Identification of Drosophila heart-specific Cis-Regulatory Modules under Hox control

Cardiac morphogenesis is a complex process governed by evolutionarily conserved transcription factors and signaling molecules. The Drosophila cardiac tube is linear, made of 52 pairs of cardiomyocytes (CMs), which express specific transcription factor genes that have human homologues implicated in Congenital Heart Diseases (CHDs) (NKX2-5, GATA4 and TBX5). The Drosophila cardiac tube is linear and composed of a rostral portion named aorta and a caudal one called heart, distinguished by morphological and functional differences controlled by Hox genes, key regulators of axial patterning. Overexpression and inactivation of the Hox gene abdominal-A (abd-A), which is expressed exclusively in the heart, revealed that abd-A controls heart identity. The aim of our work is to isolate the heart-specific cisregulatory sequences of abd-A direct target genes, the realizator genes granting heart identity. In each segment of the heart, four pairs of cardiomyocytes (CMs) express tinman (tin), homologous to NKX2-5, and acquire strong contractile and automatic rhythmic activities. By tyramide amplified FISH, we found that seven genes, encoding ion channels, pumps or transporters, are specifically expressed in the Tin-CMs of the heart. We initially used online available tools to identify their heart-specific cisregutatory modules by looking for Conserved Non-coding Sequences containing clusters of binding sites for various cardiac transcription factors, including Hox proteins. Based on these data we generated several reporter gene constructs and transgenic embryos, but none of them showed reporter gene expression in the heart. In order to identify additional abd-A target genes, we performed microarray experiments comparing the transcriptomes of aorta versus heart and identified 144 genes overexpressed in the heart. In order to find the heart-specific cis-regulatory regions of these target genes we developed a new bioinformatic approach where prediction is based on pattern matching and ordered statistics. We first retrieved Conserved Noncoding Sequences from the alignment between the D.melanogaster and D.pseudobscura genomes. We scored for combinations of conserved occurrences of ABD-A, ABD-B, TIN, PNR, dMEF2, MADS box, T-box and E-box sites and we ranked these results based on two independent strategies. On one hand we ranked the putative cis-regulatory sequences according to best scored ABD-A biding sites, on the other hand we scored according to conservation of binding sites. We integrated and ranked again the two lists obtained independently to produce a final rank. We generated nGFP reporter construct flies for in vivo validation. We identified three 1kblong heart-specific enhancers. By in vivo and in vitro experiments we are determining whether they are direct abd-A targets, demonstrating the role of a Hox gene in the realization of heart identity. The identified abd-A direct target genes may be targets also of the NKX2-5, GATA4 and/or TBX5 homologues tin, pannier and Doc genes, respectively. The identification of sequences coregulated by a Hox protein and the homologues of transcription factors causing CHDs, will provide a mean to test whether these factors function as Hox cofactors granting cardiac specificity to Hox proteins, increasing our knowledge on the molecular mechanisms underlying CHDs. Finally, it may be investigated whether these Hox targets are involved in CHDs.

L'influenza del sonno sull'andamento temporale della consolidazione delle abilità procedurali

Background: It is well known, since the pioneristic observation by Jenkins and Dallenbach (Am J Psychol 1924;35:605-12), that a period of sleep provides a specific advantage for the consolidation of newly acquired informations. Recent research about the possible enhancing effect of sleep on memory consolidation has focused on procedural memory (part of non-declarative memory system, according to Squire’s taxonomy), as it appears the memory sub-system for which the available data are more consistent. The acquisition of a procedural skill follows a typical time course, consisting in a substantial practice-dependent learning followed by a slow, off-line improvement. Sleep seems to play a critical role in promoting the process of slow learning, by consolidating memory traces and making them more stable and resistant to interferences. If sleep is critical for the consolidation of a procedural skill, then an alteration of the organization of sleep should result in a less effective consolidation, and therefore in a reduced memory performance. Such alteration can be experimentally induced, as in a deprivation protocol, or it can be naturally observed in some sleep disorders as, for example, in narcolepsy. In this research, a group of narcoleptic patients, and a group of matched healthy controls, were tested in two different procedural abilities, in order to better define the size and time course of sleep contribution to memory consolidation. Experimental Procedure: A Texture Discrimination Task (Karni & Sagi, Nature 1993;365:250-2) and a Finger Tapping Task (Walker et al., Neuron 2002;35:205-11) were administered to two indipendent samples of drug-naive patients with first-diagnosed narcolepsy with cataplexy (International Classification of Sleep Disorder 2nd ed., 2005), and two samples of matched healthy controls. In the Texture Discrimination task, subjects (n=22) had to learn to recognize a complex visual array on the screen of a personal computer, while in the Finger Tapping task (n=14) they had to press a numeric sequence on a standard keyboard, as quickly and accurately as possible. Three subsequent experimental sessions were scheduled for each partecipant, namely a training session, a first retrieval session the next day, and a second retrieval session one week later. To test for possible circadian effects on learning, half of the subjects performed the training session at 11 a.m. and half at 17 p.m. Performance at training session was taken as a measure of the practice-dependent learning, while performance of subsequent sessions were taken as a measure of the consolidation level achieved respectively after one and seven nights of sleep. Between training and first retrieval session, all participants spent a night in a sleep laboratory and underwent a polygraphic recording. Results and Discussion: In both experimental tasks, while healthy controls improved their performance after one night of undisturbed sleep, narcoleptic patients showed a non statistically significant learning. Despite this, at the second retrieval session either healthy controls and narcoleptics improved their skills. Narcoleptics improved relatively more than controls between first and second retrieval session in the texture discrimination ability, while their performance remained largely lower in the motor (FTT) ability. Sleep parameters showed a grater fragmentation in the sleep of the pathological group, and a different distribution of Stage 1 and 2 NREM sleep in the two groups, being thus consistent with the hypothesis of a lower consolidation power of sleep in narcoleptic patients. Moreover, REM density of the first part of the night of healthy subjects showed a significant correlation with the amount of improvement achieved at the first retrieval session in TDT task, supporting the hypothesis that REM sleep plays an important role in the consolidation of visuo-perceptual skills. Taken together, these results speak in favor of a slower, rather than lower consolidation of procedural skills in narcoleptic patients. Finally, an explanation of the results, based on the possible role of sleep in contrasting the interference provided by task repetition is proposed.

