Analisi di espressione di microRNA in Tumore a Cellule Giganti: identificazione dei target con ruolo di potenziali biomarcatori

Il Tumore a Cellule Giganti dell’osso (TCG) è una rara neoplasia che rappresenta il 5% dei tumori di natura ossea; sebbene venga considerato un tumore a decorso benigno può manifestare caratteri di aggressività locale dando origine a recidive locali nel 10-25% dei casi, e nel 2-4% dei casi metastatizza a livello polmonare. In questo studio è stata valutata l’espressione dei miRNA mediante miRNA microarray in 10 pazienti affetti da TCG, 5 con metastasi e 5 liberi da malattia; sono stati riscontrati miRNA differenzialmente espressi tra i 2 gruppi di pazienti e la successiva validazione mediante Real Time PCR ha confermato una differenza significativa per il miR-136 (p=0.04). Mediante analisi bioinformatica con il software TargetScan abbiamo identificato RANK e NF1B come target del miR-136 e ne abbiamo studiato l’espressione mediante Real Time PCR su una più ampia casistica di pazienti affetti da TCG, metastatico e non, evidenziando una maggior espressione di NF1B nel gruppo di pazienti metastatici, mentre RANK non ha dimostrato una differenza significativa. L’analisi di Western Blot ha rilevato una maggiore espressione di entrambe le proteine nei pazienti metastatici rispetto ai non metastatici. Successivamente è stato condotto uno studio di immunoistochimica su TMA di 163 campioni di pazienti affetti da TCG a diverso decorso clinico che ha dimostrato una maggiore e significativa espressione di entrambe i target nei pazienti con metastasi rispetto ai non metastatici; le analisi di popolazione mediante Kaplan-Meier hanno confermato la correlazione tra over-espressione di RANK, NF1B e ricaduta con metastasi (p=0.001 e p<0.0005 rispettivamente). Lo studio di immunoistochimica è stato ampliato alle proteine maggiormente coinvolte nell’osteolisi che risultano avere un significato prognostico; tuttavia mediante analisi di ROC, la co-over-espressione di RANK, RANKL e NF1B rappresenta il migliore modello per predire la comparsa di metastasi (AUC=0.782, p<0.0005).

Analisi di immagini aerofotogrammetriche e telerilevate per la caratterizzazione qualitativa e quantitativa di ecosistemi forestali e della loro evoluzione

