Biological analysis of the in-vitro effects of homologous recombination inhibition and its use in cancer treatment through synthetic lethality

Synthetic lethality represents an anticancer strategy that targets tumor specific gene defects. One of the most studied application is the use of PARP inhibitors (e.g. olaparib) in BRCA1/2-less cancer cells. In BRCA2-defective tumors, olaparib (OLA) inhibits DNA single-strand break repair, while BRCA2 mutations hamper homologous recombination (HR) repair. The simultaneous impairment of those pathways leads BRCA-less cells to death by synthetic lethality. The projects described in this thesis were aimed at extending the use of OLA in cancer cells that do not carry a mutation in BRCA2 by combining this drug with compounds that could mimic a BRCA-less environment via HR inhibition. We demonstrated the effectiveness of our “fully small-molecule induced synthetic lethality” by using two different approaches. In the direct approach (Project A), we identified a series of neo-synthesized compounds (named RAD51-BRCA2 disruptors) that mimic BRCA2 mutations by disrupting the RAD51-BRCA2 interaction and thus the HR pathway. Compound ARN 24089 inhibited HR in human pancreatic adenocarcinoma cell line and triggered synthetic lethality by synergizing with OLA. Interestingly, the observed synthetic lethality was triggered by tackling two biochemically different mechanisms: enzyme inhibition (PARP) and protein-protein disruption (RAD51-BRCA2). In the indirect approach (Project B), we inhibited HR by interfering with the cellular metabolism through inhibition of LDH activity. The obtained data suggest an LDH-mediated control on HR that can be exerted by regulating either the energy supply needed to this repair mechanism or the expression level of genes involved in DNA repair. LDH inhibition also succeeded in increasing the efficiency of OLA in BRCA-proficient cell lines. Although preliminary, these results highlight a complex relationship between metabolic reactions and the control of DNA integrity. Both the described projects proved that our “fully small-molecule-induced synthetic lethality” approach could be an innovative approach to unmet oncological needs.

Liquid biopsy for prostate cancer molecular characterization: homologous recombination system and genomic profile

Pathogenic aberrations in homologous recombination DNA repair (HRR) genes occur in approximately 1 to 4 men with advanced prostate cancer (PCa). Treatment with PARP inhibitors (PARPi) has recently been introduced for metastatic castration-resistant PCa patients, increasing clinicians' interest in the molecular characterization of all PCa patients. The limitations of using old, low-quality tumor tissue for genetic analysis, which is very common for PCa, can be overcome by using liquid biopsy as an alternative biomarker source. In this study, we aimed to evaluate the detection of molecular alterations in HRR genes on liquid biopsy compared with tumor tissue from PCa patients. Secondarily, we explored the genomic instability score (GIS), and a broader range of gene alterations for in-depth characterization of the PCa cohort. Plasma samples were collected from 63 patients with PCa. Sophia Homologous Recombination Solution (targeting 16 HRR genes) and shallow whole genome sequencing (sWGS) were used for genomic analysis of tissue DNA and circulating tumor DNA (ct). A total of 33 alterations (mainly on TP53, ATM, CHEK2, CDK12, and BRCA1/2) were identified in 28,5% of PCa plasma patients. By integrating the mutational and sWGS data, the HRR status of PCa patients was determined and a concordance agreement of 85,7% was identified with tumor tissue. A median GIS of 15 was obtained, reaching a score of 63 in 2 samples with double alterations, BRCA1 and TP53. We explored the PCa mutation landscape, and the most significant enriched pathways identified were the sphingosine 1-phosphate (S1P) receptor signaling and the PI3K-AKT-mTOR pathway. HRR analysis on FFPE and liquid biopsy samples show high concordance, demonstrating that the noninvasive ctDNA-enriched plasma can be an optimal alternative source for molecular SNV and CNV analysis. In addition, the evaluation of GIS and pathway interaction should be considered for more comprehensive molecular characterization in PCa patients.

