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.

Myc-mediated control of gene transcription in cancer cells

The Myc oncoproteins belong to a family of transcription factors composed by Myc, N-Myc and L-Myc. The most studied components of this family are Myc and N-Myc because their expressions are frequently deregulated in a wide range of cancers. These oncoproteins can act both as activators or repressors of gene transcription. As activators, they heterodimerize with Max (Myc associated X-factor) and the heterodimer recognizes and binds a specific sequence elements (E-Box) onto gene promoters recruiting histone acetylase and inducing transcriptional activation. Myc-mediated transcriptional repression is a quite debated issue. One of the first mechanisms defined for the Myc-mediated transcriptional repression consisted in the interaction of Myc-Max complex Sp1 and/or Miz1 transcription factors already bound to gene promoters. This interaction may interfere with their activation functions by recruiting co-repressors such as Dnmt3 or HDACs. Moreover, in the absence of , Myc may interfere with the Sp1 activation function by direct interaction and subsequent recruitment of HDACs. More recently the Myc/Max complex was also shown to mediate transcriptional repression by direct binding to peculiar E-box. In this study we analyzed the role of Myc overexpression in Osteosarcoma and Neuroblastoma oncogenesis and the mechanisms underling to Myc function. Myc overexpression is known to correlate with chemoresistance in Osteosarcoma cells. We extended this study by demonstrating that c-Myc induces transcription of a panel of ABC drug transporter genes. ABCs are a large family trans-membrane transporter deeply involved in multi drug resistance. Furthermore expression levels of Myc, ABCC1, ABCC4 and ABCF1 were proved to be important prognostic tool to predict conventional therapy failure. N-Myc amplification/overexpression is the most important prognostic factor for Neuroblastoma. Cyclin G2 and Clusterin are two genes often down regulated in neuroblastoma cells. Cyclin G2 is an atypical member of Cyclin family and its expression is associated with terminal differentiation and apoptosis. Moreover it blocks cell cycle progression and induces cell growth arrest. Instead, CLU is a multifunctional protein involved in many physiological and pathological processes. Several lines of evidences support the view that CLU may act as a tumour suppressor in Neuroblastoma. In this thesis I showed that N-Myc represses CCNG2 and CLU transcription by different mechanisms. • N-Myc represses CCNG2 transcription by directly interacting with Sp1 bound in CCNG2 promoter and recruiting HDAC2. Importantly, reactivation of CCNG2 expression through epigenetic drugs partially reduces N-Myc and HDAC2 mediated cell proliferation. • N-Myc/Max complex represses CLU expression by direct binding to a peculiar E-box element on CLU promoter and by recruitment of HDACs and Polycomb Complexes, to the CLU promoter. Overall our findings strongly support the model in which Myc overexpression/amplification may contribute to some aspects of oncogenesis by a dual action: i) transcription activation of genes that confer a multidrug resistant phenotype to cancer cells; ii), transcription repression of genes involved in cell cycle inhibition and cellular differentiation.

A novel specific genetic translocation in epithelioid hemangioendotelioma, showing a fusion of the WWTR1 and CAMTA1 genes, supports the monoclonality of multifocal epithelioid hemangioendotelioma.

Like other vascular tumors, epithelioid hemangioendothelioma (EHE) is multifocal in approximately 50% of cases, and it is unclear whether the separate lesions represent multifocal disease or metastases. We hypothesized that the identification of an identical WWTR1-CAMTA1 rearrangement in different EHEs from the same patient supports the monoclonal origin of EHE. To test our hypothesis, we undertook a molecular analysis of two multicentric EHEs of the liver, including separate tumor samples from each patient. Matherial and Methods: We retrieved two cases of EHE with available tissue for molecular analysis. In both cases, fluorescence in situ hybridization (FISH) was performed to identify the presence of the WWTR1-CAMTA1 rearrangement to confirm the histologic diagnosis of EHE, as previously described. The reverse transcription-polymerase chain reaction (RT-PCR) products were analyzed by electrophoresis and the RT-PCR–amplified products were sequenced using the Sanger method. Results: FISH analysis revealed signal abnormalities in both WWTR1 and CAMTA1. Combined results confirmed the presence of the t(1;3)(1p36.23;3q25.1) translocation in both cases of EHE. Using RT-PCR analysis, we found that the size of the rearranged bands was identical in the different tumors from each patient. The sequence of the fusion gene confirmed a different WWTR1-CAMTA1 rearrangement in each patient, but an identical WWTR1-CAMTA1 rearrangement in the different lesions from each patient. Discussion: Because of its generally indolent clinical course, EHE is commonly classified as a multifocal, rather than metastatic, disease. In this study, we examined two cases of multifocal liver EHE and found an identical WWTR1-CAMTA1 rearrangement in each lesion from the same patient, but not between the two patients. These findings suggest that multifocal EHE arises from metastasis of the same neoplastic clone rather than from the simultaneous formation of multiple neoplastic clones, which supports the monoclonal origin of multifocal EHE.

