a key role. In recent decades, in fact, next to the definition of cancer as a genetic disease, it has outlined the fundamental importance of the interrelationships that exist between the tumor epithelium and tissue microenvironment in the process of tumorigenesis. The tumor has long been considered a complex tissue, the cells of which do not act alone during the progression of cancer, but they can recruit normal surrounding cells as active collaborators to establish a neoplastic phenotype. While the genomic instability of tumors guarantees them a continuous evolutionary advantage, allowing more and more aggressive and resistant cells to survive the therapies, their malleable nature has highlighted the need to implement multiple strategies: many pre-clinical and clinical studies have suggested the efficacy of combination of therapies targeting not only the tumor cells, but also the components of the microenvironment, in particular those of the stroma.

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.