L’obiettivo della tesi riguarda l’utilizzo di immagini aerofotogrammetriche e telerilevate per la caratterizzazione qualitativa e quantitativa di ecosistemi forestali e della loro evoluzione. Le tematiche affrontate hanno riguardato, da una parte, l’aspetto fotogrammetrico, mediante recupero, digitalizzazione ed elaborazione di immagini aeree storiche di varie epoche, e, dall’altra, l’aspetto legato all’uso del telerilevamento per la classificazione delle coperture al suolo. Nel capitolo 1 viene fatta una breve introduzione sullo sviluppo delle nuove tecnologie di rilievo con un approfondimento delle applicazioni forestali; nel secondo capitolo è affrontata la tematica legata all’acquisizione dei dati telerilevati e fotogrammetrici con una breve descrizione delle caratteristiche e grandezze principali; il terzo capitolo tratta i processi di elaborazione e classificazione delle immagini per l’estrazione delle informazioni significative. Nei tre capitoli seguenti vengono mostrati tre casi di applicazioni di fotogrammetria e telerilevamento nello studio di ecosistemi forestali. Il primo caso (capitolo 4) riguarda l’area del gruppo montuoso del Prado- Cusna, sui cui è stata compiuta un’analisi multitemporale dell’evoluzione del limite altitudinale degli alberi nell’arco degli ultimi cinquant’anni. E’ stata affrontata ed analizzata la procedura per il recupero delle prese aeree storiche, definibile mediante una serie di successive operazioni, a partire dalla digitalizzazione dei fotogrammi, continuando con la determinazione di punti di controllo noti a terra per l’orientamento delle immagini, per finire con l’ortorettifica e mosaicatura delle stesse, con l’ausilio di un Modello Digitale del Terreno (DTM). Tutto ciò ha permesso il confronto di tali dati con immagini digitali più recenti al fine di individuare eventuali cambiamenti avvenuti nell’arco di tempo intercorso. Nel secondo caso (capitolo 5) si è definita per lo studio della zona del gruppo del monte Giovo una procedura di classificazione per l’estrazione delle coperture vegetative e per l’aggiornamento della cartografia esistente – in questo caso la carta della vegetazione. In particolare si è cercato di classificare la vegetazione soprasilvatica, dominata da brughiere a mirtilli e praterie con prevalenza di quelle secondarie a nardo e brachipodio. In alcune aree sono inoltre presenti comunità che colonizzano accumuli detritici stabilizzati e le rupi arenacee. A questo scopo, oltre alle immagini aeree (Volo IT2000) sono state usate anche immagini satellitari ASTER e altri dati ancillari (DTM e derivati), ed è stato applicato un sistema di classificazione delle coperture di tipo objectbased. Si è cercato di definire i migliori parametri per la segmentazione e il numero migliore di sample per la classificazione. Da una parte, è stata fatta una classificazione supervisionata della vegetazione a partire da pochi sample di riferimento, dall’altra si è voluto testare tale metodo per la definizione di una procedura di aggiornamento automatico della cartografia esistente. Nel terzo caso (capitolo 6), sempre nella zona del gruppo del monte Giovo, è stato fatto un confronto fra la timberline estratta mediante segmentazione ad oggetti ed il risultato di rilievi GPS a terra appositamente effettuati. L’obiettivo è la definizione del limite altitudinale del bosco e l’individuazione di gruppi di alberi isolati al di sopra di esso mediante procedure di segmentazione e classificazione object-based di ortofoto aeree in formato digitale e la verifica sul campo in alcune zone campione dei risultati, mediante creazione di profili GPS del limite del bosco e determinazione delle coordinate dei gruppi di alberi isolati. I risultati finali del lavoro hanno messo in luce come le moderne tecniche di analisi di immagini sono ormai mature per consentire il raggiungimento degli obiettivi prefissi nelle tre applicazioni considerate, pur essendo in ogni caso necessaria una attenta validazione dei dati ed un intervento dell’operatore in diversi momenti del processo. In particolare, le operazioni di segmentazione delle immagini per l’estrazione di feature significative hanno dimostrato grandi potenzialità in tutti e tre i casi. Un software ad “oggetti” semplifica l’implementazione dei risultati della classificazione in un ambiente GIS, offrendo la possibilità, ad esempio, di esportare in formato vettoriale gli oggetti classificati. Inoltre dà la possibilità di utilizzare contemporaneamente, in un unico ambiente, più sorgenti di informazione quali foto aeree, immagini satellitari, DTM e derivati. Le procedure automatiche per l’estrazione della timberline e dei gruppi di alberi isolati e per la classificazione delle coperture sono oggetto di un continuo sviluppo al fine di migliorarne le prestazioni; allo stato attuale esse non devono essere considerate una soluzione ottimale autonoma ma uno strumento per impostare e semplificare l’intervento da parte dello specialista in fotointerpretazione.

Contributo della modellistica idrologica all'analisi di suscettività alle frane superficiali in argilla

