Plants have developed elaborate defence mechanisms to protect themselves against pathogens. Recently, the ubiquitin-proteasome pathway has been proven to play important roles in regulating plant disease resistance. Previously, the tobacco (Nicotiana tabacum) ACRE276 and its Arabidopsis homolog AtPUB17 have been identified as E3 ligases that are positive regulators of the Cf-9/Avr9- and N/p50-elicited hypersensitive response (HR) in tobacco. In addition, AtPUB17 is required for the RPM1- and RPS4-mediated resistance responses in Arabidopsis. The identification of AtPUB17 signalling partners would allow us to understand the mode of action of AtPUB17 during plant defence. AtPOB1, a BTB/POZ-domain protein was isolated as an AtPUB17 interactor in a yeast-two-hybrid screen. The aim of this study was to confirm this interaction and to investigate the potential involvement of AtPOB1 in mediating disease resistance responses. The analysis of the Atpob1 knock out plants revealed a novel BTB/POZ protein implicated in plant defence. Atpob1 plants rapidly accumulated reactive oxygen species (ROS), induced the expression of pathogenesis related (PR) genes and developed spontaneous necrotic lesions upon infection with a virulent pathogen. AtPOB1 transcript and protein levels were induced by virulent Pseudomonas syringae. And transient overexpression of AtPOB1 in Cf-9 tobacco compromised the Avr9-triggered HR. In addition, Atpob1 plants showed signs of premature senescence. These results indicate that AtPOB1 is a negative regulator of plant defence- and senescence-associated pathways. The Nicotiana benthamiana AtPOB1 homolog was also identified and its cDNA sequence was used to investigate the role of NbPOB1 and its close relative NtPOB1 in disease resistance signalling. Transient overexpression of NbPOB1 and RNA interference (RNAi)-based silencing of NtPOB1 in Cf-9 tobacco compromised and accelerated the Avr9-triggered HR, respectively. Moreover, virus induced gene silencing (VIGS) of NbPOB1 accelerated the dark-induced senescence in N. benthamiana leaves. These observations identify NbPOB1 and NtPOB1 as the orthologs of AtPOB1. The subcellular localization of AtPOB1 and NbPOB1 was analyzed by transiently overexpressing GFP-AtPOB1 and GFP-NbPOB1 fusion proteins in tobacco leaf tissue. Analysis by confocal microscopy revealed that GFP fluorescence was localized in the nucleus of leaf tissue tested. The overexpression of AtPOB1 fused with a nuclear export signal (NES) failed to compromise the Avr9-triggered HR in Cf-9 tobacco, indicating the nuclear localization of AtPOB1 is crucial for its function. The BTB/POZ domain is a highly conserved protein-protein interaction interface that mediates homo- and/or hetero-dimerization of BTB/POZ proteins. The D146A and D141A mutation in the BTB/POZ domain of AtPOB1 and NbPOB1, respectively reduces their dimerization efficiency. These mutants fail to negatively regulate the Cf-9/Avr9-mediated HR, supporting the importance of an intact BTB/POZ interface for the function of AtPOB1 and NbPOB1. Finally, yeast-two-hybrid and immunoprecipitation assays indicate that AtPOB1 interacts with AtCUL3A, a component of E3 ligase complexes, in which AtPOB1 would confer substrate-specificity. We propose that AtPOB1 (and Nicotiana POB1) negatively regulate cell death and senescence possibly through ubiquitin-mediated pathways.