The photosynthetic bacterial reaction center in native and artificial envirnoments: effects on light-induced electron transfer

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Additionally in BNYVV/BSBMV mixed infections, a competition was previous described in sugar beet, where BNYVV infection reduces BSBMV accumulation in both susceptible and resistant cultivars. Considering all this observations we hypothesized that BNYVV and BSBMV crossed study, exploiting their similarities and divergences, can improve investigation of molecular interactions between sugar beets and Benyviruses. The main achievement of our research is the production of a cDNA biologically active clones collection of BNYVV and BSBMV RNAs, from which synthetic copies of both Benyviruses can be transcribed. Moreover, through recombination experiments we demonstrated, for the first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that 85 foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C. quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our research. These results confirm the close correlation among the two viruses. Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26 than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA- 3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid sequence identity with BNYVV p25 but identity increase to 43% when compared with sequence of BNYVV RNA-5 p26. Based on our results the essential sequence (Core region) for the longdistance movement of BSBMV and BNYVV in B. macrocarpa, is not only carried by RNA3s species but other regions, perhaps located on the RNA 1 and 2, could play a fundamental role in this matter. Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any protein, but it is replicated and transcript in planta. It could represent an important tool to study the interactions between Benyvirus and plant host. In conclusion different tools, comprising a method to study synthetic viruses under natural conditions of inoculum through P. Betae, have been produced and new knowledge are been acquired that will allow to perform future investigation of the molecular interactions between sugar beets and Benyviruses.

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

EoR in alternative cosmological scenarios

The aim of this Thesis is to investigate the possibility that the observations related to the epoch of reionization can probe not only the evolution of the IGM state, but also the cosmological background in which this process occurs. In fact, the history of the IGM ionization is indeed affected by the evolution of the sources of ionizing photons that, under the assumption of a structure formation paradigm determined by the hierarchic growth of the matter uctuations, results strongly dependent on the characteristics of the background universe. For the purpose of our investigation, we have analysed the reionization history in innovative cosmological frameworks, still in agreement with the recent observational tests related to the SNIa and the CMB probes, comparing our results with the reionization scenario predicted by the commonly used LCDM cosmology. In particular, in this Thesis we have considered two different alternative universes. The first one is a at universe dominated at late epochs by a dynamic dark energy component, characterized by an equation of state evolving in time. The second cosmological framework we have assumed is a LCDM characterized by a primordial overdensity field having a non-Gaussian probability distribution. The reionization scenario have been investigated, in this Thesis, through semi-analytic approaches based on the hierarichic growth of the matter uctuations and on suitable assumptions concerning the ionization and the recombination of the IGM. We make predictions for the evolution and the distribution of the HII regions, and for the global features of reionization, that can be constrained by future observations. Finally, we brie y discuss the possible future prospects of this Thesis work.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Ricerca, mediante tecniche di proteomica, di biomarcatori proteici utili nella valutazione della qualità degli alimenti di origine animale

