Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

The aim of this PhD thesis " Simulation Guided Navigation in cranio- maxillo- facial surgery : a new approach to Improve intraoperative three-dimensional accuracy and reproducibility during surgery ." was at the center of its attention the various applications of a method introduced by our School in 2010 and has as its theme the increase of interest of reproducibility of surgical programs through methods that in whole or in part are using intraoperative navigation. It was introduced in Orthognathic Surgery Validation a new method for the interventions carried out according to the method Simulation Guided Navigation in facial deformities ; was then analyzed the method of three-dimensional control of the osteotomies through the use of templates and cutting of plates using the method precontoured CAD -CAM and laser sintering . It was finally proceeded to introduce the method of piezonavigated surgery in the various branches of maxillofacial surgery . These studies have been subjected to validation processes and the results are presented . / Obiettivo di questa tesi di Dottorato “Simulation Guided Navigation in cranio-maxillo-facial surgery: a new approach to improve intraoperative three-dimensional accuracy and reproducibility during surgery.” ha avuto al centro delle proprie attenzioni le varie applicazioni di una metodica introdotta dalla ns. Scuola nel 2010 e che ha come tema di interesse l’aumento delle riproducibilità dei programmi chirurgici mediante metodiche che in toto o in parte utilizzano il navigatore intraoperatorio. Si è introdotto in Chirurgia Ortognatica un nuovo Metodo di Validazione per gli interventi effettuati secondo la metodica Simulation Guided Navigation nelle malformazioni facciali ; si è poi analizzata la metodica di controllo tridimensionale delle osteotomie mediante l’utilizzo delle dime di taglio e delle placche premodellate mediante metodica CAD-CAM e sinterizzazione laser. Si è infine proceduto ad introdurre la metodica di chirurgia piezonavigata alle varie branche di chirurgia maxillo-facciale. Tali studi sono stati sottoposti a processi di validazione ed i risultati vengono presentati.

The Notch1 signaling pathway has a pivotal role in cell fate regulation and has been found to be critically deranged in different cancers, including colorectal cancer (CRC). Inflammation is known to play an important role in the pathogenesis of CRC and a prominent function of Notch1 during inflammation has been recently demonstrated. Epithelial to Mesenchymal Transition (EMT), a crucial process in the malignant transformation, is modulated by inflammation and Metalloproteinase-9 (MMP9) is involved in this interaction. Eicosapentaenoic Acid is an omega-3 polyunsaturated fatty acid (omega-3 PUFA) known for its anti-inflammatory properties as well as for its capability in preventing colon cancer development both in sporadic and in hereditary settings. In particular, our group has demonstrated that an extra-pure formulation of Eicosapentaenoic Acid as the free fatty acid (EPA-FFA) protects from CRC development in a mouse model of colitis-associated cancer (CAC) by modulating the Notch signaling pathway. In the present work, we re-created an in vitro model of inflammatory-driven CRC by exposing colon cancer cells to a cytokine-enriched conditioned medium (CM) obtained from THP-1-differentiated macrophages. We found, for the first time, that CM strongly induces Notch1 signaling and EMT markers, increasing the capability of cells to invade. Importantly we found that, upon CM exposure, Notch1 signaling is dependent on MMP9 expression. Finally, we show that a non-cytotoxic pre-treatment with 50 μM of EPA-FFA for 72h counteracts the effect of inflammation on Notch1 signaling and EMT, leading to a reduction of invasiveness. Taken together, our data demonstrate that in CRC inflammation up-regulates Notch1 signaling through MMP9 and that this mechanism can be effectively counteracted by EPA-FFA.

