La chirurgia della trachea è sempre stata una sfida per il chirurgo in considerazione delle notevoli difficoltà nel ripristinare la anatomia e la funzione dell’organo. Attualmente il trapianto di trachea umano rivascolarizzato non è stato ancora realizzato, principalmente a causa della mancanza di un peduncolo vascolare tracheale noto. Nel 2008 l’otorinolaringoiatra belga Paul Delaere ha descritto una tecnica di trapianto di trachea da cadavere mediante il confezionamento di un lembo composito. Il lembo antibrachiale utilizzato è una ottima alternativa, ma oggi sono più utilizzati i lembi perforanti rispetto ai lembi assiali, in base a una serie di vantaggi ben noti. La possibilità di utilizzare il lembo perforante anterolaterale di coscia al posto del lembo “cinese” come base per il trapianto composito sfrutta i vantaggi di questo tipo di lembo. Nello studio è stata valutata la fattibilità tecnica del lembo composito mediante una valutazione anatomica su cadavere. / Trachea surgery has always been a challenge for the surgeon in view of remarkable difficulty in restoring the anatomy and the function. Currently revascularized human trachea transplantation is not possible due to the lack of a single vascular pedicle. In 2008 Paul Delaere described a tracheal transplantation technique using a cadaveric donor. The chinese flap used by Delaere to harvest the compiste flap is a good alternative, but today perforator flaps represent a better choice. The possibility to use the anterolateral thigh perforator flap in place of the chinese flap was studied on cadavers in the present stduy.

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.

Background: cognitive impairment is one of the non motor features widely descripted in parkinsonian syndrome, it has been related to the motor characteristics of the parkinsonian syndrome, associated with neuropsychiatric dysfunction and the characteristic sleep and autonomic features. It has been shown to be highly prevalent at all disease stages and to contribute significantly to disability. Objectives: aim of this study is to evaluate longitudinally the cognitive and behavioral characteristics of patients with a parkinsonian syndrome at onset; to describe the cognitive and behavioral characteristics of each parkinsonian syndrome; to define in PD patients at onset the presence of MCI or Parkinson disease dementia; to correlate the cognitive and behavioral characteristics with the features of the parkinsonian syndrome and with the associated sleep and autonomic features. Results: we recruited 55 patients, 22 did not present cognitive impairment both at T0 and at T1. 18 patients presented a progression of cognitive impairment. Progressive cognitively impaired patients were older and presented the worst motor phenotype. Progression of cognitive impairment was not associated to sleep and autonomic features. Conclusion: the evaluation of cognitive impairment could not be useful as a predictor of a correct diagnosis but each non motor domain will help to clarify and characterize the motor syndrome. The diagnosis of parkinsonian disorders lies in building a clinical profile in conjunction with other clinical characteristics such as mode of presentation, disease progression, response to medications, sleep and autonomic features.

Background. Ageing and inflammation are critical for the occurrence of aortic diseases. Extensive inflammatory infiltrate and excessive ECM proteloysis, mediated by MMPs, are typical features of abdominal aortic aneurysm (AAA). Mesenchymal Stromal Cells (MSCs) have been detected within the vascular wall and represent attractive candidates for regenerative medicine, in virtue of mesodermal lineage differentiation and immunomodulatory activity. Meanwhile, many works have underlined an impaired MSC behaviour under pathological conditions. This study was aimed to define a potential role of vascular MSCs to AAA development. Methods. Aortic tissues were collected from AAA patients and healthy donors. Our analysis was organized on three levels: 1) histology of AAA wall; 2) detection of MSCs and evaluation of MMP-9 expression on AAA tissue; 3) MSC isolation from AAA wall and characterization for mesenchymal/stemness markers, MMP-2, MMP-9, TIMP-1, TIMP-2 and EMMPRIN. AAA-MSCs were tested for immunomodulation, when cultured together with activated peripheral blood mononuclear cells (PBMCs). In addition, a co-colture of both healthy and AAA MSCs was assessed and afterwards MMP-2/9 mRNA levels were analyzed. Results. AAA-MSCs showed basic mesenchymal properties: fibroblastic shape, MSC antigens, stemness genes. MMP-9 mRNA, protein and enzymatic activity were significantly increased in AAA-MSCs. Moreover, AAA-MSCs displayed a weak immunosuppressive activity, as shown by PBMC ongoing along cell cycle. MMP-9 was shown to be modulated at the transcriptional level through the direct contact as well as the paracrine action of healthy MSCs. Discussion. Vascular injury did not affect the MSC basic phenotype, but altered their function, a increased MMP-9 expression and ineffective immunmodulation. These data suggest that vascular MSCs can contribute to aortic disease. In this view, the study of key processes to restore MSC immunomodulation could be relevant to find a pharmacological approach for monitoring the aneurysm progression.

