MARCATORI MOLECOLARI AD IMPATTO TRASLAZIONALE NEL CARCINOMA EPATOCELLULARE

L\u2019epatocarcinoma (EC) \ue8 un tumore maligno ad elevata morbilit\ue0 e mortalit\ue0 la cui speranza di cura \ue8 basata su diagnosi precoce e terapia individualizzata. Ad oggi il trattamento di elezione \ue8 rappresentato dalla chirurgia e l\u2019unico farmaco con comprovata efficacia terapeutica \ue8 un presidio biologico di recente introduzione ad azione anti-tirosinochinasica (Sorafenib). La gestione ottimale del paziente con EC \ue8 sempre pi\uf9 influenzata dalla possibilit\ue0 di disporre di marcatori molecolari da utilizzarsi nella pratica clinica a significato diagnostico, prognostico e predittivo. Il presente lavoro si propone come contributo all\u2019identificazione e validazione di specifici indicatori biologici nella diagnostica del piccolo EC, predizione della recidiva del tumore resecato e della sensibilit\ue0 individuale alla risposta al trattamento farmacologico nella forma avanzata. E gi\ue0 stato dimostrato che l\u2019accuratezza della diagnosi precoce di EC su biopsia epatica puo\u2019 essere migliorata in maniera significativa dalla valuatazione dell\u2019espressione combinata di alcuni marcatori recentmente proposti e gi\ue0 utilizzati nella pratica clinica. Abbiamo cercato di ottimizzare questo pannello valutando il contributo aggiuntivo della catena pesante della clatrina (CHC) che \ue8 componente essenziale nel sistema di trasporto vescicolare intra ed extracellulare, gi\ue0 segnalato come sovraregolato negli epatociti tumorali maligni. Utilizzando una casistica di EC di piccole dimensioni e di lesioni precancerose di controllo e documentando l\u2019espressione della molecola con tecnica immunocitochimica, abbiamo verificato la sua efficacia in termini di accuratezza diagnostica. In particolare l\u2019introduzione di CHC si \ue8 dimostrata in grado di incrementare la sensibilit\ue0 del pannello dal 46,8% al 63,8% a fronte del mantenimento di una specificit\ue0 assoluta. E\u2019 noto come il processo di carcinogenesi epatica origini anche sulla base di alterazioni delle vie molecolari che governano i processi di tipo ossidativo, con importante ruolo di protezione svolto dalla famiglia dei citocromi. Alcuni studi di espressione genica hanno gi\ue0 evidenziato che la recidiva di malattia tumorale nel fegato sia influenzata dalla disregolazione di alcuni citocromi, tra cui CYP1A2. Abbiamo inteso verificare e validare il ruolo del citocromo CYP1A2 nella qualit\ue0 di possibile marcatore ad impatto traslazionale nella recidiva dell\u2019EC attraverso il suo dosaggio immuncitochimico nel parenchima epatico non tumorale. Utilizzando una casistica omogenea di pazienti portatori di EC ad eziologia virale (virus C dell\u2019epatite) e a follow-up noto, il dosaggio di CYP1A2 \ue8 risultato associato in maniera statisticamente significativa, sia in analisi univariata che multivariata al tempo libero da recidiva tumorale (p=0.012). In particolare la ridotta espressione del citocromo negli epatociti non tumorali, interpretabile come background pi\uf9 suscettibile alla trasformazione neoplastica \u201cde novo\u201d, risultava in grado di predire una pi\uf9 rapida ripresa della malattia. La terapia farmacologica dell\u2019EC \ue8 attualmente somministrata a pazienti portatori di forme avanzate senza tenere conto della reale e individuale suscettibilit\ue0 all\u2019azione del farmaco. Vi \ue8 una forte esigenza clinica di selezionare i pazienti candidabili al trattamento anche per gli elevati costi della terapia, ma nessun marcatore, sia esso tissutale o sierologico, si \ue8 rivelato ad oggi capace di soddisfare questi requisiti. Abbiamo inteso esplorare se i miRNA, che costituiscono una famiglia eterogenea ma fondamentale di molecole regolatrici della traduzione, potessero a loro volta candidarsi a possibili marcatori biologici di predizione della sensibilit\ue0 al trattamento farmacologico dell\u2019EC. A questo scopo, partendo da un pool amplissimo di miRNA, abbiamo proceduto ad una loro progressiva selezione sulla base della loro disregolazione nonch\ue8 associazione con il tempo di progressione della malattia dopo trattamento. Lo studio \ue8 stato condotto su biopsie epatiche pretrattamento di EC di pazienti omogeneamente trattati con Sorafenib. La selezione delle molecole e la validazione delle stesse \ue8 stata condotta in due casistiche distinte di cui la seconda utilizzata come coorte di validazione. Abbiamo cosi potuto individuare un unico miRNA con meccanismo di azione e geni targets poco conosciuti, il cui sovradosaggio valutato con tecnica di real-time PCR risultava fortemente e statisticamente associato ad una prolungata stabilizzazione della malattia tumorale (p=0.009). L\u2019insieme di questi risultati dimostra che la crescente domanda di marcatori di medicina personalizzata per ottimizzare la gestione del paziente oncologico (con EC nel nostro caso), possa essere soddisfatta attraverso l\u2019applicazione di tecniche relativamente semplici e riproducibili quali l\u2019immunocitochimica e l\u2019analisi quantitativa del messaggero, condotte sia su tessuti tumorali che non tumorali. Naturalmente affinch\ue8 il dosaggio di una molecola possa entrare nella pratica clinica corrente, candidandosi come un autentico marcatore molecolare, \ue8 necessaria, come nei nostri casi, un\u2019ulteriore validazione in studi di tipo prospettico.Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality worldwide. The treatment is based on early diagnosis and individualized therapy. Today surgery is the primary strategy for patients with HCC and Sorafenib, an anti tyrosine kinase drug, represents the only available drug with proven efficacy. The discovery of new molecular markers (diagnostic, prognostic and predictive) that can be translate in the clinical practice represents the improvement in the management of these patients. The present research is aimed at identifying and validating biological markers related to the early HCC diagnosis and to predict HCC relapse. In addition the individual sensibility to pharmacological treatment in patients with advanced HCC is also evaluated. It has been shown that the accuracy of the HCC early diagnosis on the liver biopsy can be significantly improved by combining histological features with molecular markers, recently proposed and applied in the clinical practice. We have optimized this molecular panel for evaluating the contribution of the clathrin heavy chain (CHC), which is an essential component of intra and extracellular vesicular transport, and previously reported as an up-regulated molecule in malignant liver cells. Using a series of small HCC and control precancerous lesions we tested, by immunohistochemical analysis, whether CHC expression contribute to improve the histopathological diagnostic accuracy. We found that the introduction of CHC increases the sensitivity from 46,8% to 63,8% and maintains an absolute specificity. It is now ascertained that hepatocarcinogenesis is based on alterations in a wide variety of molecular pathways controlling oxidative processes and that cytochromes family plays a protective role in this context. Additionally, gene expression profiles have shown that relapse of HCC is caused by some cytochromes deregulations (i.e. CYP1A2). We verified and validated the CYP1A2 role in relation to relapse of disease by immunohistochemistry and its quantification in non-tumoral liver parenchyma. By investigating a homogeneous series of HCC patients with HCV infection and known follow-up, we demonstrated that the CYP1A2 dosage was associated with the recurrence-free survival (p=0,012) in both univariate and multivariate analyses. Moreover, CYP1A2 expression decreased in non-tumoral hepatocytes, the most susceptible background to \u201cde novo\u201d neoplastic transformation, predicted a faster recurrence of disease. The pharmacological therapy of HCC is currently administered to patients with advanced disease, without any discrimination according to the individual drug susceptibility. There is a compelling clinical need to select patients more suitable to underwent pharmacological treatment, because of the elevated cost. Furthemore, there is no still histological and/or serological biomarker, capable to satisfy these requirements. For this reason, we explored whether miRNA, heterogeneous family of regulatory molecules of translation, can be used as new molecular predictive markers of sensitivity to HCC treatment. For this purpose, starting from a wide pool of miRNA, we progressively selected those deregulated and associated with time to progression of disease after treatment. This research was conducted on liver biopsies sampled before treatment in patients treated with Sorafenib. The selection and validation of miRNA were conducted on two distinct series of patients. Among these the second group of patients has been used as a validation set. We identified a single miRNA, with mechanism of action ad genes target unknown, which when appears overexpressed, by real-time PCR analysis, was strongly and statistically associated with prolonged stabilization of tumor disease (p=0,009). The above findings all suggest that the increasing demand for customized markers to optimized the management of HCC patients can be investigated through the application of relatively simple and reproducibly techniques (including immunohistochemistry and real-time PCR) on both tumoral and non-tumoral tissue. Further validation in prospective studies is required to pursue a personalized dosage of new molecular markers to be introduced in the current clinical practice