Obiettivo Primo lavoro (1): convalidare il ruolo del nuovo Child-Pugh 0 in pazienti con HCC. Secondo lavoro (2): esplorare il possibile ruolo del Child-Pugh nel rilevare l'impatto funzionale a breve termine in pazienti cirrotici trattati con DAA. Metodi (1) sono stati analizzati 5456 pazienti con prima diagnosi di HCC, divisi in gruppi in base alla classe CP: 0 (343 pts), A (3143 punti), B (1628 punti), C (342 pts). (2) 53 pazienti cirrotici HCV trattati con DAA sono stati analizzati. Durante il trattamento e alla fine è stato registrato il cambiamento del CP. Risultati (1) 10,9% dei pazienti CP A sono stati riclassificati come CP 0. La sopravvivenza mediana globale differiva significativamente tra i gruppi (mesi; 95% CI): CP 0 64 (54,5-73,4), A 43 (40.7-45.3), B 21 (19,1-22,8), C 8 (6,7-9,2), p <0,0001. Il confronto tra sopravvivenze del CP 0 vs A, B e C era significativamente differente (p <0,0001 a tutte le associazioni). La prognosi dei pazienti in BCLC-B differiva in base alla funzione epatica (0 vs A vs B, p <0,0001). (2) Pazienti con miglioramento del CP di almeno un punto: 41,5% (dopo 12 settimane di trattamento), 38% (alla fine del trattamento) e 54,4% (in SVR12); pazienti con miglioramento della classe CP: 32,1% (dopo 12 settimane di trattamento), 30% (alla fine del trattamento) e 46,9% (in SVR12); pazienti che sono passati dal CP A alla CP 0: 20,6% (dopo 12 settimane di trattamento), 22,6% (alla fine del trattamento) e 44,4% (in SVR12). Conclusioni CP 0 identifica un diverso sottogruppo di pazienti con HCC con una migliore prognosi. Ciò si ripercuote non solo sulla previsione dell’outcome, ma anche sull’allocazione al trattamento della neoplasia, meglio stratificando il BCLC-B. L'andamento del CP durante il trattamento DAA esprime il guadagno funzionale che il paziente HCV trattato ottiene sul breve termine. / Aim First work (1): to validate the role of new CP 0 in HCC patients. Second work (2): to explore the possible role of CP to detect the functional impact on the short term in cirrhotic patients treated with DAA. Methods (1) 5456 patients with first diagnosis of HCC were analyzed. They were divided in groups according to CP class: 0 (343 pts), A (3143 pts), B (1628 pts), C (342 pts). (2) 53 cirrhotic HCV patients treated with DAA were analyzed. During treatment and at the end of it the change of CP was recorded. Results (1) 10.9% of CP A patients were reclassified as CP 0. Median overall survivals statistically differed among groups (months; 95% CI): CP 0 64 (54.5-73.4), A 43 (40.7-45.3), B 21 (19.1-22.8), C 8 (6.7-9.2), p<0.0001. Comparisons between survivals of CP 0 vs A, B and C were also statistically different (p<0.0001 in all associations). The prognosis of patients in BCLC-B stage differed according to liver function (0 vs A vs B, p<0.0001). (2) Patients with CP score improvement of at least one point: 41,5% (after 12 weeks of treatment), 38% (at the end of treatment) and 54.4% (at SVR12); patients with CP class improvement: 32,1% (after 12 weeks of treatment), 30% (at the end of treatment) and 46,9% (at SVR12); patients who have passed from CP A to CP 0: 20,6% (after 12 weeks of treatment), 22,6% (at the end of treatment) and 44,4% (at SVR12). Conclusions CP class 0 identifies a different subgroup of HCC patients with better prognosis. It impacts not only on outcome prediction but also on treatment allocation, better stratifying the heterogeneous BCLC-B stage. The trend of CP score during DAA treatment expresses the functional gain and the prognostic impact that HCV treated patient gets on the short term.