In the last decades, the increase of industrial activities and of the request for the world food requirement, the intensification of natural resources exploitation, directly connected to pollution, have aroused an increasing interest of the public opinion towards initiatives linked to the regulation of food production, as well to the institution of a modern legislation for the consumer guardianship. This work was planned taking into account some important thematics related to marine environment, collecting and showing the data obtained from the studies made on different marine species of commercial interest (Chamelea gallina, Mytilus edulis, Ostrea edulis, Crassostrea gigas, Salmo salar, Gadus morhua). These studies have evaluated the effects of important physic and chemical parameters variations (temperature, xenobiotics like drugs, hydrocarbons and pesticides) on cells involved in the immune defence (haemocytes) and on some important enzymatic systems involved in xenobiotic biotransformation processes (cytochrome P450 complex) and in the related antioxidant defence processes (Superoxide dismutase, Catalase, Heat Shock Protein), from a biochemical and bimolecular point of view. Oxygen is essential in the biological answer of a living organism. Its consume in the normal cellular breathing physiological processes and foreign substances biotransformation, leads to reactive oxygen species (ROS) formation, potentially toxic and responsible of biological macromolecules damages with consequent pathologies worsening. Such processes can bring to a qualitative alteration of the derived products, but also to a general state of suffering that in the most serious cases can provoke the death of the organism, with important repercussions in economic field, in the output of the breedings, of fishing and of aquaculture. In this study it seemed interesting to apply also alternative methodologies currently in use in the medical field (cytofluorimetry) and in proteomic studies (bidimensional electrophoresis, mass spectrometry) with the aim of identify new biomarkers to place beside the traditional methods for the control of the animal origin food quality. From the results it’s possible to point out some relevant aspects from each experiment: 1. The cytofluorimetric techniques applied to O. edulis and C. gigas could bring to important developments in the search of alternative methods that quickly allows to identify with precision the origin of a specific sample, contributing to oppose possible alimentary frauds, in this case for example related to presence of a different species, also under a qualitative profile, but morpholgically similar. A concrete perspective for the application in the inspective field of this method has to be confirmed by further laboratory tests that take also in account in vivo experiments to evaluate the effect in the whole organism of the factors evaluated only on haemocytes in vitro. These elements suggest therefore the possibility to suit the cytofluorimetric methods for the study of animal organisms of food interest, still before these enter the phase of industrial working processes, giving useful information about the possible presence of contaminants sources that can induce an increase of the immune defence and an alteration of normal cellular parameter values. 2. C. gallina immune system has shown an interesting answer to benzo[a]pyrene (B[a]P) exposure, dose and time dependent, with a significant decrease of the expression and of the activity of one of the most important enzymes involved in the antioxidant defence in haemocytes and haemolymph. The data obtained are confirmed by several measurements of physiological parameters, that together with the decrease of the activity of 7-etossi-resourifine-O-deetilase (EROD linked to xenobiotic biotransformation processes) during exposure, underline the major effects of B[a]P action. The identification of basal levels of EROD supports the possible presence of CYP1A subfamily in the invertebrates, still today controversial, never identified previously in C. gallina and never isolated in the immune cells, as confirmed instead in this study with the identification of CYP1A-immunopositive protein (CYP1A-IPP). This protein could reveal a good biomarker at the base of a simple and quick method that could give clear information about specific pollutants presence, even at low concentrations in the environment where usually these organisms are fished before being commercialized. 3. In this experiment it has been evaluated the effect of the antibiotic chloramphenicol (CA) in an important species of commercial interest, Chamelea gallina. Chloramphenicol is a drug still used in some developing countries, also in veterinary field. Controls to evaluate its presence in the alimentary products of animal origin, can reveal ineffective whereas the concentration results to be below the limit of sensitivity of the instruments usually used in this type of analysis. Negative effects of CA towards the CYP1A- IPP proteins, underlined in this work, seem to be due to the attack of free radicals resultant from the action of the antibiotic. This brings to a meaningful alteration of the biotransformation mechanisms through the free radicals. It seems particularly interesting to pay attention to the narrow relationships in C. gallina, between SOD/CAT and CYP450 system, actively involved in detoxification mechanism, especially if compared with the few similar works today present about mollusc, a group that is composed by numerous species that enter in the food field and on which constant controls are necessary to evaluate in a rapid and effective way the presence of possible contaminations. 4. The investigations on fishes (Gadus morhua, and Salmo salar) and on a bivalve mollusc (Mytilus edulis) have allowed to evaluate different aspects related to the possibility to identify a biomarker for the evaluation of the health of organisms of food interest and consequently for the quality of the final product through 2DE methodologies. In the seafood field these techniques are currently used with a discreet success only for vertebrates (fishes), while in the study of the invertebrates (molluscs) there are a lot of difficulties. The results obtained in this work have underline several problems in the correct identification of the isolated proteins in animal organisms of which doesn’t currently exist a complete genomic sequence. This brings to attribute some identities on the base of the comparison with similar proteins in other animal groups, incurring in the possibility to obtain inaccurate data and above all discordant with those obtained on the same animals by other authors. Nevertheless the data obtained in this work after MALDI-ToF analysis, result however objective and the spectra collected could be again analyzed in the future after the update of genomic database related to the species studied. 4-A. The investigation about the presence of HSP70 isoforms directly induced by different phenomena of stress like B[a]P presence, has used bidimensional electrophoresis methods in C. gallina, that have allowed to isolate numerous protein on 2DE gels, allowing the collection of several spots currently in phase of analysis with MALDI-ToF-MS. The present preliminary work has allowed therefore to acquire and to improve important methodologies in the study of cellular parameters and in the proteomic field, that is not only revealed of great potentiality in the application in medical and veterinary field, but also in the field of the inspection of the foods with connections to the toxicology and the environmental pollution. Such study contributes therefore to the search of rapid and new methodologies, that can increase the inspective strategies, integrating themselves with those existing, but improving at the same time the general background of information related to the state of health of the considered animal organism, with the possibility, still hypothetical, to replace in particular cases the employment of the traditional techniques in the alimentary field.

Studio genetico ed epidemiologico su Erysiphe necator agente eziologico dell'Oidio della vite

