Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

Several coralligenous reefs occur in the soft bottoms of the northern Adriatic continental shelf. Mediterranean coralligenous habitats are characterised by high species diversity and are intrinsically valuable for their biological diversity and for the ecological processes they support. The conservation and management of these habitats require quantifying spatial and temporal variability of their benthic assemblages. This PhD thesis aims to give a relevant contribution to the knowledge of the structure and dynamics of the epibenthic assemblages on the coralligenous subtidal reefs occurring in the northern Adriatic Sea. The epibenthic assemblages showed a spatial variation larger compared to temporal changes, with a temporal persistence of reef-forming organisms. Assemblages spatial heterogeneity has been related to morphological features and geographical location of the reefs, together with variation in the hydrological conditions. Manipulative experiments help to understand the ecological processes structuring the benthic assemblages and maintaining their diversity. In this regards a short and long term experiment on colonization patterns of artificial substrata over a 3-year period has been performed in three reefs, corresponding to the three main types of assemblages detected in the previous study. The first colonisers, largely depending by the different larval supply, played a key role in determining the heterogeneity of the assemblages in the early stage of colonisation. Lateral invasion, from the surrounding assemblages, was the driver in structuring the mature assemblages. These complex colonisation dynamics explained the high heterogeneity of the assemblages dwelling on the northern Adriatic biogenic reefs. The buildup of these coralligenous reefs mainly depends by the bioconstruction-erosion processes that has been analysed through a field experiment. Bioconstruction, largely due to serpulid polychaetes, prevailed on erosion processes and occurred at similar rates in all sites. Similarly, the total energy contents in the benthic communities do not differ among sites, despite being provided by different species. Therefore, we can hypothesise that both bioconstruction processes and energetic storage may be limited by the availability of resources. Finally the major contribution of the zoobenthos compared to the phytobenthos to the total energetic content of assemblages suggests that the energy flow in these benthic habitats is primarily supported by planktonic food web trough the filter feeding invertebrates.

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Il presente studio si concentra sull’analisi degli aspetti traslazionali nella ricerca farmacologica applicata alla Gastroenterologia. La trattazione si articola in due parti: una prima elaborazione teorica, che permette di inquadrare nel contesto della ricerca traslazionale il razionale scientifico ed etico alla base delle attività sperimentali eseguite durante il triennio; una seconda parte, nella quale si riportano i metodi, i risultati e le osservazioni conclusive derivanti dallo studio sperimentale. Nella prima parte vengono analizzate alcune caratteristiche delle procedure, adottate nella ricerca in ambito farmacologico gastrointestinale, che permettono di ottenere un dato verosimile derivabile da modelli diversi rispetto all’organismo umano. Sono inclusi nella trattazione gli aspetti etici dell’utilizzo di alcuni modelli animali di patologie intestinali organiche e funzionali in relazione al loro grado di predittività rispetto alla realtà sperimentale clinica. Nella seconda parte della trattazione, viene presentato uno studio esplorativo tissutale multicentrico sul ruolo del sistema oppioide e cannabinoide nella sindrome dell’intestino irritabile (IBS). Obiettivo dello studio è la valutazione dell’espressione e la localizzazione del recettore oppioide µ (µOR), del suo ligando β endorfina (β-END) e del recettore cannabinoide 2 (CB2) nei pazienti con IBS ad alvo costipato (IBS-C) e diarroico (IBS-D), ed in soggetti sani (HC). I dati ottenuti indicano un’implicazione del sistema oppioide e cannabinoide nella risposta immune alterata riscontrata nei pazienti con IBS ed in particolare nel sottogruppo IBS-C. La presente trattazione suggerisce come la creazione di nuovi sistemi di indagine sempre più validi da un punto di vista traslazionale possa dipendere, almeno in parte, dalla capacità di integrare realtà disciplinari, tecnologie ed esperienze metodologiche diverse nel contesto della ricerca in campo biomedico e farmacologico ed in particolare tramite un mutuo scambio di informazioni tra realtà clinica e ricerca di base / The present study focuses on the analysis of translational issues of pharmacological research applied to Gastroenterology. The dissertation is divided in two parts: the first one consists in a theroethical elaboration allowing to contextualize - within the framework of translational research - the scientific and ethical rationale underlying the experimental activity carried out over the last three years. The second part deals with the methods, the results and the conclusive observations deriving from the experimental study presented. In the first part some features of the procedures adopted in the field of pharmacology applied to gastroenterology are presented. These allow to obtain a reliable data deriving from models other than human one. This section also addresses the ethical issues concerning the use of animal models of both organic and functional intestinal pathologies. Such issues are put in relation to the level of predicitivity peculiar to the model and related to the research. In the second part of the dissertation the author presents a multicenter study about the role of the opioid and cannabinoid system in the irritable bowel syndrome (IBS). The aim of the study is the assessment of the expression of µ opioid receptor (µOR), of its ligand β-endorphin (β-END) and the cannabinoid receptor 2 (CB2) in IBS patients with either constipation (IBS-C) or diarrhea (IBS-D) compared to healthy controls (HC). The data obtained suggest that the opioid and cannabinoid systems contribute to the abnormal immune response detected in IBS patients, particularly in IBS-C. The present dissertation suggests how the creation of innovative research methodologies ever more effective from a translational standpoint may depend, at least in part, from the ability to integrate different research areas, technologies and methodological expertise in the context of biomedical and pharmacological research, especially through the mutual exchange of information between clinical and basic research.