The increasing occurrence of CRC developing in young patients with no identified genetic predisposition, defined as sporadic early onset colorectal cancers (EOCRCs), demands maintaining a high index of suspicion when people below 50 years of age present with symptoms. To define the clinicopathological features and the stage at presentation of EOCRCs, as well as to understand whether the histological, immunohistochemical and molecular analyses were associated with particular clinicopathological parameters and oncologic outcome, a total of 94 cases of EOCRCs diagnoses between 2006 and 2014 at the Sant’Orsola University Hospital were studied. Indeed, EOCRCs appear frequently as an aggressive disease located in the sigmoid colon and rectum, and most patients are symptomatic at the time of presentation, mainly presenting with rectal bleeding, haematochezia or abdominal pain. The genetic basis in the majority of early onset colorectal carcinomas remains unknown, however, most EOCRCs, not related hereditary syndromes, appear to arise through the same pathways as sporadic CRCs, such as the classical adenoma-carcinoma sequence, but with only rare involvement of the methylator pathway. Taken together, the analyses described in this study suggest that, in the absence of screening programs for patients under 50 years of age, the risk factor of a family history and the presence of symptoms may be considered as an indication for prompt endoscopic investigation in these patients, since this may reduce the stage of disease at diagnosis and likely have an impact on improving survival.

Peripheral T-cell lymphomas-not otherwise specified (PTCL/NOS) are the most common T-cell neoplasms. This study sought to reshape the PTCL/NOS sub-classification (including its two main morphological variants, Lennert lymphoma, LL, and Follicular variant, F-PTCL) based on the correspondence between their molecular features and those of different functional T-cell subsets, also assessing the clinical impact of such an approach. We found that PTCLs/NOS could be divided into groups corresponding to T-cell subsets differently reliant on transcription regulators including mTOR and FOXP3, and identified minimal gene sets discriminating among these groups. Notably, by grouping tumors according to their dependency on master regulators of T-lymphocyte fate, we identified three groups (T-cytotoxic, Treg/TFH, and other-T-helper) characterized by specific genetic patterns and significantly different clinical outcomes. Immunohistochemistry partially substituted for the molecular analysis by consistently recognizing only Treg and TFH cases. Finally, targeted inhibition of MTOR in T-helper cases (that were characterized by genetic lesions targeting the pathway) was proved to be effective ex vivo. We conclude that PTCL/NOS can be divided into subgroups corresponding to different cellular counterparts, characterized by different genetic patterns and possibly sensitivity to specific therapeutic approaches. Furthermore, we identified different gene and microRNA signatures for LL capable of differentiating it from other PTCL/NOS and enriched in cytotoxic function. Moreover, PI3K/Akt/mTOR pathway emerged as novel therapeutic targets for LL. Additionally, LL showed some differences with other PTCL/NOS in terms of clinical features, all supporting its recognition as a distinct entity. Besides, we found that F-PTCL has a distinct molecular signature more similar to PTCL/NOS rather than AITL, and therefore cannot be included among AITLs at least based on GEP, although this necessities more genetic studies. Overall, these results may impact on PTCL classification as well as on future studies aimed to define the more appropriate therapeutic strategy for each identified subgroup/entity.

