Aging is a complex phenomenon that affects organs and tissues at a different rate. With advancing age, the skeletal muscle undergoes a progressive loss of mass and strength, a process known as sarcopenia that leads to a decreased mobility and increased risk of falls and invalidity. On the other side, another organ such as the liver that is endowed with a peculiar regenerative capacity seems to be only marginally affected by aging. Accordingly, clinical data indicate that liver transplantation from aged subjects has, in specific conditions, function and duration comparable to those achievable with grafts of liver from young donors. The molecular mechanisms involved in these peculiar aging patterns are still largely unknown, but it is conceivable that protein degradation machineries might play an important role, as they are responsible for the maintenance of cellular homeostasis. Indeed, it has been suggested that alteration of proteostasis may contribute to the onset and progression of several age-related pathological conditions, including skeletal muscle wasting and sarcopenia, as well as to the aging phenotypes. The ubiquitin-proteasome system (UPS) is one of the most important cellular pathways for intracellular degradation of short-lived as well as damaged proteins. To date, studies on the age-related modifications of proteasomes in liver and skeletal muscle were performed prevalently in rodents, with controversial results, while only preliminary observations have been obtained in human liver and skeletal muscle. In this scenario, we want to investigate and characterize in humans the age-related modifications of proteasomes of these two different organs.

It is well known that ageing and cancer have common origins due to internal and environmental stress and share some common hallmarks such as genomic instability, epigenetic alteration, aberrant telomeres, inflammation and immune injury. Moreover, ageing is involved in a number of events responsible for carcinogenesis and cancer development at the molecular, cellular, and tissue levels. Ageing could represent a “blockbuster” market because the target patient group includes potentially every person; at the same time, oncology has become the largest therapeutic area in the pharmaceutical industry in terms of the number of projects, clinical trials and research and development (R&D) spending, but cancer remains one of the leading causes of mortality worldwide. The overall aim of the work presented in this thesis was the rational design of new compounds able to modulate activity of relevant targets involved in cancer and aging-related pathologies, namely proteasome and immunoproteasome, sirtuins and interleukin 6. These three targets play different roles in human cells, but the modulation of its activity using small molecules could have beneficial effects on one or more aging-related diseases and cancer. We identified new moderately active and selective non-peptidic compounds able to inhibit the activity of both standard and immunoproteasome, as well as novel and selective scaffolds that would bind and inhibit SIRT6 selectively and can be used to sensitize tumor cells to commonly used anticancer agents such gemcitabine and olaparib. Moreover, our virtual screening approach led us also to the discovery of new putative modulators of SIRT3 with interesting in-vitro and cellular activity. Although the selectivity and potency of the identified chemical scaffolds are susceptible to be further improved, these compounds can be considered as highly promising leads for the development of future therapeutics.

L’osteomielite associata all’impianto è un processo infettivo a carico del tessuto osseo spesso accompagnato dalla distruzione dell’osso stesso. La patogenesi delle osteomieliti associate all’impianto si basa su due concetti fondamentali: l’internalizzazione del patogeno all’interno degli osteoblasti e la capacità dei batteri di formare il biofilm. Entrambi i meccanismi consentono infatti di prevenire l’eliminazione del batterio da parte delle difese immunitarie dell’ospite e di ostacolare l’azione della maggior parte degli antibiotici (che non penetrano e non agiscono pertanto su microrganismi intracellulari), così sostenendo ed alimentando l’infezione. Il saggio di invasione messo a punto su micropiastra ha consentito di investigare in modo approfondito e dettagliato il ruolo ed il peso dell’internalizzazione nella patogenesi delle infezioni ortopediche peri-protesiche causate da S. aureus, S. epidermidis, S. lugdunensis ed E. faecalis. Lo studio ha evidenziato che l’invasione delle cellule MG-63 non rappresenta un meccanismo patogenetico delle infezioni ortopediche associate all’impianto causate da S. epidermidis, S. lugdunensis ed E. faecalis; al contrario, in S. aureus la spiccata capacità invasiva rappresenta un’abile strategia patogenetica che consente al patogeno di sfuggire alla terapia sistemica e alla risposta immunitaria dell’ospite. È stato studiato inoltre il ruolo dell’immunità innata nella difesa contro il biofilm batterico. In seguito all’incubazione del biofilm opsonizzato di S. epidermidis con i PMN è stato possibile osservare la formazione delle NETs. Le NETs rappresentano ottime armi nella difesa contro il biofilm batterico, infatti le trappole sono in grado di limitare la diffusione batterica e quindi di confinare l’infezione. La comprensione del ruolo dell’internalizzazione nella patogenesi delle osteomieliti associate all’impianto e lo studio della risposta immunitaria innata a questo tipo di infezioni, spesso caratterizzate dalla presenza di biofilm, sono presupposti per identificare e affinare le migliori strategie terapeutiche necessarie ad eradicare l'infezione. / Impant-associated osteomyelitis is an infective process against bone tissue and it often leads to the destruction of the bone itself. The pathogenesis of impant-associated osteomyelitis is based on two fundamental concepts: internalization of pathogens into osteoblasts and bacterial ability to form biofilm. Both mechanisms allow to prevent bacterial elimination by host immune system and to block the action of majority antibiotics (that do not penetrate and have effect on intracellular bacteria) sustaining and feeding in this way the infection. The microplate-based assay allow to investigate the role of internalization mechanism in the pathogenesis of orthopaedic implant-infections caused by S. aureus, S. epidermidis, S. lugdunensis and E. faecalis. The study demonstrated the incompetence of S. epidermidis, S. lugdunensis and E. faecalis clinical isolates to invade MG-63 cells, on the opposite S. aureus invasion ability represents an optimal pathogenetic strategy for the pathogen to elude systemic therapy and host immune response. In addition here we investigate the role of innate immune response against bacterial biofilm. The interaction between S. epidermidis opsonized biofilm and PMN allowed to visualize the NETs. Neutrophil extracellular traps represent optimal weapon against bacterial biofilm because they wrap bacteria and so confine the infection. The understanding of internalization role in the pathogenesis of implant-associated osteomyelitis and the study of innate immune response against this kind of infection, often characterized by biofilm presence, are the basis to identify the best therapeutic strategy in order to eradicate the infection.