Influence of the Antithrombotic Therapy in the Healing of Simple Post-Extraction Sockets: A Randomized Clinical Trial

Background: An adequate blood supply plays a leading role in the healing process of the post-extractive socket; its coagulation leads to fibrin clot formation, which acts as a physical barrier able to prevent postoperative bleeding and microbial infection. The purpose of this study was to evaluate the effectiveness of antiaggregant drugs in healing post-extraction sockets compared to natural wound healing. Methods: This was a single-center prospective clinical trial. Extraction sockets allocated in healthy patients and in patients assuming antiplatelet drugs were considered. Thirty consecutive patients under (treated with/in treatment with) oral antiplatelet treatment were enrolled in the test group. In order to provide a control group, 30 consecutive patients meeting all the exclusion and inclusion criteria were enrolled. The extraction of the mono-radicular tooth was atraumatically performed without gingivoplasty or osteotomy procedures that could influence the healing process. Photographs were obtained before and immediately after surgery and at 3-, 7-, 14-and 28-days follow-up. Results: All patients assumed the prescribed therapy and their postoperative recovery was uneventful without any kind of post-extractive complications. The results of inter-group comparison show that on the third and seventh days of follow-up, the antiplatelet group expressed a statistically significant higher level of healing compared to the control group (p < 0.05), while no statistically significant differences were recorded at 14-and 28-days follow-up. Conclusions: Patients treated with antiplatelet agents seemed to show that this therapy can positively affect the healing process after tooth extractions

Subseismic to Seismic Slip in Smectite Clay Nanofoliation

Smectite clays are the main constituent of slipping zones found in subduction zone faults at shallow depth (e.g., <1-km depth in the Japan Trench) and in the decollements of large landslides (e.g., 1963 landslide, Vajont, Italy). Therefore, deformation processes in smectite clays may control the mechanical behavior from slow creep to fast accelerations and slip during earthquakes and landslides. Here, we use (1) laboratory experiments to investigate the mechanical behavior of partly water-saturated smectite-rich gouges sheared from subseismic to seismic slip rates V and (2) nanoscale microscopy to study the gouge fabric. At all slip rates, deformation localizes in volumes of the gouge layer that contain a \u201cnanofoliation\u201d consisting of anastomosing smectite crystals. \u201cSeismic\u201d nanofoliations produced at V = 0.01, 0.1, and 1.3 m/s are similar to \u201csubseismic\u201d nanofoliations obtained at V = 10 125 m/s. This similarity suggests that frictional slip along water-lubricated smectite grain boundaries and basal planes may occur from subseismic to seismic slip rates in natural smectite-rich faults. Thus, if water is available along smectite grain boundaries and basal planes, nanofoliations can develop from slow to fast slip rates. Still, when nanofoliations are found highly localized in a volume, they can be diagnostic of slip that occurred at rates equal or larger than 0.01 m/s. In such a case, they could be markers of past seismic events when found in natural fault rocks

Fault Friction During Simulated Seismic Slip Pulses

Theoretical studies predict that during earthquake rupture faults slide at non-constant slip velocity, however it is not clear which source time functions are compatible with the high velocity rheology of earthquake faults. Here we present results from high velocity friction experiments with non-constant velocity history, employing a well-known seismic source solution compatible with earthquake source kinematics. The evolution of friction in experiments shows a strong dependence on the applied slip history, and parameters relevant to the energetics of faulting scale with the impulsiveness of the applied slip function. When comparing constitutive models of strength against our experimental results we demonstrate that the evolution of fault strength is directly controlled by the temperature evolution on and off the fault. Flash heating predicts weakening behavior at short timescales, but at larger timescales strength is better predicted by a viscous creep rheology. We use a steady-state slip pulse to test the compatibility of our strength measurements at imposed slip rate history with the stress predicted from elastodynamic equilibrium. Whilst some compatibility is observed, the strength evolution indicates that slip acceleration and deceleration might be more rapid than that imposed in our experiments

Introduction of seismic source directivity on hazard map

The seismic hazard maps are mainly influenced by the uncertainty associated to the ground motion predictive equation (GMPE). This uncertainty represents the unexplained part of the ground motion and it is mostly related to the choice of the model’s variables. In fact the representation of the ground motion through the GMPEs is simple compared to the complexity of the physical process involved: if only the magnitude and distance are taken into account, GMPEs predicts isoseismals curves that are expected to be isotropic around the hypocenter or along the fault. Instead, the presence of a fault plane across which a process of failure in shear develops makes this general formulation reliable only on average. In fact this failure is responsible of an asymmetry in the seismic radiation known, since Ben-Menhaem (PhD1961), as directivity effect. While the general knowledge of the earthquakes is treated explicitly in the empirical prediction, specific trends like the directivity effects are hidden in the uncertainty sigma. A way to reduce the sigma is therefore to refine the seismic seismic source description inside the GMPEs (e.g. NGA project, Power et al, Earthquake Spectra, 2008). In this framework we propose a strategy to introduce the directivity in the GMPEs and to study its effect on uncertainties and on hazard maps. For this purpose, we have used two different directivity models acting on the GMPE as corrective factors: one proposed by Somerville et al. (Seis.Res.Lett.1997) and the other one proposed by Spudich and Chiou (Earthquake Spectra 2008).The first factor depends on geometrical parameters and comes from theoretical deduction. The second one includes many source parameters and it is a hybrid factor, which functional formulation is deduced from the theory, calibrated on synthetic simulations and scaled on data. The classic hazard equation is then adapted in order to increase the number of source parameters (i.e. adding one integral over the parametric space for each new variable involved) and taking into account the corrective factors for directivity (Spagnuolo, PhD2010). We present the comparisons of hazard maps depending on the directivity factor and on the probability density functions of the fault strike and of the rupture “laterality”