The objective was to analyse population structure and to determine genetic diversity of Erysiphe necator (syn. Uncinula necator) populations obtained from some vineyards located in the South-East Po valley (Italy). Powdery mildew is one of the most important fungal diseases of grapes (Vitis vinifera L.) throughout the world. The causal agent is the haploid, heterothallic ascomycete E. necator. It is an obligate biotrophic fungus and it can be found only on green organs of plants belonging to the family Vitaceae. For this pathogen, two sympatric populations (groups A and B) have been described in Europe and Australia. The two genetic groups differ at multiple genetic loci and previous studies reported a lack of interfertility among isolates of the two groups. There are now several well documented examples of plant pathogen species, such as Leptosphaeria maculans, Gaeumannomyces graminis var. tritici, Botrytis cinerea and Erysiphe syringae, which are indeed composed of genetically differentiated clades, that have led to the description of new groups or even new species. Several studies have suggested that genetic E. necator group A and B correlated with ecological features of the pathogen; some researchers proposed that group A isolates over-winter as resting mycelium within dormant buds, and in spring originate infected shoots, known as Flag shoots, while group B isolates would survive as ascospores in overwintering cleistothecia. However, the association between genetic groups and mode of over-wintering has been challenged by recent studies reporting that flag-shoot may be originated indifferently by group A or group B isolate. Previous studies observed a strong association between the levels of disease severity at the end of the growing season and the initial compositions of E. necator populations in commercial vineyards. The frequencies of E. necator genetic groups vary considerably among vineyards, and the two groups may coexist in the same vineyard. This finding suggests that we need more information on the genetics and epidemiology of E. necator for optimize the crop management In this study we monitored E. necator populations in different vineyards in Emilia – Romagna region (Italy), where the pathogen overwinters both as flagshoots and as cleistothecia. During the grape growing season, symptomatic leaves were sampled early in the growing season and both leaves and berries later during the epidemic growth of the disease. From each sample, single-conidial isolate was obtained. Each isolates was grown on V. vinifera leaf cv. Primitivo and after harvesting the mycelium, the DNA was purified and used as template for PCR amplification with SCAR primers (Sequences Characterised Amplified Region ), -tubulin, IGS sequences and Microsatellite markers (SSR). Amplified DNA from b-tubulin and IGS loci was digested with AciI and XhoI restriction enzymes, respectively, to show single-nucleotide polymorphisms specific for the two genetic groups. The results obtained indicated that SCAR primers are not useful to study the epidemiology. of E. necator conversely the b-tubulin IGS sequences and SSR. Summarize the results obtained with b-tubulin, IGS sequences, in treated vineyards we have found individuals of group B along all grape growing season, whereas in the untreated vineyard individuals of the two genetic groups A and B coexisted throughout the season, with no significant change of their frequency. DNA amplified from ascospores of single cleistothecia showed the presence of markers diagnostic for either groups A and B and were seldom observed also the coexistence of both groups within a claistothecium. These results indicate that individuals of the two groups mated in nature and were able to produced ascospores. With SSR we showed the possibility of recombination between A and B groups in field isolates. During winter, cleistothecia were collected repeatedly in the same vineyards sampling leaves fallen on ground, exfoliating bark from trunks, and from soil. From each substrate, was assess the percentage of cleistothecia containing viable ascospores. Our results confirmed that cleisthotecia contained viable ascospores, therefore they have the potential to be an additional and important source of primary inoculum in Emilia-Romagna vineyards.

Metodi di ottimizzazione morfologica nel progetto preliminare di propulsori aeronautici avanzati