No description available.

Oggetto della tesi e' l'approfondimento su tecniche e metodiche di preservazione del polmone isolato per lo studio ecografico. E' discussa l'appropriatezza sull'uso degli ultrasuoni in corso di chirurgia mini invasiva polmonare, obiettivo di una ricerca sperimentale. Il razionale dello studio si fonda sull'indicazione all'exeresi chirurgica di noduli polmonari di diametro inferiore al centometro, ovvero di diametro superiore ma localizzati in aree centrali del polmone. Queste lesioni sono sempre piu' frequentente diagnosticate per mezzo di avanzate tecniche di imaging. L'atto chirurgico ha scopo terapeutico quando sia stata posta la diagnosi di neoplasia maligna, diagnostico-terapeutico quando non sia ancora ottenuta la tipizzazione istologica della lesione. La tecnica toracoscopica offre numerosi vantaggi rispetto alle tecniche chirurgiche tradizionali ma presenta il grave limite di non permettere la palpazione diretta del tessuto polmonare e la localizzazione della formazione tumorale quando essa non sia visibile macroscopicamente. Gli ultrasuoni sono stati utilizzati con successo per indirizzare la localizzazione del nodulo polmonare. Scopo dello studio sperimentale e' quello di confrontare tecniche diverse di preservazione del polmone isolato in un modello animale, comparando catatteristiche e prestazioni di sonde ecografiche differenti per tipologia. Del tutto recentemente, in ambito trapiantologico, sono state proposte tecniche di preservazione organica utili ai fini di uno studio anatomico sperimentale particolareggiato (EVLP) e moderna e' da considerarsi la concezione di mezzi tecnici specifici per la localizzazione di bersagli intrapolmonari. La tecnica clinica applicata allo studio del particolare ecografico, nel modello animale, ha reso comprensibile e meglio definito il ruolo delle sonde ecografiche nell'individuazione di forme tumorali suscettibili di exeresi definitiva. / The primary focus of this dissertation is research about techniques and methods of isolated lung tissue preservation for sonographic investigation. Doctoral thedis describes the use of ultrasound probes in an experimental model and its clinical application during minimally invasive pulmonary surgery. The rationale of the study is based on the indication to surgical procedure and when is clinically in the treatment of solitary pulmonary nodules (<1 cm or greater but more central in visible appearance). In the era of CT screening programs, these lesions are more frequently diagnosed with advanced imaging techniques. Surgical procedure is therapeutic when a diagnosis of malignancy is obtained, but also diagnostic if we must correct the technique to the histologic pattern or ascertain pathological growth of the lesion. Thoracoscopic techniques offers several advantages over traditional "open" procedures but shows clinical limits. These mininimally invasive approaches do not allow to direct handling of tumor when is not macroscopically visible. Ultrasound have been successfully used to guide localization of pulmonary nodules. The experimental study compare different techniques of lung tissue preservation in a porcine model, the proceed to analysis of diagnostic performances related to a panel of echographic probes. More recently, a number of different techniques, adopted in lung transplantation programs, has been considered as useful for experimental anatomical study (EVLP) and several instruments of scientific research has been comceived for intrapulmomary localization of neoplastic lesions. These clinical techniques were originally applied in an animal porcine model, studied for scientific purpose, and described in a personal review of cases. Results of surgical interesr were related to scientific Literature in order to identify the possible role of echographic probes on clinical context.

The arousal scoring in Obstructive Sleep Apnea Syndrome (OSAS) is important to clarify the impact of the disease on sleep but the currently applied American Academy of Sleep Medicine (AASM) definition may underestimate the subtle alterations of sleep. The aims of the present study were to evaluate the impact of respiratory events on cortical and autonomic arousal response and to quantify the additional value of cyclic alternating pattern (CAP) and pulse wave amplitude (PWA) for a more accurate detection of respiratory events and sleep alterations in OSAS patients. A retrospective revision of 19 polysomnographic recordings of OSAS patients was carried out. Analysis was focused on quantification of apneas (AP), hypopneas (H) and flow limitation (FL) events, and on investigation of cerebral and autonomic activity. Only 41.1% of FL events analyzed in non rapid eye movement met the AASM rules for the definition of respiratory event-related arousal (RERA), while 75.5% of FL events ended with a CAP A phase. The dual response (EEG-PWA) was the most frequent response for all subtypes of respiratory event with a progressive reduction from AP to H and FL. 87.7% of respiratory events with EEG activation showed also a PWA drop and 53,4% of the respiratory events without EEG activation presented a PWA drop. The relationship between the respiratory events and the arousal response is more complex than that suggested by the international classification. In the estimation of the response to respiratory events, the CAP scoring and PWA analysis can offer more extensive information compared to the AASM rules. Our data confirm also that the application of PWA scoring improves the detection of respiratory events and could reduce the underestimation of OSAS severity compared to AASM arousal.