A clustering method for robust and reliable large scale functional and structural protein sequence annotation

Bioinformatics, in the last few decades, has played a fundamental role to give sense to the huge amount of data produced. Obtained the complete sequence of a genome, the major problem of knowing as much as possible of its coding regions, is crucial. Protein sequence annotation is challenging and, due to the size of the problem, only computational approaches can provide a feasible solution. As it has been recently pointed out by the Critical Assessment of Function Annotations (CAFA), most accurate methods are those based on the transfer-by-homology approach and the most incisive contribution is given by cross-genome comparisons. In the present thesis it is described a non-hierarchical sequence clustering method for protein automatic large-scale annotation, called “The Bologna Annotation Resource Plus” (BAR+). The method is based on an all-against-all alignment of more than 13 millions protein sequences characterized by a very stringent metric. BAR+ can safely transfer functional features (Gene Ontology and Pfam terms) inside clusters by means of a statistical validation, even in the case of multi-domain proteins. Within BAR+ clusters it is also possible to transfer the three dimensional structure (when a template is available). This is possible by the way of cluster-specific HMM profiles that can be used to calculate reliable template-to-target alignments even in the case of distantly related proteins (sequence identity < 30%). Other BAR+ based applications have been developed during my doctorate including the prediction of Magnesium binding sites in human proteins, the ABC transporters superfamily classification and the functional prediction (GO terms) of the CAFA targets. Remarkably, in the CAFA assessment, BAR+ placed among the ten most accurate methods. At present, as a web server for the functional and structural protein sequence annotation, BAR+ is freely available at http://bar.biocomp.unibo.it/bar2.0.

La lucha del Derecho contra el negacionismo: una peligrosa frontera. Particular estudio de los ordenamientos italiano y español

En la sociedad europea crece la preocupación por el retorno de tendencias fascistas y neonazis y por la extensión de ideologías xenófobas y antisemitas, algunas de ellas alimentadas a partir de tesis de negacionistas de aquellos trágicos eventos de nuestra historia reciente. La lucha frente a los discursos negacionistas se ha llevado más allá del ámbito social y académico, y se ha propuesto la incorporación en los ordenamientos jurídicos europeos de tipos penales específicos que incriminan este tipo de discurso: negar, banalizar, o justificar el Holocausto u otros genocidios o graves crímenes contra la humanidad. Esta legislación, que encuentra su mayor expresión en la Decisión marco 2008/913/JAI, aunque castiga un discurso socialmente repugnante, sin embargo presenta dudas en cuanto a su legitimidad con un sistema de libertades erigido sobre el pilar del pluralismo propio de los Estados democráticos. Surge así la cuestión de si pueden estar surgiendo «nuevos» delitos de opinión y a ello se dedica entonces la presente tesis. El objetivo concreto de este trabajo será analizar esta política-criminal para proponer una configuración del delito de negacionismo compatible con la libertad de expresión, aunque se cuestionará la conveniencia de castigar penalmente a través de un específico delito este tipo de conductas. En particular se pretende responder a tres preguntas: en primer lugar, ¿el discurso negacionista debe ampararse prima facie por la libertad de expresión en un ordenamiento abierto y personalista y cuáles podrían ser las «reglas» que podrían servir como criterio para limitar este género de manifestaciones? La segunda pregunta sería entonces: ¿Cómo podría construirse un tipo penal respetuoso con los principios constitucionales y penales que específicamente incriminara este género de conductas? Y, como última pregunta: ¿Es conveniente o adecuada una política criminal que lleve a crear un específico delito de negacionismo?

