Il contributo del telerilevamento multi ed iperspettrale per la caratterizzazione del territorio e la sostenibilità ambientale

L’evoluzione dei sensori multispettrali e la prospettiva di sviluppo dei sensori iperspettrali nel campo del telerilevamento ottico offrono nuovi strumenti per l’indagine del territorio e rinnovano la necessità di ridefinire potenzialità, limiti e accuratezza delle metodologie tradizionali. Nel caso delle immagini iperspettrali, in particolare, l’elevatissima risoluzione spettrale apre nuove possibilità di sviluppo di modelli fisicamente basati per correlare grandezze radiometriche con indicatori fisico-chimici caratteristici delle superfici osservate, a prezzo però di maggiori oneri nella gestione del dato. Il presente lavoro mira appunto ad esaminare, per alcune applicazioni di carattere ambientale e attraverso casi di studio specifici, le criticità del problema del rilevamento da remoto nel suo complesso: dai problemi di correzione radiometrica delle immagini, all'acquisizione di dati di calibrazione sul campo, infine all'estrazione delle informazioni di interesse dal dato telerilevato. A tal fine sono stati sperimentati diversi modelli di trasferimento radiativo ed è stata sviluppata un’interfaccia per la gestione del modello 6SV. Per quest’ultimo sono state inoltre sviluppate routine specifiche per il supporto dei sensori Hyperion e World View 2. La ricerca svolta intende quindi offrire un contributo alla definizione di procedure operative ripetibili, per alcune applicazioni intimamente connesse all’indagine conoscitiva ed al monitoraggio dei processi in atto sul territorio. Nello specifico, si è scelto il caso di studio dell’oasi del Fayyum, in Egitto, per valutare il contenuto informativo delle immagini satellitari sotto tre diversi profili, soltanto in apparenza distinti: la classificazione della litologia superficiale, la valutazione dello stato di qualità delle acque ed il monitoraggio delle opere di bonifica. Trattandosi di un’oasi, le aree coltivate del Fayyum sono circondate dai suoli aridi del deserto libico. La mancanza di copertura vegetale rappresenta una condizione privilegiata per l’osservazione della litologia superficiale da remoto, auspicabile anche per la scarsa accessibilità di alcune aree. Il fabbisogno idrico dell’oasi è garantito dall’apporto di acque del fiume Nilo attraverso una rete di irrigazione che ha, come recettore finale, il lago Qarun, situato nella porzione più depressa dell’oasi. Questo lago, privo di emissari, soffre enormi problemi di salinizzazione, visto il clima iper-arido in cui si trova, e di inquinamento da fertilizzanti agricoli. Il problema della sostenibilità ambientale dello sfruttamento agricolo intensivo dell’oasi è un problema di deterioramento della qualità dell’acqua e della qualità dei suoli. È un problema che richiede una adeguata conoscenza del contesto geologico in cui questi terreni sono inseriti ed una capacità di monitoraggio degli interventi di bonifica ed estensione delle coltivazioni in atto; entrambe conoscenze necessarie alla definizione di un piano di sviluppo economico sostenibile. Con l’intento di contribuire ad una valutazione delle effettive potenzialità del telerilevamento come strumento di informazione territoriale, sono state sperimentate tecniche di classificazione di immagini multispettrali ASTER ed iperspettrali Hyperion di archivio per discriminare la litologia superficiale sulle aree adiacenti al lago Qarun nell’oasi del Fayyum. Le stesse immagini Hyperion di archivio più altre appositamente acquisite sono state utilizzate, assieme ad immagini multispettrali ALI, per la valutazione qualitativa e quantitativa di parametri di qualità delle acque, attraverso l’applicazione di modelli empirici di correlazione. Infine, per valutare l’ipotesi che il deterioramento della qualità delle acque possa essere correlato ai processi di bonifica ed estensione delle coltivazioni in atto negli ultimi decenni, le immagini dell’archivio Landsat sono state utilizzate per analisi di change detection. Per quanto riguarda il problema della validazione dei risultati, si è fatto uso di alcuni dati di verità a terra acquisiti nel corso di un survey preliminare effettuato nell’Ottobre 2010. I campioni di roccia prelevati e le misure di conducibilità elettrica delle acque del lago, benché in numero estremamente limitato per la brevità della missione e le ovvie difficoltà logistiche, consentono alcune valutazioni preliminari sui prodotti ottenuti dalle elaborazioni. Sui campioni di roccia e sabbie sciolte, in particolare, sono state effettuate misure di riflettività in laboratorio ed analisi mineralogiche dettagliate. Per la valutazione della qualità delle acque, più precisamente delle concentrazioni di clorofilla, la metodologia utilizzata per il caso di studio egiziano è stata applicata anche sul tratto costiero adriatico antistante le foci dei fiumi Tronto e Salinello. In questo sito sono state effettuate misure in situ di conducibilità elettrica ed il prelievo di campioni di acqua per la determinazione in laboratorio delle concentrazioni di clorofilla. I risultati ottenuti hanno evidenziato le potenzialità offerte dall’informazione spettrale contenuta nelle immagini satellitari e consentono l’individuazione di alcune pratiche operative. D’altro canto hanno anche messo in luce le carenze dei modelli attualmente esistenti, nonché le criticità legate alla correzione atmosferica delle grandezze radiometriche rilevate.