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

Obbiettivo: Valutazione delle eventuali differenze nel trattamento ortodontico di un gruppo di bambini con particolari necessità sanitarie (SHCN) rispetto ad un gruppo di bambini non diagnosticati con SHCN. Materiali e Metodi: Il gruppo campione (SHCN) è costituito da 50 bambini con SHCN. Il gruppo di controllo (NO SHCN) è costituito da 50 bambini non diagnosticati con SHCN pienamente corrispondenti per età, genere e tipo di apparecchio ortodontico utilizzato con i pazienti del gruppo di studio. I dati riguardanti i gruppi SHCN e NO SHCN sono stati analizzati in modo retrospettivo, valutando: - il punteggio pre- e post-trattamento e la riduzione finale dei valori dell'indice PAR (Peer Assessment Rating), della componente DHC (Dental Health Component) e della componente AC (Aesthetic Component) dell'indice IOTN (Orthodontic Treatment Need Index), - il numero di appuntamenti, - il numero di sedute semplici e complesse, - la durata complessiva del trattamento, - l'età all’inizio ed alla fine della terapia. Risultati: Non sono state rilevate differenze statisticamente significative tra i due gruppi per quanto concerne il numero di appuntamenti, la durata complessiva del trattamento, l'età all’inizio ed alla fine della terapia ortodontica (valori del p-value:0.682, 0.458, 0.535, 0.675). Sono state rilevate differenze statisticamente significative tra i due gruppi per quanto riguarda i punteggi dell’indice PAR, delle componenti DHC e AC dello IOTN pre- e post-trattamento, il numero di sedute semplici e complesse (valori del p-value:0.030, 0.000, 0.020, 0.023, 0.000, 0.000, 0.043, 0.037). Per quanto concerne la riduzione finale del valore dell’indice PAR, della componente DHC e di quella AC dello IOTN non sono state riscontrate differenze statisticamente significative tra i due gruppi (valori del p-value:0.060, 0.765, 0.825). Conclusioni: Lo studio incoraggia gli ortodontisti a trattare i bambini con SHCN nell'obiettivo di migliorarne la qualità di vita, pur evidenziando la necessità di un maggior numero di sedute complesse. / Purpose: To analyze any differences in the orthodontic treatment between a group of children with special health care needs (SHCN) and a group of children not diagnosed with SHCN. Materials and Methods: The study sample (SHCN) consisted of 50 orthodontically treated children with SHCN. The control group (NO SHCN) consisted of 50 orthodontically treated children not diagnosed with SHCN fully matched for age, gender and type of appliance used with patients of the study sample. The differences between the SHCN and NO SHCN groups were analyzed retrospectively: - pre-, post-treatment scores and score reduction of the Peer Assessment Rating index (PAR), the Dental Health (DHC) and Aesthetic (AC) Components of the Orthodontic Treatment Need Index (IOTN), - number of appointments, - number of simple or complex chair time appointments, - overall treatment time, - age at treatment start and end. Results: There were no statistically significant differences between the two groups for the number of appointments, overall treatment time, age at treatment start and end (p values: 0.682, 0.458, 0.535 and 0.675). There were statistically significant differences between the two groups in PAR, DHC, AC pre- and post-treatment, and the number of simple and complex chair time appointments (p values: 0.030, 0.000, 0.020, 0.023, 0.000, 0.000, 0.043 and 0.037). The reductions of PAR, DHC and AC scores were not significantly different between the two groups (p values: 0.060, 0.765 and 0.825). Conclusions: This study, while reporting a greater number of complex chair time appointments during the orthodontic treatment of children with special health care needs, encourages orthodontists to implement the treatment of patients with SHCN, in an attempt to improve their quality of life.