Il linguaggio figurato e la cabina di simultanea: Il progetto IMITES(Interpretación de la Metáfora entre ITaliano y ESpañol)

Il progetto di dottorato IMITES (Interpretación de la Metáfora entre ITaliano y ESpañol) si pone come obiettivo quello di analizzare l’interpretazione simultanea del linguaggio figurato nelle combinazioni italiano-spagnolo e spagnolo-italiano. Prevede l’analisi di una serie di dati estratti da discorsi pronunciati in italiano e spagnolo in occasione di conferenze tenutesi presso la Commissione europea, e le loro versioni interpretate in spagnolo e italiano rispettivamente. Le espressioni figurate contenute nei discorsi originali sono state allineate e messe a confronto con le versioni fornite dagli interpreti, con il duplice obiettivo di a) capire quali causano maggiori problemi agli interpreti e b) analizzare le strategie di interpretazione applicate da professionisti quali quelli della Direzione Generale Interpretazione (DG SCIC) della Commissione europea nell’interpretare metafore. Il progetto prevede anche la somministrazione di un questionario agli interpreti delle cabine spagnola e italiana del DG SCIC, con l’obiettivo di sondare la loro percezione delle difficoltà che sottendono all’interpretazione del linguaggio figurato, le indicazioni metodologiche ricevute (se del caso) dai loro docenti a tale riguardo e le strategie applicate nella pratica professionale. Infine, l’ultima fase del progetto di ricerca prevede la sperimentazione di una proposta didattica attraverso uno studio caso-controllo svolto su studenti del secondo anno della Laurea Magistrale in Interpretazione delle Scuole Interpreti di Forlì e Trieste. Il gruppo-caso ha ricevuto una formazione specifica sull'interpretazione delle metafore, mentre gruppo-controllo è stato monitorato nella sua evoluzione. L’obiettivo di questa ultima fase di ricerca è quello di valutare, da una parte, l’ “insegnabilità” di strategia per affrontare il linguaggio figurato in interpretazione simultanea, e, dall’altra, l’efficacia dell’unità didattica proposta, sviluppata in base all’analisi svolta su IMITES.

Catálogo y estudio de las gramáticas de italiano para hispanohablantes: siglos XVIII y XIX

El objetivo de la presente investigación, el catálogo y estudio de las gramáticas de italiano destinadas a hispanohablantes de los siglos XVIII y XIX, se encuadra en el macrosector gramaticográfico de la historiografía lingüística, en el cual el estudio de las gramáticas de las lenguas dirigidas a hablantes nativos y a hablantes extranjeros, con los consiguientes cruces y trasvases de tradiciones gramaticales, es de significativo interés como destacan: (i) las tesis doctorales defendidas en los últimos quince años; (ii) los proyectos de investigación dirigidos y coordinados por prestigiosos estudiosos del sector; (iii) los congresos organizados para destacar y compartir las principales actualizaciones en torno a los estudios gramaticográficos; y (iv) las publicaciones que surgen de los tres puntos anteriores. El estudio presenta dos partes centrales: la primera (constituida por los capítulos 2 y 3) es la de catálogo y estudio de las diecinueve gramáticas que conforman el corpus en base a ocho áreas descriptivas (1. información catalográfica, 2. autor, 3. editor, 4. hiperestructura, 5. elementos peritextuales, gramaticales y didácticos, 6. variedad de textos y su secuencia didáctica, 7. caracterización, fuentes e influencias, y 8. localización); la segunda (capítulo 4) es la de estudio gramaticográfico de conjunto de los datos más relevantes de las areas de estudio utilizadas en las dos primeras partes. De este modo, daremos un panorama de conjunto sobre (i) la cronología de las obras y sus ediciones y rempresiones; (ii) la nacionalidad, profesión, condición religiosa, etc. de autores; (iii) la geografía de ediciones y editores; (iv) la descripción hiperestructural de las obras; (v) la estructura de los elementos peritextuales; (vi) las partes gramaticales y elementos que las componen; (vii) el verbo: definiciones y paradigma verbal; (vii) los elementos didácticos; (viii) las líneas de descripción gramatical; (ix) la localización de las gramáticas en las bibliotecas españolas.