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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Author: search.filters.author.Tota, Bruno

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    Effetti del cadmio sulle branchie del teleosteo marino Thalassoma pavo: uno studio morfo-funzionale
    (2014-03-28) Corapi, Barbara; Tota, Bruno; Brunelli, Elvira
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    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 Rosaria
    In 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 process
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    La Catestatina come nuovo modulatore autocrino/paracrino dell’attività cardiaca
    (2014-03-26) Pasqua, Teresa; Cerra, Maria Carmela; Tota, Bruno
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    Ruolo neuroprotettivo del sistema istaminergico e delle HSPs nella risposta allo stress ambientale nell’encefalo del Teleosteo Thalassoma pavo
    (2014-03-25) Giusi, Giuseppina; Tota, Bruno; Facciolo,Rosa Maria
    At date, a plethora of evidence regarding adverse morpho-functional and neurobiological aspects provoked by environmental stressors has been considered. Following exposure to stress factors, the activation of both specific neurosignaling mechanisms and molecular pathways account for the modulation of complex adaptative processes in animal targets. In this context, the aim of the present work is to analyze the neuroprotective role of histaminergic system and heat shock proteins towards environmental neurotoxicants such as heavy metals and pesticides in the Teleost Thalassoma pavo. Such environmental stressors account for significative alterations on motor and feeding behaviors, which are tightly correlated to neurodegenerative processes in key brain regions. In this work, the molecular characterization of H2R and H3R permits to demonstrate a conservation of specific sequences, which appear to be determinant for the function of such subtypes in phylogenetically distant Vertebrates. Moreover, the inactivation of H2R and H3R, via the application of selective antagonists (Cimetidine and Thioperamide, respectively), induces in Thalassoma pavo abnormal behaviors and trascriptional alterations, suggesting a clear physiological role of this neuronal system in our model. The expression pattern of histaminergic system results to be highly modified following exposure to environmental stressors in a region-dependent manner. In particular, the heavy metals induce downregulations of H2R mRNA in some brain regions such as mesencephalon, which is involved in the regulation of motor activities. On the other hand, both heavy metal and pesticides account for an increasement of H3R trascriptional levels in hypothalamic and telencephalic areas. From the concomitant exposure to histaminergic antagonists and environmental stressors, it was possible to demonstrate that H2R blockade is responsible for enhanced stressors-dependent neurotoxic effects. On the contrary, the inhibition of H3R activities accounts for an amelioration of both abnormal motor behaviors and neuronal damage induced by such environmental stressors. Consistent with the effects on histaminergic system, heavy metals and pesticides also promote the activation of cellular defence processes through the stimulation of heat shock proteins trascription, i.e. HSP90 e HSP70. The histaminergic antagonists are able to influence heat shock proteins expression, inducing a heterogeneous pattern of HSP90 trascription levels, while in the case of HSP70 an enhanced expression is typical of all encephalic areas. The results of the present work demonstrate, for the first time in an aquatic Vertebrate, a possible interactions between histaminergic system-dependent neurosignaling activities and HSPs network, which could be represent an important neurophysiological mechanism operating during neuronal stress conditions.
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    Identificazione e caratterizzazione di FoxP nel sistema nervoso centrale di Octopus vulgaris (Mollusca, Cephalopoda)
    (2014-03-24) Sirakov, Maria; Tota, Bruno; Borra, Marco; Fiorito, Graziano
    In 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).
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    Le patelle delle coste campane: aspetti sistematici, cromosomici e valutazione delle stress genomico
    (2014-03-20) Petraccioli, Agnese; Tota, Bruno; Fiorino, Graziano; Odierna, Gaetano
    By 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
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    Regolazione dell'orologio circadiano nei mammiferi mediante SUMOylazione dell'attivatore trascrizionale BMAL1
    (2006) Giordano, Francesca; Tota, Bruno
    4 Abstract The molecular machinery that governs circadian rhythmicity is based on clock proteins organized in regulatory feedback loops. Although posttranslational modification of clock proteins is likely to finely control their circadian functions, only limited information is available to date. Here, we show that BMAL1, an essential transcription factor component of the clock mechanism, is SUMOylated on a highly conserved lysine residue (Lys259) in vivo. BMAL1 shows a circadian pattern of SUMOylation that parallels its activation in the mouse liver. SUMOylation of BMAL1 requires and is induced by CLOCK, the heterodimerization partner of BMAL1. Ectopic expression of a SUMOdeficient BMAL1 demonstrates that SUMOylation plays an important role in BMAL1 circadian expression and clock rhythmicity. This reveals an additional level of regulation within the core mechanism of the circadian clock.
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