Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

Comparison between analytical and numerical methods for the assessment of masonry arch bridges: Case study of Clemente Bridge on Savio river

This research has focused on the study of the behavior and of the collapse of masonry arch bridges. The latest decades have seen an increasing interest in this structural type, that is still present and in use, despite the passage of time and the variation of the transport means. Several strategies have been developed during the time to simulate the response of this type of structures, although even today there is no generally accepted standard one for assessment of masonry arch bridges. The aim of this thesis is to compare the principal analytical and numerical methods existing in literature on case studies, trying to highlight values and weaknesses. The methods taken in exam are mainly three: i) the Thrust Line Analysis Method; ii) the Mechanism Method; iii) the Finite Element Methods. The Thrust Line Analysis Method and the Mechanism Method are analytical methods and derived from two of the fundamental theorems of the Plastic Analysis, while the Finite Element Method is a numerical method, that uses different strategies of discretization to analyze the structure. Every method is applied to the case study through computer-based representations, that allow a friendly-use application of the principles explained. A particular closed-form approach based on an elasto-plastic material model and developed by some Belgian researchers is also studied. To compare the three methods, two different case study have been analyzed: i) a generic masonry arch bridge with a single span; ii) a real masonry arch bridge, the Clemente Bridge, built on Savio River in Cesena. In the analyses performed, all the models are two-dimensional in order to have results comparable between the different methods taken in exam. The different methods have been compared with each other in terms of collapse load and of hinge positions.

Interaction between APOE4 genotype and environmental risk factors in Alzheimer's disease

Alzheimer's disease (AD) is probably caused by both genetic and environmental risk factors. The major genetic risk factor is the E4 variant of apolipoprotein E gene called apoE4. Several risk factors for developing AD have been identified including lifestyle, such as dietary habits. The mechanisms behind the AD pathogenesis and the onset of cognitive decline in the AD brain are presently unknown. In this study we wanted to characterize the effects of the interaction between environmental risk factors and apoE genotype on neurodegeneration processes, with particular focus on behavioural studies and neurodegenerative processes at molecular level. Towards this aim, we used 6 months-old apoE4 and apoE3 Target Replacement (TR) mice fed on different diets (high intake of cholesterol and high intake of carbohydrates). These mice were evaluated for learning and memory deficits in spatial reference (Morris Water Maze (MWM)) and contextual learning (Passive Avoidance) tasks, which involve the hippocampus and the amygdala, respectively. From these behavioural studies we found that the initial cognitive impairments manifested as a retention deficit in apoE4 mice fed on high carbohydrate diet. Thus, the genetic risk factor apoE4 genotype associated with a high carbohydrate diet seems to affect cognitive functions in young mice, corroborating the theory that the combination of genetic and environmental risk factors greatly increases the risk of developing AD and leads to an earlier onset of cognitive deficits. The cellular and molecular bases of the cognitive decline in AD are largely unknown. In order to determine the molecular changes for the onset of the early cognitive impairment observed in the behavioural studies, we performed molecular studies, with particular focus on synaptic integrity and Tau phosphorylation. The most relevant finding of our molecular studies showed a significant decrease of Brain-derived Neurotrophic Factor (BDNF) in apoE4 mice fed on high carbohydrate diet. Our results may suggest that BDNF decrease found in apoE4 HS mice could be involved in the earliest impairment in long-term reference memory observed in behavioural studies. The second aim of this thesis was to study possible involvement of leptin in AD. There is growing evidence that leptin has neuroprotective properties in the Central Nervous System (CNS). Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. However, there are still numerous questions, regarding the molecular mechanism by which leptin acts, that remain unanswered. Thus, given to the importance of the involvement of leptin in AD, we wanted to clarify the function of leptin in the pathogenesis of AD and to investigate if apoE genotype affect leptin levels through studies in vitro, in mice and in human. Our findings suggest that apoE4 TR mice showed an increase of leptin in the brain. Leptin levels are also increased in the cerebral spinal fluid of AD patients and apoE4 carriers with AD have higher levels of leptin than apoE3 carriers. Moreover, leptin seems to be expressed by reactive glial cells in AD brains. In vitro, ApoE4 together with Amyloid beta increases leptin production by microglia and astrocytes. Taken together, all these findings suggest that leptin replacement might not be a good strategy for AD therapy. Our results show that high leptin levels were found in AD brains. These findings suggest that, as high leptin levels do not promote satiety in obese individuals, it might be possible that they do not promote neuroprotection in AD patients. Therefore, we hypothesized that AD brain could suffer from leptin resistance. Further studies will be critical to determine whether or not the central leptin resistance in SNC could affect its potential neuroprotective effects.