High sensitivity analysis of BRAF mutations in neoplastic and non-neoplastic thyroid lesions

The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

Seismic tomographic full-waveform inversion for the Vrancea sinking lithosphere structure using the adjoint method.

The Vrancea region, at the south-eastern bend of the Carpathian Mountains in Romania, represents one of the most puzzling seismically active zones of Europe. Beside some shallow seismicity spread across the whole Romanian territory, Vrancea is the place of an intense seismicity with the presence of a cluster of intermediate-depth foci placed in a narrow nearly vertical volume. Although large-scale mantle seismic tomographic studies have revealed the presence of a narrow, almost vertical, high-velocity body in the upper mantle, the nature and the geodynamic of this deep intra-continental seismicity is still questioned. High-resolution seismic tomography could help to reveal more details in the subcrustal structure of Vrancea. Recent developments in computational seismology as well as the availability of parallel computing now allow to potentially retrieve more information out of seismic waveforms and to reach such high-resolution models. This study was aimed to evaluate the application of a full waveform inversion tomography at regional scale for the Vrancea lithosphere using data from the 1999 six months temporary local network CALIXTO. Starting from a detailed 3D Vp, Vs and density model, built on classical travel-time tomography together with gravity data, I evaluated the improvements obtained with the full waveform inversion approach. The latter proved to be highly problem dependent and highly computational expensive. The model retrieved after the first two iterations does not show large variations with respect to the initial model but remains in agreement with previous tomographic models. It presents a well-defined downgoing slab shape high velocity anomaly, composed of a N-S horizontal anomaly in the depths between 40 and 70km linked to a nearly vertical NE-SW anomaly from 70 to 180km.

Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.

Interactions of G-quadruplex binders and Topoisomerase I inhibitors in cancer cells

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

Performing inclusion and exclusion in the peer group. Children's social organization and peer socializing practices in the classroom

This PhD thesis investigates children’s peer practices in two primary schools in Italy, focusing on the ordinary and the Italian L2 classroom. The study is informed by the paradigm of language socialization and considers peer interactions as a ‘double opportunity space’, allowing both children’s co-construction of their social organization and children’s sociolinguistic development. These two foci of attention are explored on the basis of children’s social interaction and of the verbal, embodied, and material resources that children agentively deploy during their mundane activities in the peer group. The study is based on a video ethnography that lasted nine months. Approximately 30 hours of classroom interactions were video-recorded, transcribed, and analyzed with an approach that combines the micro-analytic instruments of Conversation Analysis and the use of ethnographic information. Three main social phenomena were selected for analysis: (a) children’s enactment of the role of the teacher, (b) children’s reproduction of must-formatted rules, and (c) children’s argumentative strategies during peer conflict. The analysis highlights the centrality of the institutional frame for children’s peer interactions in the classroom. Moreover, the study illustrates that children socialize their classmates to the linguistic, social, and moral expectations of the context in and through various practices. Notably, these practices are also germane to the local negotiation of children’s social organization and hierarchy. Therefore, the thesis underlines that children’s peer interactions are both a resource for children’s sociolinguistic development and a potentially problematic locus where social exclusion is constructed and brought to bear. These insights are relevant for teachers’ professional practice. Children’s peer interactions are a resource that can be integrated in everyday didactics. Nevertheless, the role of the teacher in supervising and steering children’s peer practices appears crucial: an acritical view of children’s autonomous work, often implied in teaching methods such as peer tutoring, needs to be problematized.