Preliminary estimates of tritium permeation and retention in the first wall of DEMO due to ion bombardment

Tritium self-sufficiency presents a critical engineering challenge for DEMO, requiring efficient breeding and extraction systems, as well as minimizing tritium losses to the surrounding systems, such as plasma-facing components, vacuum vessel, cooling system, etc. Structural and plasma-facing components will act as a tritium sink, as tritium will be accumulated in the bulk of these components due to energetic particle bombardment and may permeate out of the vacuum system. The design of the plasma-facing components will consequently directly influence the plant lifetime, operational safety and cost of any future power plant. Therefore, modeling of tritium retention and permeation in these components is required for the engineering designs of the tritium breeding and safety systems. In this work, the diffusion-transport code TESSIM-X is benchmarked against the well-established TMAP7 code and a comparison with a simplified DEMO-relevant test case is performed. The use of either code for modeling of DEMO conditions is discussed. Following this, TESSIM-X is used to provide a preliminary assessment of tritium permeation and retention in the DEMO first wall, based on the current WCLL (Water Cooled Lithium Lead) and HCPB (Helium Cooled Pebble Bed) breeding blanket designs

Structural assessment of the EU-DEMO water-cooled lead lithium central outboard blanket segment adopting the sub-modelling technique

The development of a sound conceptual design of the Water-Cooled Lead Lithium Breeding Blanket (WCLL BB) is pivotal to make a breakthrough towards the selection of the driver blanket concept for the EU-DEMO. To achieve this goal, an intense research campaign has been performed at the University of Palermo, in cooperation with ENEA Brasimone, under the umbrella of EUROfusion. In this paper, structural analyses of different poloidal regions of the WCLL BB Central Outboard Blanket (COB) segment are reported. In particular, starting from the results of the thermo-mechanical analysis of the whole WCLL BB COB segment, the sub-modelling technique has been applied to the most significant poloidal regions, located at the top, middle and bottom of the segment. The aim is to focus on the stress field locally arising under purposely selected steady-state nominal and accidental loading scenarios. The nominal BB operating conditions, as well as steady-state scenarios derived from both the in-box LOCA and Vertical Plasma Disruption accidents have been considered. Thanks to the sub-modelling approach, the deformative action of the entire segment can be imposed at the boundaries of each local model to realistically assess its structural performances. Moreover, each local model reproduces structural details not included in the global one, such as the Segment Box (SB) cooling channels. Then, the structural behaviour of the selected regions has been assessed in compliance with the RCC-MRx code. The obtained results highlighted that the structural behaviour predicted by the whole segment analysis is similar to that predicted by sub-modelling calculations within the Stiffening Plates, whereas the application of the sub-modelling is a must to investigate in detail the SB structural performances. In addition, results indicate that the BB attachments should be revised, as they contribute to produce the WCLL COB large deformation originating excessive stresses, mainly within the equatorial region

Fluid pressurisation and earthquake propagation in the Hikurangi subduction zone

In subduction zones, seismic slip at shallow crustal depths can lead to the generation of tsunamis. Large slip displacements during tsunamogenic earthquakes are attributed to the low coseismic shear strength of the fluid-saturated and non-lithified clay-rich fault rocks. However, because of experimental challenges in confining these materials, the physical processes responsible for the coseismic reduction in fault shear strength are poorly understood. Using a novel experimental setup, we measured pore fluid pressure during simulated seismic slip in clay-rich materials sampled from the deep oceanic drilling of the Pāpaku thrust (Hikurangi subduction zone, New Zealand). Here, we show that at seismic velocity, shear-induced dilatancy is followed by pressurisation of fluids. The thermal and mechanical pressurisation of fluids, enhanced by the low permeability of the fault, reduces the energy required to propagate earthquake rupture. We suggest that fluid-saturated clay-rich sediments, occurring at shallow depth in subduction zones, can promote earthquake rupture propagation and slip because of their low permeability and tendency to pressurise when sheared at seismic slip velocities