Un approccio innovativo alla pianificazione della mobilità ciclabile

L’approccio innovativo di questa tesi alla pianificazione ciclabile consiste nell’integrare le linee guida per la redazione di un biciplan con aspetti, metodologie e strumenti nuovi, per rendere più efficace la programmazione di interventi. I limiti del biciplan risiedono nella fase di pianificazione e di monitoraggio, quindi, nel 1° capitolo, vengono esaminate le differenze esistenti tra la normativa americana (AASHTO) e quella italiana (D.P.R. 557/99). Nel 2° capitolo vengono analizzati gli indicatori usati nella fase di monitoraggio e la loro evoluzione fino alla definizione degli attuali indici per la determinazione del LOS delle infrastrutture ciclabili: BLOS e BCI. L’analisi è integrata con le nuove applicazioni di questi indici e con lo studio del LOS de HCM 2010. BCI e BISI sono stati applicati alla rete di Bologna per risolvere problemi di pianificazione e per capire se esistessero problemi di trasferibilità. Gli indici analizzati prendono in considerazione solo il lato offerta del sistema di trasporto ciclabile; manca un giudizio sui flussi, per verificare l’efficacia delle policy. Perciò il 3° capitolo è dedicato alla metodologia sul monitoraggio dei flussi, mediante l’utilizzo di comuni traffic counter per le rilevazioni dei flussi veicolari. Dal monitoraggio è possibile ricavare informazioni sul numero di passaggi, periodi di punta, esistenza di percorsi preferiti, influenza delle condizioni climatiche, utili ai progettisti; si possono creare serie storiche di dati per controllare l’evoluzione della mobilità ciclabile e determinare l’esistenza di criticità dell’infrastruttura. L’efficacia della pianificazione ciclabile è legata al grado di soddisfazione dell’utente e all’appetibilità delle infrastrutture, perciò il progettista deve conoscere degli elementi che influenzano le scelte del ciclista. Nel 4° capitolo sono analizzate le tecniche e gli studi sulle scelte dell’itinerario dei ciclisti, e lo studio pilota fatto a Bologna per definire le variabili che influenzano le scelte dei ciclisti e il loro peso.

Sviluppo di una metodologia per la modellazione geologica-geomeccanica ed il monitoraggio di reservoir adibiti a siti di stoccaggio di gas naturale

La valutazione dei rischi associati all’operatività dei sistemi di stoccaggio, quali la sismicità indotta e la subsidenza, è requisito basilare per una loro corretta gestione e progettazione, e passa attraverso la definizione dell’influenza sullo stato tensionale delle variazioni di pressione di poro nel sottosuolo. Principale scopo di questo progetto è lo sviluppo di una metodologia in grado di quantificare le deformazioni dei reservoir in funzione della pressione di poro, di tarare i modelli utilizzati con casi studio che presentino dati di monitoraggio reali, tali da consentire un confronto con le previsioni di modello. In questa tesi, la teoria delle inomogeneità è stata utilizzata, tramite un approccio semianalitico, per definire le variazioni dei campi elastici derivanti dalle operazioni di prelievo e immissione di fluidi in serbatoi geologici. Estensione, forma e magnitudo delle variazioni di stress indotte sono state valutate tramite il concetto di variazione dello sforzo critico secondo il criterio di rottura di Coulomb, tramite un’analisi numerica agli elementi finiti. La metodologia sviluppata è stata applicata e tarata su due reservoir sfruttati e riconvertiti a sistemi di stoccaggio che presentano dataset, geologia, petrofisica, e condizioni operative differenti. Sono state calcolate le variazioni dei campi elastici e la subsidenza; è stata mappata la variazione di sforzo critico di Coulomb per entrambi i casi. I risultati ottenuti mostrano buon accordo con le osservazioni dei monitoraggi, suggerendo la bontà della metodologia e indicando la scarsa probabilità di sismicità indotta. Questo progetto ha consentito la creazione di una piattaforma metodologica di rapido ed efficace utilizzo, per stimare l’influenza dei sistemi di stoccaggio di gas sullo stato tensionale della crosta terrestre; in fase di stoccaggio, permette di monitorare le deformazioni e gli sforzi indotti; in fase di progettazione, consente di valutare le strategie operative per monitorare e mitigare i rischi geologici associati a questi sistemi.