L'Appenino Emiliano è tra le zone più franose al mondo; i fenomeni che la interessano sono abbastanza lenti e quindi mai catastrofici, ma, vista la loro diffusione, molto dannosi per le infrastrutture. La ricerca della pericolosità associata alle frane si distingue in previsione spaziale e previsione temporale; la prima, detta anche suscettività, è il tema del presente lavoro. La suscettività è volta alla realizzazione di carte di propensione al dissesto, relative e indipendenti dal tempo. Dall'inizio degli anni '90 sono disponibili in letteratura diversi modelli per rispondere a questa esigenza, i quali sono generalmente costituiti da una componente geo-meccanica (di solito il modello del Pendio Infinito) e una idrologica. Il presente lavoro si concentra su quest'ultima che, nei diversi modelli, presenta la maggiore varietà, e cerca di capire quale sia il contributo che questa componente può dare all'analisi di suscettività in un'area argillosa. Per valutare questo contributo, sono stati applicati ad un'area di studio rappresentativa, diversi modelli fisicamente basati noti in letteratura o creati appositamente. Le informazioni dinamiche dei modelli transitori sono state integrate nel tempo secondo diversi metodi che tengono conto della permanenza delle condizioni critiche nel versante. I risultati dell'analisi suggeriscono che, nell'area di studio, e presumibilmente nelle aree a prevalenza argillosa in genere, per la determinazione della suscettività alle frane, il contributo di un modello fisicamente basato, completo di componente geo-meccanica e componente idrologica accoppiate, è assolutamente trascurabile rispetto ad un semplice modello geo-meccanico basato sulla sola pendenza come quello del Pendio Infinito. Le indicazioni provenienti da un modello completo possono essere ridondanti o addirittura fuorvianti se questo non è adatto alle caratteristiche dell'area in studio e ben calibrato.

Altinópolis, una utopia brasiliana. Analisi di una esperienza di educazione alla pace e non violenza come pratica alternativa dell'educazione popolare

The present thesis has as its object of study a project called “Education towards Peace and No Violence”, developed in the town of Altinópolis, in the inner part of the state of São Paulo, Brazil. Based on the analysis and the studies that were carried out, it was possible to identify the difficulties in the implementation of the project, as well as the positive results evidenced by the reduction of the reports of violence in the town. Through a careful research of the historical facts, it shall be demonstrated the progress of education, particularly in Brazil, throughout the years. In addition, a panoramic view of the studies and programs for peace developed in North America and Occidental Europe will be presented. The education for peace based in the holistic concept of human development appeared as a new educational paradigm that works as a crucial instrument in the construction process towards a peaceful and more human society based in social justice.

Innovazione organizzativa e istituzionale della pubblica amministrazione. Un approccio interpretativo e l'analisi di un dispositivo partecipativo nel settore della cultura

The thesis aims at inquiring into the issue of innovation and organizational and institutional change in the public administration with regard to the increasingly massive adoption of participatory devices and practices in various arenas of public policies. The field of reference regards transformations of the types of public actions and regulation systems, concerning governance. Together with the crisis of the public function and of the role played by the insitutions what is emerging are different levels of governement, both towards an over national and a local direction, and a plurality of social interlocutors, followed by a post-bureaucratic pattern of the public administration that is opening itself in the direction of environment and citizens. The public adminstration is no longer considered an inert object within the bureaucratic paradigm but as a series of communicative processes, choices, cultures and practices that actively builds itself and the environment it interacts with. Therefore, the output of the public administration isn’t the simple service being supplied but the relationship enacted with the citizen, relationship that becomes the constituent basis of adminstrative processes. The intention of thesis is to take into consideration the relation between innovation of the public administration and participatory experimentations and implementations regarded as exchanges in which citizens and the public administration hold talks and debates. The issue of the organizational change of the public administration as output and effect of inclusive deliberative practices has been analysed starting from an institutionalist approach, in other words examining the constituent features of institutions, “rediscovering” them with regard to their public nature, their ability to elaborate collective values and meanings, the social definition of problems and solutions. The participatory device employed by the Forlì city council that involved enterprises and cultural associations of the area in order to build a participatory Table, has been studied through a qualitative methodology (participant observation and semi-strutctured interviews). The analysis inquired into the public nature both of the participatory device and the administrative action itself as well as into elements pertaining the deliberative setting, the regulative reference framework and the actors which took part in the process.