Muscular Dystrophy (MD) is a progressive muscle wasting disease which currently has no cure, and is caused by the mutation of the dystrophin gene. A multitude of approaches for the improvement of the muscular pathology caused by this condition are being investigated, one of which is gene therapy. This approach is used to deliver vectors containing therapeutic transgenes such as dystrophin to target muscle cells. One method of gene delivery utilises viral vectors, and although this has resulted in systemic delivery and efficient transgene expression, there are many safety implications which have led to the development of non-viral approaches, such as the direct delivery of naked plasmid DNA. However, the shortcomings of these vectors include an inability to replicate within host cells, resulting in the loss of vector as cells replicate, and the silencing of transgene expression. In an effort to overcome such limitations, a novel system called the ‘pEPI vector' has been developed. Here, the inclusion of the β-IFN scaffold/matrix-attachment region (S/MAR) element into the open reading frame of an actively transcribed transgene has been found to lead to sustained, long term transgene expression, and to allow the episomal propagation and maintenance of the vector in dividing cells over many generations. The aim of this thesis was to investigate the potential of this vector for use as a gene therapy vector in muscle cells in order to treat MD. In this study, the long-term expression of the eGFP reporter transgene inserted into the pEPI vector was evaluated, and the pEPI vector's episomal/integrant status was investigated, in C2C12 murine myoblasts, HeLa, and HepG2 cell lines. 60 days after transfection the vector was found not to have integrated into the host genomes of any of the cell lines. Transgene expression had declined to nearly undetectable levels in fast-replicating C2C12 and HeLa cells, but was at high levels in the relatively slow-dividing HepG2 cells. An attempt to improve long term transgene expression in C2C12 cells by changing the promoter from CMV to CAGG still led to low transgene expression after 60 days. To address this issue, this study focused on the development of a novel approach to ameliorate long-term transgene expression, based upon the origin of replication and nuclear matrix attachment properties of the S/MAR element, as well as the results obtained from testing the vector in the HeLa and HepG2 cells. It involved the arrest of C2C12 cells in the G0/G1 phase of the cell cycle post transfection with the pEPI vector in order to allow these fast-dividing cells an extended period of time to epigenetically mark pEPI prior to selection. The findings indicated that this novel method of pEPI vector establishment was superior to that which utilises selection alone. However, in spite of the improvement in long-term episomal transgene expression observed using this novel method of establishment of the pEPI vector, transgene expression levels were still relatively low after 35 days of cell proliferation, which led to the conclusion that further development of this vector is essential in order for it to be able to elicit a significant restoration of muscle function in MD patients. Additionally, two other S/MAR vectors were tested in C2C12 cells. One vector contained the ‘mini-S/MAR', a shorter version of the β-IFN S/MAR, and the other contained a novel S/MAR derived from the c-myc proto-oncogene. Transgene expression by either vector was nearly undetectable after several weeks of proliferation, and both were found to integrate into the C2C12 host genome, leading to the conclusion that not all S/MAR elements inserted within a plasmid vector can lead to long-term transgene expression, nor confer protection from vector integration.

Chapter 1 in this thesis provides a short introduction to the biochemistry and chemistry of iron-sulphur systems, particularly in relation to biological nitrogen fixation and the inhibition of nitrogenases and hydrogenases by carbon monoxide. Chapter 2 describes some results bearing on the interaction of the inhibitor CO with simple iron-sulphur and iron-sulphur-vanadium systems under reducing conditions. It is shown that in the presence of CO an Fe454-centre can be reduced to carbonylated {Fe252} and {Fe35}-fragments. The crystal structure of [Fe35(CO)g ][PPh4]2 is described. Chapter 3 describes infra-red/electrolysis studies of the [Fe4S4 (5Ph)4 ]2-/CO system and of related Fe-S species. Evidence is presented for the occurrence of [Fe454(CO)12]2- as an intermediate in the pathway of reduction of [Fe454 (5Ph)4 ]2- to [Fe252 (CO)612- under an atmosphere of co. Chapter 4 describes the fortuitous isolation of [V(52)2(terpyridine)] from a reaction of 2,2',2"-terpyridine with [VFe3S4Cl3 (dmf)]- and its rational synthesis from 2,2' ,2"-terpyridine and [V54 ]3-. Some physical and chemical and electrochemical properties of this compound are described together with a description of its X-ray crystallographic structure.

No description available.

No description available.