Aims of the study: To assess the prevalence of Antiepileptic Drug (AED) exposure in pregnant women with or without epilepsy and the comparative risk of terminations of pregnancy (TOPs), spontaneous abortions, stillbirth, major congenital malformations (MCMs) and foetal growth retardation (FGR) following intrauterine AED exposure in the Emilia Romagna region (RER), Northern Italy (4 million inhabitants). Methods: Data were obtained from official regional registries: Certificate of Delivery Assistance, Hospital Discharge Card, reimbursed prescription databases and Registry of Congenital Malformations. We identified all the deliveries, hospitalized abortions and MCMs occurred between January 2009 and December 2011. Results: We identified 145,243 pregnancies: 111,284 deliveries (112,845 live births and 279 stillbirths), 16408 spontaneous abortions and 17551 TOPs. Six hundred and eleven pregnancies (0.42% 95% Cl: 0.39-0.46) were exposed to AEDs. Twenty-one per cent of pregnancies ended in TOP in the AED group vs 12% in the non-exposed (OR:2.24; CI 1.41-3.56). The rate of spontaneous abortions and stillbirth was comparable in the two groups. Three hundred fifty-three babies (0.31%, 95% CI: 0.28-0.35) were exposed to AEDs during the first trimester. The rate of MCMs was 2.3% in the AED group (2.2% in babies exposed to monotherapy and 3.1% in babies exposed to polytherapy) vs 2.0% in the non-exposed. The risk of FGR was 12.7 % in the exposed group compared to 10% in the non-exposed. Discussion and Conclusion: The prevalence of AED exposure in pregnancy in the RER was 0.42%. The rate of MCMs in children exposed to AEDs in utero was almost superimposable to the one of the non-exposed, however polytherapy carried a slightly increased risk . The rate of TOPs was significantly higher in the exposed women. Further studies are needed to clarify whether this high rate reflects a higher rate of MCMs detected prenatally or other more elusive reasons.

The immunosuppressive therapy still remains the only therapeutic strategy to control excessive immune activation following renal transplantation, but remain the problems related to excessive immunosuppression and in particular the toxicity due to high doses of immunosuppressive drugs such as calcineurin inhibitors. The present study has the aim of documenting, in a porcine animal model, the histological damage from calcineurin inhibitors using incremental doses of Tacrolimus, achieved in a limited amount of time, until it reaches toxic blood concentrations. We perform the study under different condition, like oral administration, intravenous infusion and with or without kidney transplant. It is noted that the damage is early, predominantly vascular and that affects different organs in addition to the kidneys. We also observe that the functional damage underestimates the structural damage. The search for non-invasive methods for the identification of biomarkers of nephrotoxicity and rejection, and to better characterize the inflammation status, led us to conduct analysis of the exosomal content, allowing us to observe the presence of serum cytokines which, although in low amounts, suggest a possible role of these in the inflammatory process mediated by exosomal vesicles. Finally, following the genetic SLA typing for the determination of the donors and the recipients, in an accessory project, we got a controlled and stable colony of pigs with SLA defined in homozygosis through the coupling of specific pathogen free pigs (SPF) for DQB-1 and SLA-1 genes.