Genetica molecolare dell'autismo: studi di associazione ed analisi di geni candidati

Autism is a neurodevelpmental disorder characterized by impaired verbal communication, limited reciprocal social interaction, restricted interests and repetitive behaviours. Twin and family studies indicate a large genetic contribution to ASDs (Autism Spectrum Disorders). During my Ph.D. I have been involved in several projects in which I used different genetic approaches in order to identify susceptibility genes in autism on chromosomes 2, 7 and X: 1)High-density SNP association and CNV analysis of two Autism Susceptibility Loci. The International Molecular Genetic Study of Autism Consortium (IMGSAC) previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we evaluated the patterns of linkage disequilibrium (LD) and the distribution of haplotype blocks, utilising data from the HapMap project, across the two strongest peaks of linkage on chromosome 2 and 7. More than 3000 SNPs have been selected in each locus in all known genes, as well as SNPs in non-genic highly conserved sequences. All markers have been genotyped to perform a high-density association analysis and to explore copy number variation within these regions. The study sample consisted of 127 and 126 multiplex families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Association and CNV analysis implicated several new genes, including IMMP2L and DOCK4 on chromosome 7 and ZNF533 and NOSTRIN on the chromosome 2. Particularly, my contribution to this project focused on the characterization of the best candidate gene in each locus: On the AUTS5 locus I carried out a transcript study of ZNF533 in different human tissues to verify which isoforms and start exons were expressed. High transcript variability and a new exon, never described before, has been identified in this analysis. Furthermore, I selected 31 probands for the risk haplotype and performed a mutation screen of all known exons in order to identify novel coding variants associated to autism. On the AUTS1 locus a duplication was detected in one multiplex family that was transmitted from father to an affected son. This duplication interrupts two genes: IMMP2L and DOCK4 and warranted further analysis. Thus, I performed a screening of the cohort of IMGSAC collection (285 multiplex families), using a QMPSF assay (Quantitative Multiplex PCR of Short fluorescent Fragments) to analyse if CNVs in this genic region segregate with autism phenotype and compare their frequency with a sample of 475 UK controls. Evidence for a role of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family. 2)Analysis of X chromosome inactivation. Skewed X chromosome inactivation (XCI) is observed in females carrying gene mutations involved in several X-linked syndromes. We aimed to estimate the role of X-linked genes in ASD susceptibility by ascertaining the XCI pattern in a sample of 543 informative mothers of children with ASD and in a sample of 164 affected girls. The study sample included families from different european consortia. I analysed the XCI inactivation pattern in a sample of italian mothers from singletons families with ASD and also a control groups (144 adult females and 40 young females). We observed no significant excess of skewed XCI in families with ASD. Interestingly, two mothers and one girl carrying known mutations in X-linked genes (NLGN3, ATRX, MECP2) showed highly skewed XCI, suggesting that ascertainment of XCI could reveal families with X-linked mutations. Linkage analysis was carried out in the subgroup of multiplex families with skewed XCI (≥80:20) and a modest increased allele sharing was obtained in the Xq27-Xq28 region, with a peak Z score of 1.75 close to rs719489. In this region FMR1 and MECP2 have been associated in some cases with austim and therefore represent candidates for the disorder. I performed a mutation screen of MECP2 in 33 unrelated probands from IMGSAC and italian families, showing XCI skewness. Recently, Xq28 duplications including MECP2, have been identified in families with MR, with asymptomatic carrier females showing extreme (>85%) skewing of XCI. For these reason I used the sample of probands from X-skewed families to perform CNV analysis by Real-time quantitative PCR. No duplications have been found in our sample. I have also confirmed all data using as alternative method the MLPA assay (Multiplex Ligation dependent Probe Amplification). 3)ASMT as functional candidate gene for autism. Recently, a possible involvement of the acetylserotonin O-methyltransferase (ASMT) gene in susceptibility to ASDs has been reported: mutation screening of the ASMT gene in 250 individuals from the PARIS collection revealed several rare variants with a likely functional role; Moreover, significant association was reported for two SNPs (rs4446909 and rs5989681) located in one of the two alternative promoters of the gene. To further investigate these findings, I carried out a replication study using a sample of 263 affected individuals from the IMGSAC collection and 390 control individuals. Several rare mutations were identified, including the splice site mutation IVS5+2T>C and the L326F substitution previously reported by Melke et al (2007), but the same rare variants have been found also in control individuals in our study. Interestingly, a new R319X stop mutation was found in a single autism proband of Italian origin and is absent from the entire control sample. Furthermore, no replication has been found in our case-control study typing the SNPs on the ASMT promoter B.

