Localizzazione della topoisomerasi IB sui telomeri di S. cerevisiae e ruolo della sua attività catalitica sulle modificazioni istoniche

Il superavvolgimento del DNA nelle cellule, regolato dalle DNA Topoisomerasi, influenza molti processi biologici, quali la trascrizione, la replicazione, la ricombinazione ed il rimodellamento della cromatina. La DNA Topoisomerasi IB eucariotica, (Top1), è un enzima efficiente nella rimozione dei superavvolgimenti del DNA in vitro e la sua principale funzione cellulare è la rimozione dei superavvolgimenti positivi e negativi generati durante la trascrizione e la replicazione. Risultati recenti hanno fornito evidenze sperimentali del coinvolgimento di Top1 in meccanismi multipli di regolazione dell’espressione genica eucariotica, in particolare nella fase di inizio e maturazione dei trascritti. Tuttavia, le funzioni di Top1 non sono ancora state stabilite a livello globale. Pertanto, nella presente tesi di dottorato abbiamo risposto a questa domanda con l’analisi dei profili di trascrizione genica globale e con studi di immunoprecipitazione della cromatina (ChIP) in cellule di S. cerevisiae. Circa il 9% dei geni sono influenzati da Top1, e l’analisi dei profili di espressione mostra che Top1 wt aumenta l’utilizzo del glucosio e dei pathway per la produzione di energia, con specifica diminuzione della trascrizione dei geni telomerici e subtelomerici. Abbiamo inoltre dimostrato che Top1 wt, ma non il suo mutante inattivo, aumenta la velocità di crescita cellulare nelle cellule di lievito studiate. Le analisi di ChIP mostrano che, in confronto all’assenza dell’enzima, Top1 wt diminuisce l’acetilazione dell’istone H4, compresa quella specifica della lisina 16, nel telomero destro del cromosoma XIV mentre la mutazione che inattiva l’enzima aumenta in maniera marcata l’acetilazione dell’istone H4 e la di-metilazione della lisina 4 dell’istone H3. Top1 wt incrementa anche il reclutamento di Sir3 nelle regioni di confine della cromatina silenziata dello stesso telomero. Studi di immunoprecipitazione indicano che l’enzima interagisce direttamente con la struttura della cromatina telomerica poichè entrambe le proteine, quella wt e quella inattiva, sono localizzate sulle ripetizioni telomeriche dei cromosomi di lievito. Questi risultati dimostrano che Top1, una proteina non essenziale in lievito, ottimizza i livelli globali dei trascritti per una crescita più efficiente di cellule in fase esponenziale. Indagando il meccanismo che è alla base della specifica repressione dei geni telomerici, abbiamo dimostrato che Top1 favorisce delle modifiche posttraduzionali degli istoni che indicano una struttura della cromatina repressa nelle regioni telomeriche.

Sviluppo di metodologie di localizzazione nel contesto del Comprehensive Nuclear Test-Ban Treaty

Since the first underground nuclear explosion, carried out in 1958, the analysis of seismic signals generated by these sources has allowed seismologists to refine the travel times of seismic waves through the Earth and to verify the accuracy of the location algorithms (the ground truth for these sources was often known). Long international negotiates have been devoted to limit the proliferation and testing of nuclear weapons. In particular the Treaty for the comprehensive nuclear test ban (CTBT), was opened to signatures in 1996, though, even if it has been signed by 178 States, has not yet entered into force, The Treaty underlines the fundamental role of the seismological observations to verify its compliance, by detecting and locating seismic events, and identifying the nature of their sources. A precise definition of the hypocentral parameters represents the first step to discriminate whether a given seismic event is natural or not. In case that a specific event is retained suspicious by the majority of the State Parties, the Treaty contains provisions for conducting an on-site inspection (OSI) in the area surrounding the epicenter of the event, located through the International Monitoring System (IMS) of the CTBT Organization. An OSI is supposed to include the use of passive seismic techniques in the area of the suspected clandestine underground nuclear test. In fact, high quality seismological systems are thought to be capable to detect and locate very weak aftershocks triggered by underground nuclear explosions in the first days or weeks following the test. This PhD thesis deals with the development of two different seismic location techniques: the first one, known as the double difference joint hypocenter determination (DDJHD) technique, is aimed at locating closely spaced events at a global scale. The locations obtained by this method are characterized by a high relative accuracy, although the absolute location of the whole cluster remains uncertain. We eliminate this problem introducing a priori information: the known location of a selected event. The second technique concerns the reliable estimates of back azimuth and apparent velocity of seismic waves from local events of very low magnitude recorded by a trypartite array at a very local scale. For the two above-mentioned techniques, we have used the crosscorrelation technique among digital waveforms in order to minimize the errors linked with incorrect phase picking. The cross-correlation method relies on the similarity between waveforms of a pair of events at the same station, at the global scale, and on the similarity between waveforms of the same event at two different sensors of the try-partite array, at the local scale. After preliminary tests on the reliability of our location techniques based on simulations, we have applied both methodologies to real seismic events. The DDJHD technique has been applied to a seismic sequence occurred in the Turkey-Iran border region, using the data recorded by the IMS. At the beginning, the algorithm was applied to the differences among the original arrival times of the P phases, so the cross-correlation was not used. We have obtained that the relevant geometrical spreading, noticeable in the standard locations (namely the locations produced by the analysts of the International Data Center (IDC) of the CTBT Organization, assumed as our reference), has been considerably reduced by the application of our technique. This is what we expected, since the methodology has been applied to a sequence of events for which we can suppose a real closeness among the hypocenters, belonging to the same seismic structure. Our results point out the main advantage of this methodology: the systematic errors affecting the arrival times have been removed or at least reduced. The introduction of the cross-correlation has not brought evident improvements to our results: the two sets of locations (without and with the application of the cross-correlation technique) are very similar to each other. This can be commented saying that the use of the crosscorrelation has not substantially improved the precision of the manual pickings. Probably the pickings reported by the IDC are good enough to make the random picking error less important than the systematic error on travel times. As a further justification for the scarce quality of the results given by the cross-correlation, it should be remarked that the events included in our data set don’t have generally a good signal to noise ratio (SNR): the selected sequence is composed of weak events ( magnitude 4 or smaller) and the signals are strongly attenuated because of the large distance between the stations and the hypocentral area. In the local scale, in addition to the cross-correlation, we have performed a signal interpolation in order to improve the time resolution. The algorithm so developed has been applied to the data collected during an experiment carried out in Israel between 1998 and 1999. The results pointed out the following relevant conclusions: a) it is necessary to correlate waveform segments corresponding to the same seismic phases; b) it is not essential to select the exact first arrivals; and c) relevant information can be also obtained from the maximum amplitude wavelet of the waveforms (particularly in bad SNR conditions). Another remarkable point of our procedure is that its application doesn’t demand a long time to process the data, and therefore the user can immediately check the results. During a field survey, such feature will make possible a quasi real-time check allowing the immediate optimization of the array geometry, if so suggested by the results at an early stage.

