La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF. / Protein kinases C (PKC) are known to be ubiquitously distributed and to have pleiotropic effects. Isoforms epsilon (PKCε) and delta (PKCδ) are involved in the regulation of cell growth, survival and differentiation; in particular, they have been also investigated for their role in the hematopoiesis and in aberrant processes of differentiation along the erythroid and megakaryocytic lineages. In this PhD thesis, the results of an in vitro study about the role of these two kinases in models of megakaryocytic (MK) differentiation, both normal and pathological, are presented. The observations about PKCε and PKCδ kinetics show how these proteins have a specific modulation during the MK differentiation that results in an opposite pattern of expression and, in the murine model if compared with the human model, also a reciprocal one. In particular, in human megakaryocytopoiesis, PKCε results down-modulated, whereas in mouse its levels increase. Instead, PKCδ shows a high and steady expression in maturing CD34+ MK committed, but it is strongly down-modulated during the latest phases of platelet maturation in the murine model. The study also elucidates the different pathways PKCε and PKCδ work through, being an inhibitory action of PKCε on RhoA during proplatelets (ppt) formation in the mouse model while, in the human MK differentiation, platelets production is regulated by PKCδ through Bcl-xL. In this dissertation it is also demonstrated how in an aberrant megakaryocytopoiesis, as in the pathologic model of primary myeloproliferative neoplasm (PMF), PKCε is strongly deregulated and it results in an altered Bcl-xL expression. A forced down-modulation of this kinase restores a normal MK differentiation and ppt maturation. Therefore, the data presented show that PKCε and PKCδ play a key role in proper megakaryocyte maturation and that PKCε could be a potential new therapeutic target for PMF.

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Animal neocentromeres are defined as ectopic centromeres that have formed in non-centromeric locations and avoid some of the features, like the DNA satellite sequence, that normally characterize canonical centromeres. Despite this, they are stable functional centromeres inherited through generations. The only existence of neocentromeres provide convincing evidence that centromere specification is determined by epigenetic rather than sequence-specific mechanisms. For all this reasons, we used them as simplified models to investigate the molecular mechanisms that underlay the formation and the maintenance of functional centromeres. We collected human cell lines carrying neocentromeres in different positions. To investigate the region involved in the process at the DNA sequence level we applied a recent technology that integrates Chromatin Immuno-Precipitation and DNA microarrays (ChIP-on-chip) using rabbit polyclonal antibodies directed against CENP-A or CENP-C human centromeric proteins. These DNA binding-proteins are required for kinetochore function and are exclusively targeted to functional centromeres. Thus, the immunoprecipitation of DNA bound by these proteins allows the isolation of centromeric sequences, including those of the neocentromeres. Neocentromeres arise even in protein-coding genes region. We further analyzed if the increased scaffold attachment sites and the corresponding tighter chromatin of the region involved in the neocentromerization process still were permissive or not to transcription of within encoded genes. Centromere repositioning is a phenomenon in which a neocentromere arisen without altering the gene order, followed by the inactivation of the canonical centromere, becomes fixed in population. It is a process of chromosome rearrangement fundamental in evolution, at the bases of speciation. The repeat-free region where the neocentromere initially forms, progressively acquires extended arrays of satellite tandem repeats that may contribute to its functional stability. In this view our attention focalized to the repositioned horse ECA11 centromere. ChIP-on-chip analysis was used to define the region involved and SNPs studies, mapping within the region involved into neocentromerization, were carried on. We have been able to describe the structural polymorphism of the chromosome 11 centromeric domain of Caballus population. That polymorphism was seen even between homologues chromosome of the same cells. That discovery was the first described ever. Genomic plasticity had a fundamental role in evolution. Centromeres are not static packaged region of genomes. The key question that fascinates biologists is to understand how that centromere plasticity could be combined to the stability and maintenance of centromeric function. Starting from the epigenetic point of view that underlies centromere formation, we decided to analyze the RNA content of centromeric chromatin. RNA, as well as secondary chemically modifications that involve both histones and DNA, represents a good candidate to guide somehow the centromere formation and maintenance. Many observations suggest that transcription of centromeric DNA or of other non-coding RNAs could affect centromere formation. To date has been no thorough investigation addressing the identity of the chromatin-associated RNAs (CARs) on a global scale. This prompted us to develop techniques to identify CARs in a genome-wide approach using high-throughput genomic platforms. The future goal of this study will be to focalize the attention on what strictly happens specifically inside centromere chromatin.