Credit-Default-Swaps and the 2008 Financial Crisis: An Inquiry into the Underlying Legal Origins and the European Union’s Regulatory Reforms

After the 2008 financial crisis, the financial innovation product Credit-Default-Swap (CDS) was widely blamed as the main cause of this crisis. CDS is one type of over-the-counter (OTC) traded derivatives. Before the crisis, the trading of CDS was very popular among the financial institutions. But meanwhile, excessive speculative CDSs transactions in a legal environment of scant regulation accumulated huge risks in the financial system. This dissertation is divided into three parts. In Part I, we discussed the primers of the CDSs and its market development, then we analyzed in detail the roles CDSs had played in this crisis based on economic studies. It is advanced that CDSs not just promoted the eruption of the crisis in 2007 but also exacerbated it in 2008. In part II, we asked ourselves what are the legal origins of this crisis in relation with CDSs, as we believe that financial instruments could only function, positive or negative, under certain legal institutional environment. After an in-depth inquiry, we observed that at least three traditional legal doctrines were eroded or circumvented by OTC derivatives. It is argued that the malfunction of these doctrines, on the one hand, facilitated the proliferation of speculative CDSs transactions; on the other hand, eroded the original risk-control legal mechanism. Therefore, the 2008 crisis could escalate rapidly into a global financial tsunami, which was out of control of the regulators. In Part III, we focused on the European Union’s regulatory reform towards the OTC derivatives market. In specific, EU introduced mandatory central counterparty clearing obligation for qualified OTC derivatives, and requires that all OTC derivatives shall be reported to a trade repository. It is observable that EU’s approach in re-regulating the derivatives market is different with the traditional administrative regulation, but aiming at constructing a new market infrastructure for OTC derivatives.

Miglioramento delle performance termiche e energetiche degli edifici rurali: valutazione del retrofit di locali per la conservazione del vino su casi studio

Il presente studio si colloca all’interno di una ricerca più ampia volta alla definizione di criteri progettuali finalizzati all’ottimizzazione delle prestazioni energetiche delle cantine di aziende vitivinicole, di dimensioni produttive medio - piccole. Nello specifico la ricerca riguarda la riqualificazione di fabbricati rurali esistenti di modeste dimensioni, da convertire a magazzini per la conservazione del vino in bottiglia. Lo studio si pone come obiettivo la definizione di criteri di analisi per la valutazione di interventi di retrofit di tali fabbricati, volto sia al miglioramento delle prestazioni energetiche dell’involucro edilizio, sia alla riduzione del fabbisogno energetico legato al funzionamento di eventuali impianti di controllo termico. La ricerca è stata condotta mediante l’utilizzo del software di simulazione termica Energy Plus, per ottenere i valori simulati di temperatura interna relativi ai diversi scenari migliorativi ipotizzati, e mediante la successiva definizione di indicatori che esplicitino l’influenza delle principali variabili progettuali sull’andamento delle temperature interne dei locali di conservazione e sul fabbisogno energetico del fabbricato necessario a garantire l’intervallo di temperatura di comfort del vino. Tra tutti gli interventi possibili per il miglioramento della prestazione energetica degli edifici, quelli analizzati in questo studio prevedono l’aggiunta di un isolamento a cappotto delle pareti esterne, l’isolamento della copertura e l’aggiunta di una struttura ombreggiante vegetale esterna. I risultati ottenuti danno una prima indicazione sugli interventi più efficaci in termini di miglioramento energetico e mettono in luce l’utilità del criterio proposto nell’evidenziare le criticità degli interventi migliorativi ipotizzati. Il metodo definito nella presente ricerca risulta quindi un valido strumento di valutazione a supporto della progettazione degli interventi di retrofit dei fabbricati rurali da convertire a magazzini per la conservazione del vino.