La Valvola Aortica Bicuspide (BAV) rappresenta la più comune anomalia cardiaca congenita, con un’incidenza dello 0,5%-2% nella popolazione generale. Si caratterizza per la presenza di due cuspidi valvolari anziché tre e comprende diverse forme. La BAV è frequentemente associata agli aneurismi dell’aorta toracica (TAA). La dilatazione dell’aorta espone al rischio di sviluppare le complicanze aortiche acute. Materiali e metodi Sono stati reclutati 20 probandi consecutivi sottoposti a chirurgia della valvola aortica e dell'aorta ascendente presso l'Unità di Chirurgia Cardiaca di Policlinico S.Orsola-Malpighi di TAA associata a BAV. Sono stati esclusi individui con una condizione sindromica predisponente l’aneurisma aortico. Ciascun familiare maggiorenne di primo grado è stato arruolato nello studio. L’analisi di mutazioni dell’intero gene ACTA2 è stata eseguita con la tecnica del “bidirectional direct sequencing”. Nelle forme familiari, l’intera porzione codificante del genoma è stata eseguita usando l’exome sequencing. Risultati Dopo il sequenziamento di tutti i 20 esoni e giunzioni di splicing di ACTA2 nei 20 probandi, non è stata individuata alcuna mutazione. Settantasette familiari di primo grado sono stati arruolati. Sono state identificate cinque forme familiari. In una famiglia è stata trovata una mutazione del gene MYH11 non ritenuta patogenetica. Conclusioni La mancanza di mutazioni, sia nelle forme sporadiche sia in quelle familiari, ci suggerisce che questo gene non è coinvolto nello sviluppo della BAV e TAA e, l’associazione che è stata riportata deve essere considerata occasionale. L’architettura genetica della BAV verosimilmente dovrebbe consistere in svariate differenti varianti genetiche che interagiscono in maniera additiva nel determinare un aumento del rischio. / The bicuspid aortic valve (BAV) is the most common congenital cardiac abnormality, with an incidence of 0.5% -2% in the general population. BAV is characterized by the presence of two valve cusps rather than three, and includes various forms. BAV is frequently associated with thoracic aorta aneurysms (TAA).The pathological dilatation of the aorta exposes these patients at risk of acute aortic complications associated with aneurysms, including dissection or rupture. Materials and methods We recruited 20 consecutive probands who underwent surgery of the aortic valve and ascending aorta at the Cardiac Surgery Unit of Policlinico S.Orsola-Malpighi for TAA associated with BAV, and who had their first degree relatives available. Individuals with a syndromic condition predisposing to aortic aneurysm were excluded. Each first degree family member (age> 18 years old) was recruited. Mutational analysis of the entire ACTA2 gene by bidirectional direct sequencing of amplified genomic DNA fragment with intron-based was performed. In the familiar forms, the entire coding portion of the genoma was done using the exome sequencing. Results After sequencing all 20 exons and splice junctions of ACTA2 in our 20 probands, no mutation was detected.Seventy-seven first-degree familiar members were enrolled in our study.Five familiar cases are identified: 2 of these underwent surgery for BAV and one for BAV+TAA.In one family we found a mutation on MYH11 gene but it was considered not pathogenic. Conclusion The lack of ACTA2 mutations, both in sporadic and in familiar forms, suggests that this gene is not involved in the development of BAV and TAA and their association, which was reported, has to be considerate occasional. In conclusion, the genetic architecture of the BAV would likely consist of several genetic variants that interact in an additive in determining an increased risk of development of BAV and dilatation of the thoracic aorta.