Caratterizzazione di EPC in pazienti con malattia renale cronica. Background: Gli effetti del recettore della vitamina D (VDR)e dell'espressione dell' osteocalcina (OCN), così come VDR agonista terapia (VDRA) su circolanti cellule progenitrici endoteliali (EPCs) non è stato ancora chiarito. Metodi: sono state analizzat EPCs in 23 controlli sani e 53 pazienti sottoposti a dialisi. La percentuale di EPCs (CD34+KDR+CD133+/-CD45-) VDR+/- o OCN+/- è stata analizzata in citometria a flusso e correlata con molecole coinvolte nelle malattie. Risultati: EPCs aumentano nei pazienti con CKD trattati con VDRAs. EPCsOCN +/- in pazienti non trattati con VDRAs correlano positivamente con il calcio sierico e reticolociti, e negativamente con DKK1. La percentuale di EPCsVDR +/- correla negativamente con OPN.  EPCsOCN + nei pazienti trattati con VDRAs correla positivamente con la vitamina D. La percentuale di EPCsVDR +/- positivamente correla con IL-6. Conclusioni: la terapia VDRA influenza l'espressione VDR e di OCN su EPCs circolanti. Dal momento che l'espressione OCN può contribuire alla calcificazione vascolare, ipotizziamo un putativo effetto pro-calcificazione di VDRA. Effetti immunomodulatori di EPO. Background: Il cloruro di sodio spinge l'induzione di cellule TH17 patogeni e TH1. La correzione dell'anemia con eritropoietina (EPO) è associato ad un miglioramento della tolleranza del trapianto di rene. Prove emergenti indicano che queste osservazioni possano essere eritropoiesi-indipendente e che l'EPO presenta proprietà immunosoppressive. Metodi: esaminato gli effetti del trattamento con EPO e sale su cellule T umana, apoptosi e la produzione di INF-γ, l'effetto sulla iTh17 e iFoxP3. Le cellule sono state analizzate con l'analisi di citometria a flusso. Risultati: NaCl aumenta la proliferazione di CD4+ e CD8+, iTh17 e la produzione di INFγ. Questo effetto è prevenuto da EPO. EPO aumenta l'induzione di Foxp3. Non cambia l'apoptosi e la stabilità di FoxP3. EPO è in grado di contiene l'effetto pro infiammatorio del sale / First project: Characterization of EPCs in patients with CKD. Background: The effects of vitamin D receptor (VDR) and osteocalcin (OCN) expression as well as VDR agonist (VDRA) therapy on circulating endothelial progenitor cells (EPCs) has not been elucidated yet. Methods: we therefore analyzed EPCs in 23 healthy controls and 53 patients undergoing dialysis. The percentage of EPCs (CD34+CD133+/-KDR+CD45-) expressing VDR or OCN were analyzed using flow cytometry and correlated with molecules involved in diseases. Results: EPCs increase in CKD patients treated with VDRAs. EPCsOCN+/- in patients untreated with VDRAs correlated positively with serum calcium and reticulocytes, and negatively with DKK1. The percentage of EPCsVDR+/- correlated negatively with OPN. EPCsOCN+ in patients treated with VDRAs correlated positively with vitamin D. The percentage of EPCsVDR+/- correlated positively with IL-6. Conclusions: our data suggest that VDRA therapy influence VDR and OCN expression on circulating EPCs. Since OC expression may contribute to vascular calcification, we hypothesize a putative pro-calcifying effect of VDRA. Second project: Immunomodulatory effects of EPO. Background: Sodium chloride drives the induction of pathogenic TH17 cells and TH1. Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. Methods: examined the effects of treatment with EPO and salt on human T-cell alloimmunity, the apoptosis and the production to INF-γ, the effect on iTh17 and iFoxP3. The cells were analyzed with flow cytometry analysis. Results: NaCl increases the proliferation of CD4+ and CD8+ cells, iTh17 and the production of INFγ. This effect is prevented by EPO. EPO increases the induction of Foxp3. It does not change the apoptosis and stability of FoxP3. Epo contains the inflammatory effect of NaCl. EPO in the kidney, where NaCl is high, may have a tolerance effect.

Cell competition describes the result of a mechanism of fitness comparison undertaken by cells inhabiting the same tissue, that leads to the elimination of the weakest cells and, in the physiology, to the formation of a homogeneous organ. Over the years, many molecules have been identified that are involved in cell competition and among them MYC oncoprotein: from Drosophila to mammals, cell populations characterised by higher expression of MYC induce apoptotic death of the neighbours, allowing the fittest to acquire an advantage in space occupancy. My work defined the presence of markers of MYC-mediated cell competition in primary and secondary human carcinomas and demonstrated through experiments in human cancer cell lines that MYC modulation is per se sufficient to induce competitive behaviours in both genetically distant and identical cells. Noteworthy, MYC under-regulation in the fittest cell line is sufficient to undermine its competitive status, suggesting a role for MYC-mediated cell competition in the selective growth of tumour clones and, as a consequence, in cancer evolution. In addition, I was able to demonstrate a functional cooperation between MYC and p53 in this phenomenon. The data obtained in the Drosophila model, where MYC over-expressing and MYC knock-down clones have been induced within a growing tumour, suggest that MYC-mediated cell competition is normally at work in these malignant cells, and it shapes cancer evolution through the elimination of the less fit cells (with lower levels of MYC) and the expansion of the most performant ones (with higher levels of MYC), demonstrating an evolutionary role played in defining the composition and the size of the final mass.

