Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato
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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento Dipartimento di Biologia, Ecologia e Scienze della Terra dell'Università della Calabria.
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Item Effetti del cadmio sulle branchie del teleosteo marino Thalassoma pavo: uno studio morfo-funzionale(2014-03-28) Corapi, Barbara; Tota, Bruno; Brunelli, ElviraItem Pathway infiammatorio del Sistema del Complemento nelle ascidie: sequenziamento e caratterizzazione funzionale del recettore dell’anafilatossina C3a di Ciona intestinalis(2014-03-26) Melillo, Daniela; Tota, Bruno; Panno, Maria Luisa; Pinto, Maria RosariaIn mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, a C3a-mediated chemotaxis of hemocytes has been demonstrated in the deuterostome invertebrate Ciona intestinalis and an important role for this molecule in inflammatory processes has been suggested. In this study, we have cloned and characterized the CiC3aR molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence of CiC3aR, encoding a 95,394 Da seventransmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent, in the ovaries, hemocytes, and larvae. Three polyclonal antibodies raised against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop, react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific antibodies revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the antibodies against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis.expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory processItem La Catestatina come nuovo modulatore autocrino/paracrino dell’attività cardiaca(2014-03-26) Pasqua, Teresa; Cerra, Maria Carmela; Tota, BrunoItem Identificazione e caratterizzazione di FoxP nel sistema nervoso centrale di Octopus vulgaris (Mollusca, Cephalopoda)(2014-03-24) Sirakov, Maria; Tota, Bruno; Borra, Marco; Fiorito, GrazianoIn this study, I searched and was able to identify FoxP in the transcriptome of the cephalopod mollusc Octopus vulgaris, an invertebrate. In addition, I attempted to analyze the expression of Ov-FoxP in the brain of this animal. The results of this analysis are preliminary at this stage. Fox proteins are a set of transcription factors highly conserved in metazoans. They are characterized by a typical DNA binding domain (Forkhead) that, among others, allows to identify 15 different classes of Fox genes. Fox proteins are reported to act as activators/repressors of transcription during both development (including differentiation) and the adult life (e.g. lung, brain, etc.). In vertebrates, FoxP2 (together with FoxP1), in particular, are known to be involved in the development of the neural circuit controlling bird-song and human speech. Our interest for the octopus derives from the fact that this animal, together with other cephalopods, is considered as the most evolved among molluscs. The complexity of the architecture and wiring of the cephalopod nervous system stems from the simpler nervous systems of other taxa belonging to the phylum. In addition, cephalopods show a highly rich behavioral repertoire including the unique capability of changing the appearance of their body (through body patterning) in fractions of seconds and for both mimetic and communicative purposes. Taken all together, these features allow these animals to be considered analogous to higher vertebrates. In the first part of my project, a detailed analysis of the aminoacidic and nucleotidic sequences available for FoxP2 (vertebrates) and FoxP (invertebrates), allowed us to design FoxP in Octopus vulgaris 1 appropriate oligos that were utilized in subsequent PCR experiments to identify the gene of interest in the transcriptome of the brain of O. vulgaris. FoxP resulted in a fragment of 220 bp that corresponded to the Forkhead domain. Further efforts allowed us to identify a 1111bp mRNA sequence of Ov-FoxP corresponding to almost the entire part of the mature mRNA codifying for this protein (the 5’ extremity of the gene results unidentified at this stage). During the second part of my project, I attempted to analyze the expression pattern of Ov- FoxP in the octopus brain using Real Time qPCR and in-situ hybridization. This was carried out with the aim of investigating the possible variability of expression of the gene in different parts of the brain (i.e. supra-, sub-esophageal masses and optic lobes) relative to another tissue (muscular tissue of the mantle) here considered as control. Other genes (16S, tubulin, actin) were also cloned for the aims of this project and their expression was taken as reference; an analysis that is carried out for the first time in O. vulgaris. By Real-Time qPCR I was able to recognize a different pattern of expression in different parts of the brain (N = 10). The data allowed to identify a gradient in the expression levels of FoxP (relative to reference genes) in the subesophageal mass, when the smallest individual of my sample (30 g body weight) was compared with the others (150-2100 g body weight). In situ hybridization (N=6) allowed to localize the expression of FoxP in the lobes of the octopus brain. Ov-FoxP transcripts were identified in neurons of: i. the optic lobes (several sparse cells possibly related with visual input processing); ii. the superior buccal and the lateral part of the basal lobes (high-order motor centers of the supraesophageal mass), and iii. the pedal tracts and anterior and posterior chromatophore lobes (subesophageal mass). FoxP in Octopus vulgaris 2 An elevated number of cells was revealed through in-situ hybridization in the last two lobes. It is noteworthy to mention that these structures are known to play a key role in the neural control of the chromatic expression of the skin of O. vulgaris (and other cephalopods): namely the animal’s body pattern. Our data seems to suggest that Ov-FoxP is expressed during different phases of the life of the octopus. In addition the localized expression in definite lobes and the variability among individuals of its expression in the same brain parts allows us to formulate the working hypothesis of the role of Ov-FoxP in the plasticity and/or maintainance of neural networks. My project in O. vulgaris confirms similar results deduced from other studies in both invertebrates (i.e. motor neurons in C. elegans) and vertebrates (i.e. song-birds, mouse, etc).Item Le patelle delle coste campane: aspetti sistematici, cromosomici e valutazione delle stress genomico(2014-03-20) Petraccioli, Agnese; Tota, Bruno; Fiorino, Graziano; Odierna, GaetanoBy applying advanced techniques that are available now days, it is relatively feasible to determine and monitor the degree of pollution of marine systems. However, it is still difficult to evaluate the effects that pollution may have on marine organisms and, in general, on the environment. A way to overcome this limit is by biomonitoring, i.e. by monitoring the biological responses of selected organisms (namely biomarkers) to pollutants. Many species of molluscs have been labelled as efficient biomarkers. In my research project the aim was to verify the appropriateness of limpets (Patella caerulea) as biomarkers by analyzing specimens of Patella collected in sites with different degrees of pollution along the coasts of the Campania region (Italy). To reach my goal, I measured the potential damage of pollutants at two levels: somatic (by monitoring gill histopathologies) and germinal (by monitoring gonad chromosomic aberrations). My results suggest that pollutants caused a significant damage in natural populations of P. caerulea. The gills of specimens collected from polluted sites presented structural changes, cell and tissue degeneration (cells with vacuoles, dissociation of haemocele septa, nuclear alterations), or the development of compensative mechanisms related to environmental toxicity (epithelium swelling, hyperplasia and fusion of septa). Furthermore, although preliminary, the results suggest that the level of gill injury appeared to be related to the degree of pollution of the study site. Finally, gill aberrations were always higher in each study site compared to the reference site. 2 The gonads of individuals of polluted sites also showed comparatively higher rates of chromosome aberrations, which are supposed to be a by product of an exposition to heavy metal and organic pollutants. In conclusion, the results obtained in my research project suggest that pollutants may act on the cells both of the somatic and germinal lineage in Patella and are thus potentially dangerous for the whole natural population of limpets of the Campania region