Impiego della spettrometria di massa nell'analisi di fumonisine in alimenti e matrici di origine animale

Mycotoxins are heterogeneous chemical compounds characterized by a low molecular weight and synthesized by the secondary metabolism of different molds. Fumonisins are water-soluble mycotoxins produced by Fusarium species spoiling corn and derived produc ts. These mycotoxins can be a health hazard when consuming contaminated cereals, but they can reach humans also indirectly through the consumption of food products derived from animals fed with contaminated feed. Fumonisins have been associated with several animal and human diseases: they are suspected risk factors for esophageal and liver cancers, neural tube defects and cardiovascular problems. Improved methods are needed to accurately assess fumonisins concentrations in food of vegetable and animal origin, in order to prevent acute and chronic human exposure. The aim of the present work was to evaluate the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis from foods and matrices of animal origin. Different methods for the identification and quantification of fumonisins and related products have been developed and validated to determine fumonisin B1 in milk, fumonisin B1, fumonisin B2 and their complete hydrolyzed products (HFB1 and HFB2) in pig liver and fumonisins B1 and B2 in complete and complementary dry dog food. The experimental procedures have been carefully studied, considering matrices features, number and type of molecules to detect. Therefore, several extraction, clean up and separation techniques were tested in order to obtain the better conditions of sample processing. The fit for purpose sample preparation, matched with high mass spectrometry sensibility and specificity, have allowed to achieve good results in any tested animal matrices. Hence, the developed methods were validated and have shown a high accuracy, sensibility and precision, fulfilling performance requirements of Decision 2002/657/EC and of European Project Standard, Measuring and Testing (SMT). In any developed method, the analytes were identified and quantified even at very low concentrations : the limits of quantification resulted lower than other similar works, performed with different detectors. These methods were applied to some commercial samples and to some samples collected for research projects in the Department of Veterinary Public Health and Animal Pathology (DVPHAP) of University of Bologna. Although the disclosed data must be considered completely preliminary and without statistical significance, they emphasize the presence of mycotoxins in animal products. The outcomes obtained from the processed samples (bovine milk, pig liver and dry dog food) suggest the efficacy of these methods also on other food matrices, confirming the versatility and the performances of mass spectrometry, coupled with liquid chromatography, in fumonisins analysis. Moreover the results underline the need to set up a large scale monitoring in order to evaluate the presence of fumonisins in food of animal origin for human consumption.

Applicazione di strumenti e metodi per la gestione ambientale di un territorio vasto - Sviluppo di un sistema di gestione ambientale in un ente pubblico secondo lo schema europeo EMAS e analisi di un sistema energetico territoriale

Environmental Management includes many components, among which we can include Environmental Management Systems (EMS), Environmental Reporting and Analysis, Environmental Information Systems and Environmental Communication. In this work two applications are presented: the developement and implementation of an Environmental Management System in local administrations, according to the European scheme "EMAS", and the analysis of a territorial energy system through scenario building and environmental sustainability assessment. Both applications are linked by the same objective, which is the quest for more scientifically sound elements; in fact, both EMS and energy planning are oftec carachterized by localism and poor comparability. Emergy synthesis, proposed by ecologist H.T. Odum and described in his book "Environmental Accounting: Emergy and Environmental Decision Making" (1996) has been chosen and applied as an environmental evaluation tool, in order complete the analysis with an assessment of the "global value" of goods and processes. In particular, eMergy syntesis has been applied in order to improve the evaluation of the significance of environmental aspects in an EMS, and in order to evaluate the environmental performance of three scenarios of future evolution of the energy system. Regarding EMS, in this work an application of an EMS together with the CLEAR methodology for environmental accounting is discussed, in order to improve the identification of the environmental aspects; data regarding environmental aspects and significant ones for 4 local authorities are also presented, together with a preliminary proposal for the integration of the assessment of the significance of environmental aspects with eMergy synthesis. Regarding the analysis of an energy system, in this work the carachterization of the current situation is presented together with the overall energy balance and the evaluation of the emissions of greenhouse gases; moreover, three scenarios of future evolution are described and discussed. The scenarios have been realized with the support of the LEAP software ("Long Term Energy Alternatives Planning System" by SEI - "Stockholm Environment Institute"). Finally, the eMergy synthesis of the current situation and of the three scenarios is shown.