L’adenocarcinoma dell’intestino tenue (SBA) è una neoplasia estremamente rara nella popolazione generale. La letteratura suggerisce che la malattia celiaca (MC) sia associata ad un aumentato rischio di sviluppare un SBA, ma non ci siano dati sulle caratteristiche di questa variante associata a MC. Lo scopo dello studio è consistito nel chiarire la prevalenza del SBA in una coorte soggetti con MC, definendo le loro caratteristiche cliniche, istologiche ed immunologiche. Sono stati studiati retrospettivamente (1995-2014) tutti i casi di SBA trovati in associazione a MC. Le biopsie dei casi identificati sono state valutate attraverso indagini immunoistochimiche impiegando anticorpi monoclonali che riconoscono markers epiteliali intestinali (e.g. MUC2, CDX2 e CD10) e gastrici (e.g. MUC5AC e MUC6). Sono inoltre state ricercate eventuali mutazioni di KRAS, NRAS e BRAF. Sono stati identificati 5 SBA su 779 pazienti con MC (0,65%), tutte di sesso femminile età media 53. La tipizzazione dell'HLA ha mostrato un DQ2+ in tutti i casi. Al momento della diagnosi di SBA il quadro clinico di questi pazienti era caratterizzato da diarrea in 3 casi e da episodi subocclusivi negli altri due casi. La più frequente localizzazione anatomica dell’SBA era il digiuno. In nessuno dei 5 casi lo SBA è stato preceduto da una malattia celiaca refrattaria. L’esame istologico eseguito mostrava la presenza in tre casi di un carcinoma di alto grado, scarsamente differenziato (grado III-IV). La sopravvivenza a 5 anni è risultata molto migliore rispetto al SBA sporadico. KRAS è stato trovato mutato in 2/5 casi. L’ SBA associato a MC sembra avere caratteristiche cliniche, istologiche e fenotipiche differenti rispetto al SBA sporadico. In particolare: a) il più frequente coinvolgimento del sesso femminile; b) l’età di esordio più giovane; c) la localizzazione digiunale; d) una migliore prognosi associata a positività per CDX2; e) presenza di neoplasie con KRAS mutato. / The small bowel adenocarcinoma (SBA) is a very rare neoplasia in the general population. Previous studies suggest that celiac disease (CD) is associated with an increased risk in developing a SBA. Unfortunatly, there are no information about the features of this cancer when associated with CD. The aims of the present study were to shed light on the prevalence of SBA in a CD patients cohort and to define its clinical, histological and immunological features. We retrospectively investigated all the cases of SBAs in a cohort of CD patients during a 19 years period (1995-2014). Biopsies from selected cases were analyzed by immunohistochemestry, looking for intestinal and gastric markers, using monoclonal antibodies against MUC2, CDX2, CD10, MUC5AC, MUC6. Moreover, we checked the presence of KRAS, NRAS and BRAF mutations. We identified 5 cases of SBA in a population of 779 CD patients (0,65%). All the SBA found were in female patients with a mean age of 53 years. The HLA genotyping revealed a positivity for the DQ2+ in all cases. At onset SBA showed a clinical picture characterized by diarrhoea in 3 cases and subocclusion in 2 cases. Refractory CD never preceded the onset of a SBA. Th histologica evaluation revealed a high grade, poorly differentiated neoplasia in 3 cases (G3-G4). Overall survival at 5 years was extremely better than that of the sporadic SBA. A mutation of KRAS was found in 2/5 cases. In conclusion, the SBA associated with CD showed different features in comparison to the sporadic one, in particular: a) a female gender predominace, b) a lower median age at diagnosis, c) a preferred jejunal localization, d) a better prognosis (in particular when associated witha CDX2 positivity) and e) for the finding of KRAS mutations.