OBIETTIVO : Quantificare le CECs/ml nei pazienti affetti da ischemia critica (IC) degli arti inferiori, eventuali correlazioni tra i fattori di rischio, lo stadio clinico con l’ aumento delle CECs. Valutare i cambiamenti strutturali (calcificazione ed infiltratto infiammatorio) e l’ angiogenesi (numero di capillari /sezione) della parete arteriosa. MATERIALI E METODI: Da Maggio 2006 ad Aprile 2008 in modo prospettico abbiamo arruolato paziente affetti da IC da sottoporre ad intervento chirurgico. In un data base abbiamo raccolto : caratteristiche demografiche, fattori di rischio, stadiazione dell'IC secondo Leriche-Fontaine (L-F), il tipo di intervento chirurgico. Per ogni paziente abbiamo effettuato un prelievo ematico di 2 ml per la quantificazione immunomagnetica delle CECs e prelievo di parete arteriosa. RISULTATI: In modo consecutivo abbiamo arruolato 33 pazienti (75.8% maschi) con età media di 71 aa (range 34-91aa), affetti da arteriopatia ostruttiva cronica periferica al IV stadio di L-F nel 84.8%, da cardiopatia ischemica cronica nel 60.6%, da ipertensione arteriosa nel 72.7% e da diabete mellito di II tipo nel 66.6%. Il valore medio di CECs/ml è risultato significativamente più elevato (p= 0.001) nei soggetti affetti da IC (CECs/ml =531.24 range 107- 3330) rispetto ai casi controllo (CECs/ml = 125.8 range 19-346 ). Le CECs/ml nei pazienti diabetici sono maggiori rispetto alle CECs/ml nei pazienti non diabetici ( 726.7 /ml vs 325.5/ml ), p< 0.05 I pazienti diabetici hanno presentato maggior incidenza di lesioni arteriose complesse rispetto ai non diabetici (66% vs 47%) e minor densità capillare (65% vs 87%). Conclusioni : Le CECs sono un marker sierologico attendibile di danno vascolare parietale, la loro quantità è maggiore nei pazienti diabetici e ipertesi. La minor capacità angiogenetica della parete arteriosa in presenza di maggior calcificazioni ed infiltrato infiammatorio nei diabetici, dimostra un danno istopatologico di parete maggiore . / OBJECTIVE: To quantify the number of circulating endothelial mature cells (EMC) in patients with critical limb ischemia (CI), and if correlations exit with risk factors, clinical stage and the number of cEMC. Evaluate the vascular structure changes (calcification and inflammatory infiltrate) and angiogenesis (number of capillary/arterial slice) of arterial wall. METHODS: Between 2006, may, and 2008, april, we’ve enrolled in a prospective study patients with CI scheduled for surgery. Demographic data, risk factors, clinical stage according to Leriche-Fontaine, type of surgery have been collected and stored in a database. For every patient 2 ml of blood have been sampled for immunomagnetic quantification of cEMC, so as a sample of the arterial wall. RESULTS: Thirtythree patients (25 males, 75,8%), aged 34-91, average 71years old, with CI underwent surgical revascularization have been enrolled. Twentyeight patients (84.8%) had a IV stage PAOD, chronic heart ischemia (60.6%), hypertension (72.7%) and diabetes mellitus type II (66.6%). The average concentration of cEMC/ml is significant higher (p=0.001) in patients with CI (cEMC=531.24, range 107-3330) than in control cases (cEMC =125.8, range 19-346). Also patients with diabetes type II have a higher concentration of cEMC than the non-diabetic patients (726.7/mm vs 325.5/ml vs.), with a p<0.05. Arterial wall of pts. with diabetes , compared with control group, revealed a higher incidence of complex arterial lesions (66% vs. 47%) and a lower capillary density (65% vs. 87%). CONCLUSION: cEMC are a reliable marker of vascular wall damage. Their concentration is higher in patients with diabetes, hypertensive disease. In patients with diabetes type 2 we’ve found a minor angiogenic capability with more calcifications and inflammatory infiltrate, showing a more serious damage