Colorectal cancer (CRC) is a multistep process that goes through adenoma-carcinoma sequence. Many forms of CRC may be prevented by routine control, which can detect precancerous neoplasm before they undergo malignant transformation (123). For this reasons we hypothesized that a combination of simple faecal tests, may help to identify patients with higher risk of adenomas and/or CRC. The aim of this study is to clarify whether FOBT, enzyme Tumor M2-PK and endocannabinoid system molecules (CB1, CB2, FAAH), could represent diagnostic non-invasive markers, alone or in combination, for early diagnosis of CRC and its precancerous lesions. In a pivotal study we analyzed a selected population, using i-FOBT and quantitative ELISA stool test for t-M2-PK detection. i-FOBT showed the highest specificity and PPV (88.8% and 52.7% respectively); M2-PK had the best sensitivity (87.2%); the best results it obtained with combination tests; in fact if our patients had been subjected only to the i-FOBT test, 33 high risk adenoma and 14 CRC would not have been diagnosed. Supported by these findings, we analyzed a consecutive population: patients with both positive tests have only 31.6% risk of developing CRC; in contrast, patients negative to both markers, cancer risk was less than 2% (VPN 98.5%). We confirmed, also with immuoistochemistry, that increase of tumour M2-PK in patients with CRC, as well as in stool samples, is correlated with pre neoplastic stages. To investigate the expression of endocannabinoid system in CRC, CB1, CB2 and FAAH markers were studied immunochemically in different stages and normal tissue. CB receptors and their ligands, as well as FAAH inhibitions, showed to have a protective role in colorectal cancer. They, in combination with other markers (such as t-M2-PK and FOBT), could be indicated to develop a non-invasive test for an early diagnosis of cancerous and pre-cancerous colorectal lesions.

