Infiammazione e cancro: l'interazione COX-2/CA-IX promuove il potenziale invasivo e la sopravvivenza in ipossia delle cellule del cancro colon-rettale

Cyclooxygenase-2/Carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells Purpose: Cyclooxygenase-2 (COX-2) is a major mediator of inflammation, playing a pivotal role in colorectal carcinogenesis. Hypoxia is an universal hallmark of solid tumour in vivo. This investigation was prompted by the observation that in colorectal cancer cells the expression of COX-2 protein is positively correlated with that of the hypoxia survival gene Carbonic Anhydrase-IX (CA-IX). Experimental Design: Since COX-2 gene expression and activity is increased in hypoxia, and that CA-IX is expressed also in normoxia in colorectal cancer cells, we tested the hypothesis that COX-2 activity in normoxia, as well as in hypoxia may be functionally linked to that of CA-IX gene. We investigated the role of COX-2 and CA-IX in colorectal cancer cell lines. In this regard, we performed RNA interference to knockdown COX-2 gene in vitro and immunohistochemistry to evaluate the protein expression of COX-2 and CA-IX in human colon cancer tissue specimens ex vivo. Results: We found that COX-2, by PGE2 production, controls CA-IX gene expression in an ERK dependent manner. In line with this finding, we also showed that the COX-2 inhibition by a specific short harpin COX-2 RNA (shCOX-2) or by a specific drug (SC-236), down-regulated CA-IX expression in colon cancer cells. We then exposed colon cancer cells to hypoxia stimuli and found that COX-2/CA-IX interplay promoted hypoxia survival. Moreover, we also report that COX-2/CA-IX interplay triggers Matrix Metalloproteinase 2/9 (MMP-2/9) activation and enhances the invasiveness of colorectal cancer cells. Thus given our above observations, we found that CA-IX and COX-2 protein expressions correlate with more aggressive stage colorectal cancer tissues ex vivo. Conclusions: Taken together these data indicate that COX-2/CA-IX interplay promotes an aggressive phenotype (hypoxia survival and invasiveness) which can be modulated in vitro by COX-2 selective inhibition and which may play a role in determining the biological aggressiveness of colorectal tumours. Moreover, in vitro and ex vivo data also suggest that the signatures of inflammation (COX-2) and hypoxia (CA-IX) may be difficult to be disentangled in colon cancer, being both responsible for the up-regulation of the same pathways.

Computational Modeling of Cardiac Excitation-Contraction Coupling in Physiological and Pathological Conditions

The cardiomyocyte is a complex biological system where many mechanisms interact non-linearly to regulate the coupling between electrical excitation and mechanical contraction. For this reason, the development of mathematical models is fundamental in the field of cardiac electrophysiology, where the use of computational tools has become complementary to the classical experimentation. My doctoral research has been focusing on the development of such models for investigating the regulation of ventricular excitation-contraction coupling at the single cell level. In particular, the following researches are presented in this thesis: 1) Study of the unexpected deleterious effect of a Na channel blocker on a long QT syndrome type 3 patient. Experimental results were used to tune a Na current model that recapitulates the effect of the mutation and the treatment, in order to investigate how these influence the human action potential. Our research suggested that the analysis of the clinical phenotype is not sufficient for recommending drugs to patients carrying mutations with undefined electrophysiological properties. 2) Development of a model of L-type Ca channel inactivation in rabbit myocytes to faithfully reproduce the relative roles of voltage- and Ca-dependent inactivation. The model was applied to the analysis of Ca current inactivation kinetics during normal and abnormal repolarization, and predicts arrhythmogenic activity when inhibiting Ca-dependent inactivation, which is the predominant mechanism in physiological conditions. 3) Analysis of the arrhythmogenic consequences of the crosstalk between β-adrenergic and Ca-calmodulin dependent protein kinase signaling pathways. The descriptions of the two regulatory mechanisms, both enhanced in heart failure, were integrated into a novel murine action potential model to investigate how they concur to the development of cardiac arrhythmias. These studies show how mathematical modeling is suitable to provide new insights into the mechanisms underlying cardiac excitation-contraction coupling and arrhythmogenesis.

Propter adhorantium auctoritatem voluntate. Legittimazione, patronage e propaganda nelle Gesta Regum Anglorum di Guglielmo di Malmesbury

L’oggetto di ricerca della presente tesi di dottorato è costituito dall’analisi dell’opera Gesta Regum Anglorum, del monaco benedettino Guglielmo di Malmesbury, all’interno della quale sono stati esplorati e verificati i temi di legittimazione, di patronage e di propaganda. L’opera, infatti, rimane senza un manifesto committente, ad eccezione di una primissima versione. Il titolo della tesi rivela fin da subito questo aspetto, giacché estrae un passaggio del prologo al I libro: «propter adhorantium auctoritatem voluntate», traducibile con «per le autorevoli esortazioni che ricevetti». Dopo un’analisi delle lettere dedicatorie premesse all’opera, si è ipotizzata la volontà dell’autore di dedicare le Gesta Regum Anglorum, nella loro versione definitiva, a Roberto conte di Gloucester, approfondendo in tal senso l’aspetto legittimatorio dell’opera e la possibilità che essa potesse servire come strumento per ottenere un patronage dal conte nei confronti dell’abbazia di Malmesbury. La seconda parte della tesi è incentrata sulla comparazione tra le due principali redazioni dell’opera – quella conclusa intorno al 1126/27 e quella rivista tra 1135 e 1140 – per analizzarne le modifiche, ipotizzandone la funzione come volta mitigare aspetti relativi ai principali antenati di Roberto di Gloucester (Guglielmo I e Guglielmo II). La terza parte della tesi si è concentrata sull’aspetto propagandistico dell’opera in favore del monastero di appartenenza di Guglielmo (Malmesbury) e soprattutto in favore del clero regolare, nella dicotomia che caratterizzò questo e il clero secolare durante gli anni in cui l’autore viveva. Nell’ultima parte della tesi, è stato ripreso l’aspetto legittimatorio delle Gesta Regum, tentando di fornire un’analisi delle tre raffigurazioni dei sovrani normanni d’Inghilterra, che punteggiano i tre libri finali dell’opera.