Negli ultimi anni la longevità è divenuto un argomento di notevole interesse in diversi settori scientifici. Le ricerche volte ad indagare i meccanismi che regolano i fattori della longevità si sono moltiplicate nell’ultimo periodo interessando, in maniera diversa, alcune regioni del territorio italiano. Lo studio presentato nella tesi ha l’obiettivo di identificare eventuali aggregazioni territoriali caratterizzate da una significativa propensione alla longevità nella regione Emilia-Romagna mediante l’impiego di metodologie di clustering spaziale, alcune delle quali di recente implementazione. La popolazione in esame è costituita dagli individui residenti in Emilia- Romagna nel quinquennio 2000-2004 suddivisa in classi di età, sesso e comune. L’analisi è di tipo puramente spaziale, in cui l’unità geografica elementare è identificata dal comune, ed è stata condotta separatamente per i due sessi. L’identificazione delle aree regionali ad elevata longevità è avvenuta utilizzando quattro metodologie di clustering spaziale, basate sulla teoria della massima verosimiglianza, che si differenziano tra loro per la modalità di ricerca dei potenziali clusters. La differenza consiste nella capacità di identificare aggregazioni territoriali di forma regolare (spatial scan statistic, Kulldorff e Nagarwalla,1995; Kulldorff,1997, 1999) o dall’andamento geometrico “libero” (flexible scan statistic, Tango e Takahashi,2005; algoritmo genetico, Duczmal et al.,2007; greedy growth search, Yiannakoulias et al.,2007). Le caratteristiche di ciascuna metodologia consentono, in tal modo, di “catturare” le possibili conformazioni geografiche delle aggregazioni presenti sul territorio e la teoria statistica di base, comune ad esse, consente di effettuare agevolmente un confronto tra i risultati ottenuti. La persistenza di un’area caratterizzata da un’elevata propensione alla longevità consente, infatti, di ritenere il cluster identificato di notevole interesse per approfondimenti successivi. Il criterio utilizzato per la valutazione della persistenza di un cluster è stato derivato dalla teoria dei grafi, con particolare riferimento ai multigrafi. L’idea è confrontare, a parità di parametri di ricerca, i grafi associati alle aggregazioni spaziali identificate con le diverse metodologie attraverso una valutazione delle occorrenze dei collegamenti esistenti tra le coppie di vertici. Alcune valutazioni di carattere demografico ed un esame della letteratura esistente sugli studi di longevità, hanno indotto alla definizione di una classe (aperta) di età per rappresentare il fenomeno nella nostra ricerca: sono stati considerati gli individui con età superiore o uguale a 95 anni (indicata con 95+). La misura di sintesi utilizzata per descrivere il fenomeno è un indicatore specifico di longevità, mutuato dalla demografia, indicato con Centenarian Rate (CR) (Robine e Caselli, 2005). Esso è definito dal rapporto tra la popolazione 95+ e la popolazione residente, nello stesso comune, al censimento del 1961. L’idea alla base del CR è confrontare gli individui longevi di un istante temporale con quelli presenti, nella stessa area, circa 40 anni prima dell’osservazione, ipotizzando che l’effetto migratorio di una popolazione possa ritenersi trascurabile oltre i 60 anni di età. La propensione alla longevità coinvolge in maniera diversa le aree del territorio dell’Emilia-Romagna. Le province della regione caratterizzate da una maggiore longevità sono Bologna, Ravenna e parte di Forlì-Cesena mentre la provincia di Ferrara si distingue per un livello ridotto del fenomeno. La distinzione per sesso non appare netta: gli uomini con età 95+, numericamente inferiori alle donne, risiedono principalmente nei comuni delle province di Bologna e Ravenna, con qualche estensione nel territorio forlivese, analogamente a quanto accade per la popolazione femminile che mostra, tuttavia, una maggiore prevalenza nei territori di Bologna e Forlì-Cesena, includendo alcune aree del riminese. Le province occidentali della regione, invece, non risultano interessate significativamente da questo fenomeno. Le metodologie di cluster detection utilizzate nello studio hanno prodotto risultati pressoché simili seppur con criteri di ricerca differenti. La spatial scan statistic si conferma una metodologia efficace e veloce ma il vincolo geometrico regolare imposto al cluster condiziona il suo utilizzo, rivelando una scarsa adattabilità nell’identificazione di aggregazioni irregolari. La metodologia FSC ha evidenziato buone capacità di ricerca e velocità di esecuzione, completata da una descrizione chiara e dettagliata dei risultati e dalla possibilità di poter visualizzare graficamente i clusters finali, anche se con un livello minimo di dettaglio. Il limite principale della metodologia è la dimensione ridotta del cluster finale: l’eccessivo impegno computazionale richiesto dalla procedura induce a fissare il limite massimo al di sotto delle 30 aree, rendendola così utilizzabile solo nelle indagini in cui si ipotizza un’estensione limitata del fenomeno sul territorio. L’algoritmo genetico GA si rivela efficace nell’identificazione di clusters di qualsiasi forma ed estensione, seppur con una velocità di esecuzione inferiore rispetto alle procedure finora descritte. Senza un’adeguata selezione dei parametri di ricerca,la procedura può individuare clusters molto irregolari ed estesi, consigliando l’uso di penalizzazione non nulla in fase di ricerca. La scelta dei parametri di ricerca non è comunque agevole ed immediata e, spesso, è lasciata all’esperienza del ricercatore. Questo modo di procedere, in aggiunta alla mancanza di informazioni a priori sul fenomeno, aumenta il grado di soggettività introdotto nella selezione dei parametri influenzando i risultati finali. Infine, la metodologia GGS richiede un carico computazionale nettamente superiore rispetto a quello necessario per le altre metodologie utilizzate e l’introduzione di due parametri di controllo favorisce una maggiore arbitrarietà nella selezione dei valori di ricerca adeguati; inoltre, la recente implementazione della procedura e la mancanza di studi su dati reali inducono ad effettuare un numero maggiore di prove durante la fase di ricerca dei clusters.

