Transcriptional responses of the Helicobacter pylori cag pathogenicity island

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

E' possibile giustificare moralmente l'obiezione di coscienza in campo medico?

Scopo di questa tesi di dottorato è stabilire se l’obiezione di coscienza in ambito medico sia moralmente giustificabile. Esistono essenzialmente tre tipi di rifiuto motivati dall’obiezione di coscienza (1) rifiuto di informare un paziente relativamente alle opzioni terapeutiche come ad esempio l’uso di un contraccettivo di emergenza o l’interruzione volontaria di gravidanza (2) rifiuto di rinviare un paziente che chiede un particolare intervento (o terapia) presso un collega non obiettore (3) rifiuto di svolgere in prima persona una certa attività richiesta dal paziente Per rispondere a questo interrogativo si è svolta un’analisi filosofico-morale dei principali argomenti utilizzati dalla letteratura su questo tema per giustificare o per negare un diritto morale all’obiezione di coscienza degli operatori sanitari. Il diritto degli operatori sanitari all’integrità morale e a non essere complici di attività ritenute immorali dev’essere infatti confrontato con il diritto dei pazienti ad avere un’assistenza sanitaria efficiente, a poter compiere scelte autonome riguardo alla propria salute e ad essere informati relativamente a tutte le opzioni terapeutiche disponibili. Nel corso dell’intero lavoro è stato dimostrato come suddetti diritti dei pazienti sono facilmente e frequentemente violati a causa dell’incidenza dell’obiezione di coscienza in ambito medico. L’analisi condotta nel corso del lavoro di tesi si concentra fondamentalmente su quattro importanti aspetti del problema quali diritto all’integrità morale dell’ operatore sanitario, obblighi professionali, cooperazione al male e laicità dello stato. Alla fine del lavoro di analisi si è giunti alla conclusione che: le obiezioni di tipo (1) e (2) non sono mai moralmente giustificabili perché comportano sempre una violazione dei diritti fondamentali del paziente. Le obiezioni di coscienza di tipo (3) sono moralmente accettabili solo quando non impongono un peso eccessivo al paziente, vale a dire quando il rinvio presso un collega non obiettore è veloce, sicuro e agevole. Tuttavia le condizioni ideali in cui vengono rispettati i criteri minimi di ammissibilità dell’obiezione di coscienza di tipo (3) non si verificano quasi mai nella realtà dei fatti (per ragioni ampiamente spiegate nel corso del lavoro), per cui tali obiezioni risultano in pratica solo raramente accettabili da un punto d vista morale.

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.

Au(I) Catalyzed Manipulation of Propargylic Alcohols: A New Route Towards the Synthesis of Indole Alkaloids

In this work we presented several aspects regarding the possibility to use readily available propargylic alcohols as acyclic precursors to develop new stereoselective [Au(I)]-catalyzed cascade reactions for the synthesis of highly complex indole architectures. The use of indole-based propargylic alcohols of type 1 in a stereoselective [Au(I)]-catalyzed hydroindolynation/immiun trapping reactive sequence opened access to a new class of tetracyclic indolines, dihydropyranylindolines A and furoindolines B. An enantioselective protocol was futher explored in order to synthesize this molecules with high yields and ee. The suitability of propargylic alcohols in [Au(I)]-catalyzed cascade reactions was deeply investigated by developing cascade reactions in which was possible not only to synthesize the indole core but also to achieve a second functionalization. Aniline based propargylic alcohols 2 were found to be modular acyclic precursors for the synthesis of [1,2-a] azepinoindoles C. In describing this reactivity we additionally reported experimental evidences for an unprecedented NHCAu(I)-vinyl specie which in a chemoselective fashion, led to the annulation step, synthesizing the N1-C2-connected seven membered ring. The chemical flexibility of propargylic alcohols was further explored by changing the nature of the chemical surrounding with different preinstalled N-alkyl moiety in propargylic alcohols of type 3. Particularly, in the case of a primary alcohol, [Au(I)] catalysis was found to be prominent in the synthesis of a new class of [4,3-a]-oxazinoindoles D while the use of an allylic alcohol led to the first example of [Au(I)] catalyzed synthesis and enantioselective functionalization of this class of molecules (D*). With this work we established propargylic alcohols as excellent acyclic precursor to developed new [Au(I)]-catalyzed cascade reaction and providing new catalytic synthetic tools for the stereoselective synthesis of complex indole/indoline architectures.