Biofeedback is an important tool in the rehabilitation of several dysphagic conditions. This thesis presents an investigation into using bio-impedance as a technique for providing biofeedback of the swallowing sequence, specifically sequencing in the pharynx. The motivation behind this project was to find an alternative rehabilitation tool for detecting pharyngeal sequencing, as the current tool of pharyngeal manometry is invasive and non-portable. This investigation included the design and creation of a bio-impedance measuring device named the Guided Utility for Latency in Pharyngeal Sequencing (GULPS). This system was continued from a previous unpublished investigation at the University of Canterbury, where an initial prototype was designed and created. It was found that this pre-existing system had numerous faults in both its hardware and software, limiting the use of the device. Electrical impedance across the throat can be determined by applying a known constant amplitude current signal across the throat and recording the corresponding voltage. This impedance has been shown to change during a swallowing sequence due to a change in the structure of the throat. The principle used in this project was to investigate if two positions of impedance measurement could be used to determine the sequencing of the pharynx during a swallow. The design of the GULPS device was influenced by the pre-existing system and several prototypes were built to obtain a system capable of providing two channels of impedance measurement. Software was adapted from the pre-existing system to interface with this hardware to provide a system that could be attached to an external computer. Various electrode positions for the final device were trialled aimed at measuring two similar, but temporally separated, impedance waveforms. It was found that positioning the electrodes close to the approximate position of the pharynx with a 40 mm gap between channels allowed for two temporally separated channels to be produced with three distinct features: two peaks and one trough in each of the GULPS waveforms. The GULPS device with these electrode positions was trialled on three `healthy' subjects and one dysphagic subject. The three features could be identified in both impedance waveforms in all four subjects. To determine if the identified features related to the sequencing of the pharynx, the GULPS device was trialled alongside the current conventional method for detecting pharyngeal sequencing, pharyngeal manometry. The results from these trials revealed a potential relationship between the temporal separation of the second peaks found in the GULPS waveforms and the temporal separation of the pressure peaks from pharyngeal manometry. The GULPS device was trialled alongside pharyngeal manometry on one `healthy' and one dysphagic subject. A linear regression between peak-to-peak latencies between the two methods had an R squared value of 0.347 for the `healthy' subject and 0.241 for the dysphagic subject. However, these peaks were often difficult to detect, and could only be detected in 64% of swallows in the `healthy' subjects using the GULPS device in a standalone fashion and in 23% of swallows when used concurrently with manometry. As the current GULPS device is unable to produce the desired results in a consistent manner, no definitive conclusions can be drawn on the ability of using bio-impedance to measure the pharyngeal sequence. Notwithstanding, substantial progress has been made towards a device for reliable measurement of pharyngeal sequencing and, together with the clinical benefits to be gained, more than justify further research and development into GULPS for dysphagia rehabilitation.

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The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

Per comprendere le vicende di una famiglia illustre e nobile il cui ruolo politico e sociale in Sicilia si data alle soglie del XIV secolo, non possiamo astenerci dal ricordare i fatti e gli eventi che hanno dominato la storia siciliana e determinato l’ascesa di Castelvetrano come centro signorile per eccellenza. E’ necessario, quindi, collocare geograficamente e storicamente l’isola per inserirla all’interno di un preciso quadro socio-politico. All’origine della sua storia sono sicuramente da individuare sia il legame intercorso nei secoli tra l’Asia e l’Europa, in particolare tra l’Asia Minore bizantina e l’area mediterranea unificata proprio dall’impero di Bisanzio, sia le lotte per l’egemonia tra Chiesa e Impero, (che abbastanza presto sarà impero d’Occidente) lotte che vedono entrambe le parti impegnate a contendersi il ruolo di guida politica, morale e spirituale dell’intera cristianità medievale, ritenendo ogni altro potere subordinato al proprio. / To understand the story of a noble and illustrious family whose political and social role in Sicily date is on the threshold of the fourteenth century, we can not refrain from remembering facts and events that have dominated the history of Sicily and led to the rise of Castelvetrano as a center for aristocratic excellence. E 'therefore necessary to place geographically and historically the island to slip into a specific socio-political context. At the origin of its history are definitely identify both the bond in the centuries that elapsed between Asia and Europe, in particular between the Byzantine Asia Minor and the area Mediterranean Unified own empire of Byzantium, and the struggles for hegemony between Church and Empire, (which will be soon enough empire of the West) that struggles see both sides committed to contend for the role of political leadership, moral and spiritual whole of Christianity medieval, considering all other powers subject to their own.

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.