EXAM-S: an Analysis tool for Multi-Domain Policy Sets

As distributed collaborative applications and architectures are adopting policy based management for tasks such as access control, network security and data privacy, the management and consolidation of a large number of policies is becoming a crucial component of such policy based systems. In large-scale distributed collaborative applications like web services, there is the need of analyzing policy interactions and integrating policies. In this thesis, we propose and implement EXAM-S, a comprehensive environment for policy analysis and management, which can be used to perform a variety of functions such as policy property analyses, policy similarity analysis, policy integration etc. As part of this environment, we have proposed and implemented new techniques for the analysis of policies that rely on a deep study of state of the art techniques. Moreover, we propose an approach for solving heterogeneity problems that usually arise when considering the analysis of policies belonging to different domains. Our work focuses on analysis of access control policies written in the dialect of XACML (Extensible Access Control Markup Language). We consider XACML policies because XACML is a rich language which can represent many policies of interest to real world applications and is gaining widespread adoption in the industry.

La diagnosi genetica preimpianto: argomenti favorevoli e contrari alla sua legittimità

Questa tesi ricostruisce la storia della giurisprudenza italiana che ha riguardato la legittimità o meno dell’impiego della diagnosi genetica preimpianto nell’ambito della procreazione medicalmente assistita, dall’emanazione della legge 40 del 2004 a tutt’oggi. Ed in particolare questa tesi si prefigge due obiettivi: uno, individuare ed illustrare le tipologie di argomenti utilizzati dal giurista-interprete per giudicare della legittimità o meno della pratica della diagnosi preimpianto degli embrioni prodotti, mediante le tecniche relative alla procreazione assistita; l’altro obiettivo, mostrare sia lo scontro fra i differenti argomenti, sia le ragioni per le quali prevalgono gli argomenti usati per legittimare la pratica della diagnosi preimpianto. Per raggiungere questi obiettivi, e per mostrare in maniera fenomenologica come avviene l’interpretazione giuridica in materia di diagnosi preimpianto, si è fatto principalmente riferimento alla visione che ha della detta interpretazione la prospettiva ermeneutica (concepita originariamente sul piano teoretico, quale ermeneutica filosofica, da H.G. Gadamer; divulgata ed approfondita sul piano giusfilosofico e della teoria dell’interpretazione giudica in Italia, fra gli altri, da F. Viola e G. Zaccaria). Così, in considerazione dei vari argomenti utilizzati per valutare la legittimità o meno della pratica della diagnosi preimpianto, i motivi per i quali in ultimo il giurista-interprete per giudicare ragionevolmente, deve ritenere legittima la pratica della diagnosi preimpianto sono i seguenti. I principi superiori dell’ordinamento e talune direttive giuridiche fondamentali dell’ordinamento, elaborate della giurisprudenza, le quali costituiscono la concretizzazione di detti principi e di una serie di disposizioni normative fondamentali per disciplinare il fenomeno procreativo, depongono per la legittimità della diagnosi preimpianto. Le tipologie degli argomenti impiegati per avallare la legittimità della diagnosi preimpianto attengono al tradizionale repertorio argomentativo a cui attinge il giurista, mentre la stessa cosa non si può dire per gli argomenti usati per negare la legittimità della diagnosi. Talune tipologie di argomenti utilizzate per negare la legittimità della diagnosi preimpianto costituiscono delle fallacie logiche, per esempio l’argomento del pendio scivoloso, e soprattutto le tipologie degli argomenti utilizzati per sostenere la legittimità della diagnosi preimpianto sono per lo più caratterizzate dalla ragionevolezza ed applicate per lo più opportunamente. Poi, si può osservare che: determinati argomenti, associati dal giurista-interprete ai principi i quali depongono per l’illegittimità della diagnosi preimpianto, facendo leva sulla categoria della possibilità, ed equiparando attualità e possibilità, privilegiano l’immaginazione alla realtà e portano a risultati interpretativi non razionalmente fondati; mentre gli argomenti associati dal giurista-interprete ai principi i quali depongono per la legittimità della diagnosi preimpianto, facendo leva sulla categoria della attualità, e tenendo ben distinte attualità e possibilità, privilegiano l’osservazione della realtà e portano a risultati razionalmente fondati.