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The main aim of this Ph.D. dissertation is the study of clustering dependent data by means of copula functions with particular emphasis on microarray data. Copula functions are a popular multivariate modeling tool in each field where the multivariate dependence is of great interest and their use in clustering has not been still investigated. The first part of this work contains the review of the literature of clustering methods, copula functions and microarray experiments. The attention focuses on the K–means (Hartigan, 1975; Hartigan and Wong, 1979), the hierarchical (Everitt, 1974) and the model–based (Fraley and Raftery, 1998, 1999, 2000, 2007) clustering techniques because their performance is compared. Then, the probabilistic interpretation of the Sklar’s theorem (Sklar’s, 1959), the estimation methods for copulas like the Inference for Margins (Joe and Xu, 1996) and the Archimedean and Elliptical copula families are presented. In the end, applications of clustering methods and copulas to the genetic and microarray experiments are highlighted. The second part contains the original contribution proposed. A simulation study is performed in order to evaluate the performance of the K–means and the hierarchical bottom–up clustering methods in identifying clusters according to the dependence structure of the data generating process. Different simulations are performed by varying different conditions (e.g., the kind of margins (distinct, overlapping and nested) and the value of the dependence parameter ) and the results are evaluated by means of different measures of performance. In light of the simulation results and of the limits of the two investigated clustering methods, a new clustering algorithm based on copula functions (‘CoClust’ in brief) is proposed. The basic idea, the iterative procedure of the CoClust and the description of the written R functions with their output are given. The CoClust algorithm is tested on simulated data (by varying the number of clusters, the copula models, the dependence parameter value and the degree of overlap of margins) and is compared with the performance of model–based clustering by using different measures of performance, like the percentage of well–identified number of clusters and the not rejection percentage of H0 on . It is shown that the CoClust algorithm allows to overcome all observed limits of the other investigated clustering techniques and is able to identify clusters according to the dependence structure of the data independently of the degree of overlap of margins and the strength of the dependence. The CoClust uses a criterion based on the maximized log–likelihood function of the copula and can virtually account for any possible dependence relationship between observations. Many peculiar characteristics are shown for the CoClust, e.g. its capability of identifying the true number of clusters and the fact that it does not require a starting classification. Finally, the CoClust algorithm is applied to the real microarray data of Hedenfalk et al. (2001) both to the gene expressions observed in three different cancer samples and to the columns (tumor samples) of the whole data matrix.

No description available.

No description available.