Sviluppo di modelli motore e veicolo per l'analisi di strategie di controllo in applicazioni Software e Hardware In the Loop

This work describes the development of a simulation tool which allows the simulation of the Internal Combustion Engine (ICE), the transmission and the vehicle dynamics. It is a control oriented simulation tool, designed in order to perform both off-line (Software In the Loop) and on-line (Hardware In the Loop) simulation. In the first case the simulation tool can be used in order to optimize Engine Control Unit strategies (as far as regard, for example, the fuel consumption or the performance of the engine), while in the second case it can be used in order to test the control system. In recent years the use of HIL simulations has proved to be very useful in developing and testing of control systems. Hardware In the Loop simulation is a technology where the actual vehicles, engines or other components are replaced by a real time simulation, based on a mathematical model and running in a real time processor. The processor reads ECU (Engine Control Unit) output signals which would normally feed the actuators and, by using mathematical models, provides the signals which would be produced by the actual sensors. The simulation tool, fully designed within Simulink, includes the possibility to simulate the only engine, the transmission and vehicle dynamics and the engine along with the vehicle and transmission dynamics, allowing in this case to evaluate the performance and the operating conditions of the Internal Combustion Engine, once it is installed on a given vehicle. Furthermore the simulation tool includes different level of complexity, since it is possible to use, for example, either a zero-dimensional or a one-dimensional model of the intake system (in this case only for off-line application, because of the higher computational effort). Given these preliminary remarks, an important goal of this work is the development of a simulation environment that can be easily adapted to different engine types (single- or multi-cylinder, four-stroke or two-stroke, diesel or gasoline) and transmission architecture without reprogramming. Also, the same simulation tool can be rapidly configured both for off-line and real-time application. The Matlab-Simulink environment has been adopted to achieve such objectives, since its graphical programming interface allows building flexible and reconfigurable models, and real-time simulation is possible with standard, off-the-shelf software and hardware platforms (such as dSPACE systems).

Tumori primitivi multipli del polmone. Profilo clinico e biologico in pazienti affetti da neoplasie polmonari multiple. Analisi di elementi clinici e marcatori biologici come possibili fattori di differenziazione dei tumori polmonari multipli

Background: MPLC represents a diagnostic challenge. Topic of the discussion is how to distinguish these patients as a metastatic or a multifocal disease. While in case of the different histology there are less doubt on the opposite in case of same histology is mandatory to investigate on other clinical features to rule out this question. Matherials and Methods: A retrospective review identified all patients treated surgically for a presumed diagnosis of SPLC. Pre-operative staging was obtained with Total CT scan and fluoro-deoxy positron emission tomography and mediastinoscopy. Patients with nodes interest or extra-thoracic location were excluded from this study. Epidermal growth factor receptor (EGFR) expression with complete immunohistochemical analisis was evaluated. Survival was estimated using Kaplan-Meyer method, and clinical features were estimated using a long-rank test or Cox proportional hazards model for categorical and continuous variable, respectively. Results: According to American College Chest Physician, 18 patients underwent to surgical resection for a diagnosis of MPLC. Of these, 8 patients had 3 or more nodules while 10 patients had less than 3 nodules. Pathologic examination demonstrated that 13/18(70%) of patients with multiple histological types was Adenocarcinoma, 2/18(10%) Squamous carcinoma, 2/18(10%) large cell carcinoma and 1/18(5%) Adenosquamosu carcinoma. Expression of EGFR has been evaluated in all nodules: in 7 patients of 18 (38%) the percentage of expression of each nodule resulted different. Conclusions: MPLC represent a multifocal disease where interactions of clinical informations with biological studies reinforce the diagnosis. EGFR could contribute to differentiate the nodules. However, further researches are necessary to validate this hypothesis.