Ingegneria di sistemi auto‐organizzanti con il paradigma multiagente

Self-organisation is increasingly being regarded as an effective approach to tackle modern systems complexity. The self-organisation approach allows the development of systems exhibiting complex dynamics and adapting to environmental perturbations without requiring a complete knowledge of the future surrounding conditions. However, the development of self-organising systems (SOS) is driven by different principles with respect to traditional software engineering. For instance, engineers typically design systems combining smaller elements where the composition rules depend on the reference paradigm, but typically produce predictable results. Conversely, SOS display non-linear dynamics, which can hardly be captured by deterministic models, and, although robust with respect to external perturbations, are quite sensitive to changes on inner working parameters. In this thesis, we describe methodological aspects concerning the early-design stage of SOS built relying on the Multiagent paradigm: in particular, we refer to the A&A metamodel, where MAS are composed by agents and artefacts, i.e. environmental resources. Then, we describe an architectural pattern that has been extracted from a recurrent solution in designing self-organising systems: this pattern is based on a MAS environment formed by artefacts, modelling non-proactive resources, and environmental agents acting on artefacts so as to enable self-organising mechanisms. In this context, we propose a scientific approach for the early design stage of the engineering of self-organising systems: the process is an iterative one and each cycle is articulated in four stages, modelling, simulation, formal verification, and tuning. During the modelling phase we mainly rely on the existence of a self-organising strategy observed in Nature and, hopefully encoded as a design pattern. Simulations of an abstract system model are used to drive design choices until the required quality properties are obtained, thus providing guarantees that the subsequent design steps would lead to a correct implementation. However, system analysis exclusively based on simulation results does not provide sound guarantees for the engineering of complex systems: to this purpose, we envision the application of formal verification techniques, specifically model checking, in order to exactly characterise the system behaviours. During the tuning stage parameters are tweaked in order to meet the target global dynamics and feasibility constraints. In order to evaluate the methodology, we analysed several systems: in this thesis, we only describe three of them, i.e. the most representative ones for each of the three years of PhD course. We analyse each case study using the presented method, and describe the exploited formal tools and techniques.

Buoni cibi per buoni pensieri. Rituali di consumo alimentare tra flussi di globalizzazione e pratiche di localizzazione

La tesi di dottorato “Buoni cibi per buoni pensieri. Rituali di consumo alimentare tra flussi di globalizzazione e pratiche di localizzazione” verte attorno allo studio e all’approfondimento della sociologia dei consumi, con particolare riferimento alle relazioni che si possono instaurare tra i comportamenti alimentari collettivi a livello locale e il sistema globale. Il focus dell’analisi si incentra sulla sostenibilità dei consumi, fenomenologia esemplificativa di una modalità di rapporto degli uomini con i cibi innovativa e al contempo nient’affatto scontata: non soltanto a livello individual-collettivo come strumento di intelligibilità sociale e costruzione di senso, ma anche sul piano sistemico come rapporto con l’ambiente e il mondo circostante. In effetti, il percorso di studi che ha condotto all’elaborazione teorica ed empirica della tesi di dottorato, pur muovendo da studi radicati nella letteratura socio-antropologica come il consumo dei cibi e la cultura da essi veicolata, viene oggi inquadrata all’interno di nuovi scenari sullo sfondo del mutamento socioeconomico: le pratiche di sostenibilità causate dalle ambivalenti ripercussioni giocate dalla globalizzazione sui sistemi sociali. Se è vero infatti che oggi si vive in una società complessa, caratterizzata dalla crescente centralità del sistema del consumo rispetto agli altri sottosistemi, è evidente come una ri-lettura critica delle sue dimensioni simboliche ed intersoggettive nel senso del ridimensionamento degli aspetti legati alla mercificazione e al materialismo, faccia sì che la tesi dottorale si orienti sulla ricerca di una sostenibilità oggi tanto diffusa, da sostanziarsi sempre più anche nelle pratiche di consumo alimentare. Oggetto specifico di questo lavoro sono dunque, senza pretesa di esaustività ma rappresentative del contesto spaziale preso in esame, fenomenologie del consumo alimentare italiano all’interno di uno scenario globale: in particolare, si affronta la nuova cultura della sostenibilità che nasce dall’incontro fra i cittadini-consumatori e le complesse tematiche emergenti del sistema sociale di riferimento.