Introduzione: l’epatite cronica C è la più comune infezione virale trasmessa per via ematica e la principale causa di mortalità tra le epatopatie. La terapia antivirale può prevenire la progressione della malattia nei pazienti HCV. Telaprevir e simeprevir sono Direct Acting Antivirals e due inibitori delle proteasi, utili nell’eradicazione del virus. Scopo: stabilire l’efficacia e sicurezza di un regime di terapia antivirale con telaprevir, pegIFN/ribavirin e di uno con simeprevir-sofosbuvir+/- ribavirina. Metodi: 35 pazienti venivano consecutivamente arruolati nel gruppo telaprevir (54.3% maschi, età mediana 61, 43-71) e confrontati con 70 controlli, selezionati random da una popolazione di pazienti trattati con simeprevir-sofosbuvir e appaiati per età, sesso e fibrosi. Erano valutati l’efficacia misurata attraverso la risposta virologica sostenuta (SVR) e il miglioramento dei parametri biochimici, e la sicurezza. Risultati: i pazienti trattati con telaprevir presentavano eventi avversi nel 94.2% dei casi contro il 28.6% del gruppo simeprevir (p.000). Gli eventi avversi di grado severo si concentravano poi tutti nel gruppo telaprevir (20% vs 0%, p.000). Il più comune evento avverso in entrambi i gruppi era rappresentato dall’anemia (77.1% nel gruppo telaprevir va 14.3% nel gruppo simeprevir, p 0.000). L’SVR era del 91.4% nei casi e del 71.4% nei controlli (p 0.01). L’utilizzo di ribavirina, il tipo di genotipo 1 e lo stadio di fibrosi, non influenzavano i tassi di SVR. Conclusioni: il nostro studio ha mostrato che il telaprevir è meno efficace e sicuro del simeprevir nei pazienti con fibrosi avanzata o cirrosi epatica. I dati confermano l’indicazione a preferire i regimi liberi da interferone a quelli che lo contengono ancora. / Introduction: Chronic hepatitis C (CHC) is the most common viral infection blood-transmitted and it is the leading cause of death from liver disease. Antiviral therapy can prevent disease progression in patients with CHC. Telaprevir and Simeprevir are Direct Acting Antivirals and two protease inhibitor, useful in the eradication of the virus. Aim: to assess the safety and efficacy of telaprevir-based antiviral therapy with pegIFN/ribavirin or simeprevir-based antiviral therapy with sofosbuvir ± ribavirin. Methods: consecutive 35 CHC patients (54.3% males, median age 61, range 43-71) were enrolled in telaprevir group and compared with 70 controls, randomly selected from the population of patients treated with simeprevir-sofosbuvir and matched by age ± 5 years, sex and degree of fibrosis. Efficacy by sustained virological response (SVR) and improvement of laboratory tests and safety were evaluated. Results: patients treated with telaprevir had adverse events in 94.2% of cases while occurred in 28.6% of patients treated with simeprevir (p.000). Severe adverse events occurred all in telaprevir group (20% vs 0%, p 0.000). The most common adverse event in both groups was anemia (77.1% in telaprevir treatment vs 14.3% in simeprevir treatment, p 0.000). SVR was 91.4% in cases and 71.4% in controls (p 0.01). Use of ribavirin, type of genotype 1 and stage of fibrosis did not affect SVR rates. Conclusion: our study showed the telaprevir is less effective and safe compared to simeprevir in patients with advanced fibrosis or cirrhosis. The data confirm the indication to prefer interferon free regimens to those still based on interferon in this setting.