Nella presente tesi, abbiamo caratterizzato un modello di ratto trattato con dieta iperlipidica e streptozotocina (D), utilizzato per valutare i potenziali effetti anti-steatotici di una miscela contenente 5 estratti vegetali (caigua, soia, erba medica, carciofo e riso rosso fermentato) somministrata a due differenti dosaggi (0,3 [A] e 1 mg/Kg [B]) e confrontata con il controllo positivo eugenolo (E) ed il veicolo di somministrazione latte di soia (M). I valori concernenti colesterolo, LDL, glucosio, ALT ed il contenuto di lipidi nel tessuto epatico sono risultati aumentati in D, A ed M, rispetto ai CTR, e ripristinati significativamente in B ed E. Le attività marcatrici del CYP2E1 (p-nitrofenolo idrossilasi ed anilina-idrossilasi) e le analisi di Western blotting per le proteine CYP2E1 e CYP4A hanno evidenziato una significativa induzione su D, A ed M ed un parziale ripristino dei valori di controllo in B. Dal dosaggio del GSH e della carbonilazione delle proteine è stata evidenziata la presenza di stress ossidativo nei ratti iperlipidici, significativamente ridotta nel B. La presenza del colesterolo nella dieta iperlipidica è stata determinante per la significativa riduzione dei geni HMGCR ed LDLr. Dall’analisi di geni coinvolti nell’infiammazione e nello stress mitocondriale si è evidenziato un significativo aumento nei gruppi D, A e M rispetto al CTR, mentre una significativa riduzione si è osservata in B ed E. Per estendere la caratterizzazione del ratto HFD/STZ, in un nuovo esperimento abbiamo analizzato geni chiave del metabolismo del colesterolo e del trasporto degli acidi biliari. Abbiamo osservato un’induzione dei geni responsabili della formazione dell’acido colico CYP7A1 e CYP8B1, ma non di quelli dell’acido chenodeossicolico CYP27A1 e CYP7B1. Inoltre è emersa una diminuita espressione di SHP (gene chiave per l’inibizione feedback del CYP7A1 e del CYP8B1) e dei geni responsabili del trasporto degli acidi biliari, del colesterolo, e della fosfatidilcolina. / In this thesis, we characterized a rat model treated with a high fat diet and low dose of streptozotocin (HFD/STZ, D), to evaluate the anti-steatotic effects of a 5 plant extracts mixture (caigua, soybean, alfalfa, artichoke and fermented red rice) administered at two different doses (0.3 [A] and 1 mg/kg [B]) and compared to the positive control eugenol (E) and the vehicle soy milk (M) administration. Cholesterol, LDL, glucose and ALT blood parameters values and liver lipids content were increased in D, A and M, with the respect to CTR, and significantly recovered in B and E. CYP2E1 specific activities (p-nitrophenol hydroxylase and aniline hydroxylase) and Western blot analysis for CYP2E1 and CYP4A proteins were induced in D, A and M, and partially restored in B. GSH and protein carbonylation analysis highlighted, compared to CTR, the presence of oxidative stress in HFD/STZ rats, which was significantly reduced in B. The analysis of genes involved in inflammation (IL6, TNFα) and mitochondrial stress revealed a significant increase in A, D, M groups, but not in E and B. The presence of 2% of cholesterol in the high fat diet determined a significant reduction of HMGCR and LDLr transcripts. To extend the characterization of our steatotic rat model, we analyzed key genes of cholesterol metabolism and bile acids transporters in a new experiment. It was observed an induction of CYP7A1 and CYP8B1 genes responsible for the formation of cholic acid (CA), but not of CYP27A1 and CYP7B1, which lead to chenodeoxycholic acid (CDCA). In addition, we observed a decreased expression of FXR-regulated SHP (the key gene for the feedback CYP7A1 and CYP8B1 inhibition) and of the genes responsible for the transport of bile acids (NTCP, MRP2/4, OATP1A1, OATP1B2, BSEP), cholesterol (ABCG5), and phosphatidylcholine (MDR3).