The aim of this Doctoral Thesis is to develop a genetic algorithm based optimization methods to find the best conceptual design architecture of an aero-piston-engine, for given design specifications. Nowadays, the conceptual design of turbine airplanes starts with the aircraft specifications, then the most suited turbofan or turbo propeller for the specific application is chosen. In the aeronautical piston engines field, which has been dormant for several decades, as interest shifted towards turboaircraft, new materials with increased performance and properties have opened new possibilities for development. Moreover, the engine’s modularity given by the cylinder unit, makes it possible to design a specific engine for a given application. In many real engineering problems the amount of design variables may be very high, characterized by several non-linearities needed to describe the behaviour of the phenomena. In this case the objective function has many local extremes, but the designer is usually interested in the global one. The stochastic and the evolutionary optimization techniques, such as the genetic algorithms method, may offer reliable solutions to the design problems, within acceptable computational time. The optimization algorithm developed here can be employed in the first phase of the preliminary project of an aeronautical piston engine design. It’s a mono-objective genetic algorithm, which, starting from the given design specifications, finds the engine propulsive system configuration which possesses minimum mass while satisfying the geometrical, structural and performance constraints. The algorithm reads the project specifications as input data, namely the maximum values of crankshaft and propeller shaft speed and the maximal pressure value in the combustion chamber. The design variables bounds, that describe the solution domain from the geometrical point of view, are introduced too. In the Matlab® Optimization environment the objective function to be minimized is defined as the sum of the masses of the engine propulsive components. Each individual that is generated by the genetic algorithm is the assembly of the flywheel, the vibration damper and so many pistons, connecting rods, cranks, as the number of the cylinders. The fitness is evaluated for each individual of the population, then the rules of the genetic operators are applied, such as reproduction, mutation, selection, crossover. In the reproduction step the elitist method is applied, in order to save the fittest individuals from a contingent mutation and recombination disruption, making it undamaged survive until the next generation. Finally, as the best individual is found, the optimal dimensions values of the components are saved to an Excel® file, in order to build a CAD-automatic-3D-model for each component of the propulsive system, having a direct pre-visualization of the final product, still in the engine’s preliminary project design phase. With the purpose of showing the performance of the algorithm and validating this optimization method, an actual engine is taken, as a case study: it’s the 1900 JTD Fiat Avio, 4 cylinders, 4T, Diesel. Many verifications are made on the mechanical components of the engine, in order to test their feasibility and to decide their survival through generations. A system of inequalities is used to describe the non-linear relations between the design variables, and is used for components checking for static and dynamic loads configurations. The design variables geometrical boundaries are taken from actual engines data and similar design cases. Among the many simulations run for algorithm testing, twelve of them have been chosen as representative of the distribution of the individuals. Then, as an example, for each simulation, the corresponding 3D models of the crankshaft and the connecting rod, have been automatically built. In spite of morphological differences among the component the mass is almost the same. The results show a significant mass reduction (almost 20% for the crankshaft) in comparison to the original configuration, and an acceptable robustness of the method have been shown. The algorithm here developed is shown to be a valid method for an aeronautical-piston-engine preliminary project design optimization. In particular the procedure is able to analyze quite a wide range of design solutions, rejecting the ones that cannot fulfill the feasibility design specifications. This optimization algorithm could increase the aeronautical-piston-engine development, speeding up the production rate and joining modern computation performances and technological awareness to the long lasting traditional design experiences.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Mg for hydrogen storage: synthesis, nanostructure and thermodynamics properties

Nowadays alternative energies are an extremely important topic and the possibility of using hydrogen as an energy carrier must be explored. Many problems infer the technological application of this abundant and powerful resource, one of them the possibility of storage. In the framework of suitable materials for hydrogen storage, magnesium has been the center of this study because it is cheap and the amount of stored hydrogen that it achieves (7.6 wt%) is extremely appealing. Nanostructure helps to overcome the slow hydrogen diffusion and the functionalization of surfaces with transition metals or oxides favors the hydrogen molecule dissociation/recombination. The aim of this research is the investigation of the metal-hydride transformation in magnesium nanoparticles synthesized by inert-gas condensation, exploiting the fact that they are a simple model system. The so produced nanostructured powder has been analyzed in response to nanoparticles surface functionalization by transition metal clusters, specifically palladium, nickel and titanium, chosen on the basis of their completely different Mg-related phase diagrams. The role of the intermetallic phases formed upon heating and hydrogenation treatments will be presented to provide a comprehensive picture of hydrogen sorption in this class of nanostructured storage materials.

Organic heterostructure approach for multifunctional light-emitting field-effect transistors

The possibility of combining different functionalities in a single device is of great relevance for further development of organic electronics in integrated components and circuitry. Organic light-emitting transistors (OLETs) have been demonstrated to be able to combine in a single device the electrical switching functionality of a field-effect transistor and the capability of light generation. A novel strategy in OLET realization is the tri-layer vertical hetero-junction. This configuration is similar to the bi-layer except for the presence of a new middle layer between the two transport layers. This “recombination” layer presents high emission quantum efficiency and OLED-like (Organic Light-Emitting Diode) vertical bulk mobility value. The key idea of the vertical tri-layer hetero-junction approach in realizing OLETs is that each layer has to be optimized according to its specific function (charge transport, energy transfer, radiative exciton recombination). Clearly, matching the overall device characteristics with the functional properties of the single materials composing the active region of the OFET, is a great challenge that requires a deep investigation of the morphological, optical and electrical features of the system. As in the case of the bi-layer based OLETs, it is clear that the interfaces between the dielectric and the bottom transport layer and between the recombination and the top transport layer are crucial for guaranteeing good ambipolar field-effect electrical characteristics. Moreover interfaces between the bottom transport and the recombination layer and between the recombination and the top transport layer should provide the favourable conditions for the charge percolation to happen in the recombination layer and form excitons. Organic light emitting transistor based on the tri-layer approach with external quantum efficiency out-performing the OLED state of the art has been recently demonstrated [Capelli et al., Nat. Mater. 9 (2010) 496-503] widening the scientific and technological interest in this field of research.