PURPOSE: The aim of this randomized clinical trial is to compare 2 different approaches for Guided Bone Regeneneration (GBR) in posterior region of atrophic mandibles regarding: I) the amount of vertical bone regeneration, II) the type and frequencies of surgical and healing complications, III) the implants primary stability and the osseointegration rate. MATERIAL AND METHODS: Forty partially edentulous patients with atrophic posterior mandible, who require a three-dimensional bone regeneration and an implant-prosthetic rehabilitation, were treated according the following protocol. Patients were randomly divided into two study group: 20 patients were treated with non-resorbable d-PTFE titanium-reinforced membranes (group A); and 20 patients with titanium mesh covered by cross-linked collagen membranes (group B). All patients received two or more double variable thread tapered implants and a mixture of autogenous bone and bone allograft according to one-stage GBR approach. In each group were evaluated the amount of vertical bone regeneration and the insertion torque value (IT) and implant stability quotient (ISQ) of all implants. In addition, all complications were analyzed, distinguishing between “surgical” and “healing” complications. RESULTS: The vertical bone regeneration was 4.3 ± 1.2 mm in the group A and 4.2 ± 1.1 in the group B. 99% of implants showed an optimal primary stability with insertion torque values > 35 Ncm and ISQ values > 60. All implants achieved a successful osseointegration. In the group A, surgical and healing complication rates were 5.0% and 15.9% respectively, In the group B, complication rates were 15.0% and 21.1% respectively. CONCLUSIONS: The preliminary results of this RCT suggested that both GBR approaches are predictability and effectiveness for the restoration of atrophic posterior mandible. Double variable thread tapered implants permitted to achieve an adequate primary stability in order to perform an one-stage GBR approach. / PURPOSE: The aim of this randomized clinical trial is to compare 2 different approaches for Guided Bone Regeneneration (GBR) in posterior region of atrophic mandibles regarding: I) the amount of vertical bone regeneration, II) the type and frequencies of surgical and healing complications, III) the implants primary stability and the osseointegration rate. MATERIAL AND METHODS: Forty partially edentulous patients with atrophic posterior mandible, who require a three-dimensional bone regeneration and an implant-prosthetic rehabilitation, were treated according the following protocol. Patients were randomly divided into two study group: 20 patients were treated with non-resorbable d-PTFE titanium-reinforced membranes (group A); and 20 patients with titanium mesh covered by cross-linked collagen membranes (group B). All patients received two or more double variable thread tapered implants and a mixture of autogenous bone and bone allograft according to one-stage GBR approach. In each group were evaluated the amount of vertical bone regeneration and the insertion torque value (IT) and implant stability quotient (ISQ) of all implants. In addition, all complications were analyzed, distinguishing between “surgical” and “healing” complications. RESULTS: The vertical bone regeneration was 4.3 ± 1.2 mm in the group A and 4.2 ± 1.1 in the group B. 99% of implants showed an optimal primary stability with insertion torque values > 35 Ncm and ISQ values > 60. All implants achieved a successful osseointegration. In the group A, surgical and healing complication rates were 5.0% and 15.9% respectively, In the group B, complication rates were 15.0% and 21.1% respectively. CONCLUSIONS: The preliminary results of this RCT suggested that both GBR approaches are predictability and effectiveness for the restoration of atrophic posterior mandible. Double variable thread tapered implants permitted to achieve an adequate primary stability in order to perform an one-stage GBR approach.