L’insufficienza renale acuta(AKI) grave che richiede terapia sostitutiva, è una complicanza frequente nelle unità di terapia intensiva(UTI) e rappresenta un fattore di rischio indipendente di mortalità. Scopo dello studio é stato valutare prospetticamente, in pazienti “critici” sottoposti a terapie sostitutive renali continue(CRRT) per IRA post cardiochirurgia, la prevalenza ed il significato prognostico del recupero della funzione renale(RFR). Pazienti e Metodi:Pazienti(pz) con AKI dopo intervento di cardiochirurgia elettivo o in emergenza con disfunzione di due o più organi trattati con CRRT. Risultati:Dal 1996 al 2011, 266 pz (M 195,F 71, età 65.5±11.3aa) sono stati trattati con CRRT. Tipo di intervento: CABG(27.6%), dissecazione aortica(33%), sostituzione valvolare(21.1%), CABG+sostituzione valvolare(12.6%), altro(5.7%). Parametri all’inizio del trattamento: BUN 86.1±39.4, creatininemia(Cr) 3.96±1.86mg/dL, PAM 72.4±13.6mmHg, APACHE II score 30.7±6.1, SOFAscore 13.7±3. RIFLE: Risk (11%), Injury (31.4%), Failure (57.6%). AKI oligurica (72.2%), ventilazione meccanica (93.2%), inotropi (84.5%). La sopravvivenza a 30 gg ed alla dimissione è stata del 54.2% e del 37.1%. La sopravvivenza per stratificazione APACHE II: <24=85.1 e 66%, 25-29=63.5 e 48.1%, 30-34=51.8 e 31.8%, >34=31.6 e 17.7%. RFR ha consentito l’interruzione della CRRT nel 87.8% (86/98) dei survivors (Cr 1.4±0.6mg/dL) e nel 14.5% (24/166) dei nonsurvivors (Cr 2.2±0.9mg/dL) con un recupero totale del 41.4%. RFR è stato osservato nel 59.5% (44/74) dei pz non oligurici e nel 34.4% dei pz oligurici (66/192). La distribuzione dei pz sulla base dei tempi di RFR è stata:<8=38.2%, 8-14=20.9%, 15-21=11.8%, 22-28=10.9%, >28=18.2%. All’analisi multivariata, l’oliguria, l’età e il CV-SOFA a 7gg dall’inizio della CRRT si sono dimostrati fattori prognostici sfavorevoli su RFR(>21gg). RFR si associa ad una sopravvivenza elevata(78.2%). Conclusioni:RFR significativamente piu frequente nei pz non oligurici si associa ad una sopravvivenza alla dimissione piu elevata. La distribuzione dei pz in rapporto ad APACHE II e SOFAscore dimostra che la sopravvivenza e RFR sono strettamente legati alla gravità della patologia. / Severe AKI requiring RRT frequently occurs in ICU and represents an independent risk factor for mortality. The aim was to prospectively evaluate, in critically ill undergoing CRRT for AKI following heart surgery, prevalence and prognostic significance of renal function recovery (RFR). Patients and Methods: Patients (pts) with AKI following elective or emergent cardiac surgery with dysfunction of 2 or more organs treated with CRRT. Results: From 1996 to 2011, 266 pts (M 195, F 71, age 65.5±11.3) underwent CRRT. Type of surgery: CABG (27.6%), aortic dissection (33%), valvular surgery (21.1%), CABG+valvular surgery (12.6%), others (5.7%). CRRT starting parameters: BUN 86.1±39.4, creatinine (Cr) 3.96±1.86 mg/dL, MAP 72.4±13.6 mmHg, APACHE II 30.7±6.1, SOFA 13.7±3. RIFLE staging: Risk (11%), Injury (31.4%), Failure (57.6%). Oliguric AKI (72.2%), ventilation (93.2%), inotropics (84.5%). At 30 days and at hospital discharge, total survival was 54.2% and 37.1% (APACHE II score clusters survival: <24= 85.1 and 66%, 25-29= 63.5 and 48.1%, 30-34= 51.8 and 31.8%, >34= 31.6 and 17.7%). RFR allowed to stop CRRT in 87.8% (86/98) of survivors (Cr 1.4±0.6 mg/dL) and in 14.5% (24/166) of nonsurvivors (Cr 2.2±0.9mg/dL) with an overall recovery of 41.4%. RFR has been observed in 59.5% (44/74) of non oliguric pts and in 34.4% of oliguric pts (66/192). Distribution of pts according to the timing of RFR (days from CRRT start): <8 (38.2%), 8-14 (20.9%), 15-21 (11.8%), 22-28 (10.9%), >28 (18.2%). Logistic regression selected occurrence of oliguria, age, CV-SOFA at 7 days from CRRT start as a prognostic factors for delayed RFR (> 21 days).RFR was associated with a high survival rate (78.2%). Conclusions: RFR, more frequently observed in pts with nonoliguric AKI, was mostly associated with a favourable outcome. Patient distribution according to APACHE II and SOFA score revealed that survival and RFR are strictly related to the severity of illness.