L'ipossia è una condizione patologica determinata da un ridotto apporto di ossigeno a livello tissutale. A partire dagli anni '60, Tomlinson e Gray hanno dimostrato la presenza di regioni di scarsa ossigenazione nel cancro del polmone e hanno osservato che questa caratteristica del microambiente tumorale è associata ad una maggiore resistenza alla radioterapia. La determinazione dell'ipossia nei tumori è pertanto della massima rilevanza clinica, in quanto l'aggressività del tumore, la deriva metastatica, il mancato controllo della neoplasia, l'aumento del rischio di recidiva e, in definitiva l'esito sfavorevole sono associati all'ipossia. Negli ultimi decenni, c'è stato un crescente interesse nello sviluppo di metodi per la valutazione dell'ossigenazione tumorale. Questi metodi possono essere invasivi, come il sensore polarografico di O2, o non invasivi, basati principalmente sulle tecniche di imaging. Le modalità di imaging sono senza dubbio le più interessanti perché garantiscono una visualizzazione onnicomprensiva del tessuto patologico e sono in grado di identificare il fenomeno anche in luoghi inaccessibili alle procedure invasive. Tra le modalità di imaging per la valutazione dell'ipossia, la tomografia ad emissione di positroni (PET) è una delle più studiate, in quanto offre: (a) ampio assortimento di radiofarmaci; (B) buona risoluzione intrinseca; (C) rappresentazione tridimensionale (3D); (D) (semi)quantificazione; (E) maggiore facilità per il paziente, e (f) riproducibilità dei dati. L'oggetto della tesi attuale è quello di studiare il ruolo dell'imaging molecolare alla PET nell'ipossia tumorale. Il testo è diviso in quattro sessioni distinte incentrate nel fornire in primis una panoramica sui radiofarmaci principali (Sessione-1), poi nella valutazione della correlazione tra l'espressione tissutale d'ipossia e l'imaging alla 18F-FDG PET/TC (Sessione-2). Le altre due sessioni analizzano l'impatto prognostico del tracciante per l'ipossia (64Cu-ATSM) nei tumori solidi (Sessione-3), seguito da una sofisticata analisi frattale di confronto fra le acquisizioni precoci e tardive alla 64Cu ATSM PET/TC nei tumori solidi (Sessione-4). / Hypoxia is a pathological condition caused by a reduced oxygen supply at the tissue level. Since the 60’s, Tomlinson and Gray have demonstrated the presence of regions of poor oxygenation in lung cancer, and noted that this characteristic of tumor microenvironment is associated to increased resistance to radiotherapy. The detection of hypoxia in tumors is therefore of utmost clinical relevance, because tumor aggressiveness, metastatic drift, failure to disease control, increased risk of recurrence and ultimately poor outcome are associated with hypoxia. In recent decades, there has been an increasing interest in developing methods for the assessment of tumor oxygenation. These methods can be invasive, such as the polarographic O2-sensor, or non-invasive, mainly based on imaging techniques. Imaging modalities are undoubtedly the most appealing techniques for this purpose, because they guarantee an all-encompassing visualization of the pathological tissue and can identify the phenomenon even at sites inaccessible to invasive procedures. Among the image-based modalities for hypoxia assessment, positron emission tomography (PET) is one of the most extensively investigated, because it offers: (a) broad assortment of radiopharmaceuticals; (b) good intrinsic resolution; (c) three-dimensional (3D) tumor representation; (d) (semi)quantification of the hypoxic burden; (e) patient friendliness, and (f) reproducibility. The object of the current thesis is to investigate the role of molecular imaging with PET in cancer hypoxia. The text is divided into four different sessions focused on giving at first an insight on principal radiopharmaceuticals applied for hypoxia imaging (Session-1), then concentrating on the correlation of tissue expression of hypoxia and imaging findings on 18F-FDG PET/CT (Session-2). The next two sessions will analyze the prognostic impact of the hypoxia-specific tracer (64Cu-ATSM) in solid tumors (Session-3), followed by a sophisticated ad hoc computer-aided fractal geometry based analysis of DICOM images for early and late acquisitions on 64Cu-ATSM PET/CT in solid tumors (Session-4).

Chronic pancreatitis is an established risk factor for pancreatic ductal adenocarcinoma (PDAC) development. Polymorphisms in the pro-inflammatory cytokine gene interleukin 1β (IL-1β), as well as high IL-1β or low IL-1 receptor antagonist (IL-1RA) serum levels, are associated to worse prognosis in PDAC patients. To characterize the role of IL-1β in pancreatic tumorigenesis, we generated a transgenic mouse model bearing KRASG12D mutation combined to chronic inflammation induced by pancreatic overexpression of human IL-1β (KC-IL-1β). We found that IL-1β overexpression induced PDAC onset in 6 out of 13 KRASG12D bearing animals (46%), with a median overall survival of 10.5 months, compared to only 1 out of 13 mice carrying KRASG12D mutation alone (KC)(7.7% p= 0.02). In primary pancreatic KRASG12D organoid cultures, IL-1β exposure increased the number of spheroids and induced gene expression changes consistent with epithelial to mesenchymal transition (EMT), as shown by increased expression of vimentin, Zeb1, Snail. All these changes were counteracted using a recombinant human IL-1receptor antagonist (IL1-RA). Consistently, immuno-histochemical analysis confirmed that in KC-IL-1β tumor epithelial cells and metastasis were strongly positive for vimentin. The relevance of these findings was confirmed in human PDAC, showing higher IL-1 receptor I (IL1-RI) and vimentin expression in tumor tissue compared with adjacent normal pancreas. Regarding the mechanism involved in EMT activation, IL-1β exposure was found to induce an up-regulation of ribosome biogenesis rate, with consequent down-regulation of p53 protein expression which has been shown to be responsible for EMT changes. The finding that IL-1β/IL1-RI inflammatory pathway stimulates acinar cell proliferation and promotes EMT provides the rationale for a therapeutic strategy based on IL-1β receptor blockade to counteract inflammation-induced pancreatic tumorigenesis