Negli ultimi decenni, in virtù delle eccellenti proprietà antiossidanti della vitamina E (VE), la possibile associazione tra questa e la riduzione dell’incidenza di patologie neoplastiche è stata studiata mediante numerosi trial clinici. I risultati sono però ambigui e l’impiego della VE come agente chemiopreventivo su larga scala è oggi più che mai al centro del dibattito scientifico. Il Selenium and Vitamin E Cancer Prevention Trial (SELECT) ha addirittura evidenziato un rischio d’incidenza più alto per il cancro alla prostata nel gruppo d’intervento in cui è stata somministrata la VE. Tuttavia, il meccanismo d’azione non è noto. Poiché la VE induce l’espressione di alcune isoforme del P450 nel fegato e poichè tale induzione è associata ad un aumento della produzione di specie reattive centrate sull’ossigeno, ci siamo posti il problema di come un’eventuale up-regulation a livello prostatico avrebbe potuto generare uno stress ossidativo responsabile del fenomeno di cui sopra. Il presente lavoro ha mostrato come la VE provochi una marcata induzione delle isoforme CY1A1, CYP1A2, e CYP1B1/2 nel rene e nella prostata di ratto trattato i.p. con VE (100 o 200 mg/kg p.c. per sette o quattordici giorni consecutivi), e nel rene una generale inattivazione degli enzimi post-ossidativi GST e UDPGT ed una riduzione della potenzialità antiossidante. La spettroscopia EPR abbinata alla tecnica radical trapping ha rilevato una generazione anomala di radicali liberi in entrambi i tessuti. I risultati sono stati confermati in vitro in cellule epiteliali di prostata umana RWPE-1 esposte alla VE. Insieme ad un aumento dell’espressione genica (mRNA) di differenti CYPs, è stato osservato un incremento di radicali liberi e della prostaglandina E2 (PGE2) rispetto al controllo. Lo studio indica che la VE induce la superfamiglia CYP e uno stress ossidativo a livello prostatico (co-cancerogenesi) e può contribuire a spiegare i risultati inaspettati del trial SELECT. / Several meta-analysis and randomized clinical trials have seriously questioned chemoprevention based on vitamins including vitamin E (VE). Recently, the Selenium and Vitamin E Cancer Prevention Trial (SELECT) has pointed out an increased risk for prostate cancer among VE long–term users. However, to date, the mechanism underlying these findings still remain unknown. Evidence from both in vitro and in vivo models reported how VE might increase the expression of hepatic cytochrome P450 (CYP). Induction may increase the biotransformation of ubiquitous pre-carcinogens and trigger an over-production of oxygen centred radicals (ROS) in the target tissue. We apotheosized that if such phenomenon occured also in the prostate, it could contribute to explain the SELECT unexpected data. Male Sprague-Dawley rats were daily treated i.p. with either 100 or 200 mg/kg b.w. for 7 or 14 consecutive days. A powerful booster effect of various CYP isoforms such as CY1A1, CYP1A2, and CYP1B1/2, coupled with a marked free radical over-generation were recorded in renal and prostate tissues. VE treatment led to a wide down-regulation of antioxidant (catalase, NAD(P)H:quinone reductase) and phase II enzymes (glutathione S-transferase, UDP-glucuronosyl transferase capability. Results observed in the in vivo study were consistent with those obtained by the use of a RWPE-1 human prostate cell based model. Compared to the control RWPE-1, cells exposed to VE reported a general CYP up-regulation associated with a higher content of free radicals. Interestingly, VE treatment also induced the cyclooxygenase (COX-2) expression with a consequently increased of the prostaglandin E2 levels. The present study suggests that VE can act as a co-carcinogen and pro-oxidant agent. If such epigenetic mechanisms occur in human, may contribute to explain the harmful outcomes raised up from the SELECT study.

In this PhD thesis I will describe different aspects of conservation genetics and genomics of two wild Canidae species, the wolf (Canis lupus) and the golden jackal (Canis aureus), through the study of two of the most variable gene families: the Major Histocompatibility Complex genes (MHC), and Olfactory Receptors genes (OR). In order to perform these studies both Sanger and next generation sequencing (NGS) DNA techniques have been used. The background of the thesis is described in the “General introduction” with phylogeny, classification and evolutionary ecology of the Canidae, with a focus on the species Canis lupus and its main conservation concerns in Italy. Moreover, I will introduce the importance to perform genetic studies as tools for wild-life conservation and management, with a description of the framework of the principal historical and currently used molecular markers that had driven to develop MHC and OR sequencing projects. The thesis is divided into two parts, “PART I – The MHC typing project” and “PART II – The OR genes typing project”. A total of four scientific papers (already published or under revision) will be introduced and illustrated as result of three years of PhD activities at ISPRA’s (Istituto Superiore per la Protezione e la Ricerca Ambientale), Laboratory of conservation genetics, in Ozzano dell’Emilia, and thankfully to a PhD fellowship granted by the Università di Bologna.

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.

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.