The conundrum of human visceral leishmaniasis in Emilia-Romagna, Italy: are wild and peridomestic animals potential reservoirs?

Leishmaniasis is a complex parasitic disease caused by intracellular protozoans of the genus Leishmania mainly transmitted by the bite of sand flies. In Italy, leishmaniasis is caused by Leishmania infantum, responsible for the human visceral and canine leishmaniases (HVL and CanL, respectively). Within Emilia-Romagna region, Italy, recent molecular studies indicated that L. infantum strains circulating in dogs and humans are different. This suggests that an animal reservoir other than dog should be evaluated in the epidemiology of HVL in Emilia-Romagna. Therefore, the main aim of this PhD project was to investigate the role of wild and peridomestic mammals as potential animal reservoirs of L. infantum in the regional zones where HVL foci are still active, also evaluating the possible role of arthropod vectors other than phlebotomine sandflies as vectors of Leishmania spp. in the sylvatic cycle of the protozoa. Overall, 206 specimens of different animal species (roe deer, rats, mice, badgers, hares, polecats, foxes, beech martens, bank voles, hedgehogs, and shrews), collected in Emilia-Romagna were screened for Leishmania with a real-time PCR, revealing a prevalence of 33% for roe deer (first report in this species). Positivity was also found in brown rats (10.6%), black rats (13.1%), mice (10%), badgers (25%), hedgehogs (80%) and bank voles (11%). To distinguish the two strains of L. infantum circulating in Emilia-Romagna, a nested PCR protocol optimized for animal tissues was developed, demonstrating that over 90% of L. infantum infections in roe deer were due to the strain isolated from humans and suggesting their possible role as reservoirs in the study area. Furthermore, the presence of Leishmania kDNA was detected in unfed larvae, nymphs and males of questing Ixodes ricinus ticks collected in regional parks of Emilia-Romagna suggesting their possible role in the transmission of L. infantum in a sylvatic or rural cycle.

Unveiling the molecular mechanism of BRCA2-RAD51 interaction to tackle cancer onset

Different kinds of lesions can occur to DNA, and among them, one of the most dangerous is the double strand breaks (DSBs). Actually, DSBs can result in mutations, chromosome translocation or deletion. For this kind of lesions, depending on cell cycle phase as well as DNA-end resection, cells have developed specific repair pathways. Among these the error-free homologous recombination (HR) plays a crucial role. HR takes place during S/G2 phases, since the sister chromatids can be used as homologous templates. In this process, hRAD51 and BRCA2 are key players. hRAD51 is a recombinase of 339 amino-acids highly conserved through evolution which displays an intrinsic tendency to form oligomeric structures. BRCA2 is a very large protein of 3418 amino-acids, essential for the recruitment and accumulation of hRAD51 in the nucleus repairing-foci. BRCA2 interacts with hRAD51 through eight, so-called, BRC repeats, composed of 35-40 amino-acids. Mutations within this region have been linked to an increased risk of ovarian cancer development. In particular, several reports highlighted that missense mutations within one BRC repeat can hamper BRCA2 activity. Considering the close homology between the BRC repeats, it is striking how these mutations cannot be counterbalanced by the other non-mutated repeats preserving the function and the interactions of BRCA2 with hRAD51. To date the only interaction that has been structurally elucidated, is the one taking place amid the fourth BRC repeat and hRAD51. Only very little biophysical information is available on the interaction of the other BRC repeats with hRAD51. This thesis aims at elucidating the mechanism of hRAD51-BRCA2 interaction, by means of biophysical and structural approaches.