Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato

Permanent URI for this collectionhttp://localhost:4000/handle/10955/34

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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Start date: 2010Subject: search.filters.subject.Biologia animale

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Now showing 1 - 10 of 14
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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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    Azione cardiaca della catestatina in Rana esculenta
    (2014-03-26) Barbieri, Sandra Francesca; Canonaco, Marcello; Gattuso, Alfonsina; Mazza, Rosa
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    Molecole e correnti ioniche coinvolte nella maturazione ovocitaria in Ciona intestinalis (ascidie)
    (2014-03-25) Silvestre, Francesco; Canonaco, Marcello; De Santis, Rosaria; Tosti, Elisabetta
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    Meccanismi di Cross-talking tra i sistemi Orexinergico e GABAergico nei teleostei
    (2014-03-25) Crudo, Michele; Canonaco, Marcello; Facciolo, Maria
    The neuropeptides hypocretins/orexins (ORX) are known to control state-dependent activities such as sleep-wakefulness, energy homeostasis and reward/addiction processes. To date, interests regarding ORXergic neuronal functions have mostly dealt with mammals, while only recently attention is beginning to be directed towards aquatic vertebrates. On this basis, the intention of the present study was to evaluate ORX-A effects on behavioral and neuronal activities in marine (the ornate wrasse, Thalassoma pavo) and freshwater (the goldfish, Carassius auratus) teleost fishes. To achieve such aims behavioral, molecular and neurodegenerative analysis were performed following intraperitoneal injections of not only the most effective doses of ORX-A, but also of GABAAR β subunit agonist muscimol (MUS) and antagonist bicuculline (BIC) as well as a classical benzodiazepine, diazepam (DZP), and a non benzodiazepine, zolpidem (ZOL), that are instead agonists of the GABAAR α subunits. Behavioral studies on explorative behaviors (swimming towards food sources; STF), free swimming (FS) and on resting states (R) displayed that ORX-A was responsible for very great (p<0.001) enhancements of STF in both teleosts while similarly very great and great (p<0.01) reduction of R was detected in wrasse and goldfish, respectively. Interestingly, ORX-A effects showed to be strongly influenced by environmental photoperiodic conditions as pointed out by behavioral and ORXR expression relationships in wrasses during constant light/dark conditions. In this case, ORX-A/photoperiodic-dependent motor behaviors might be related to a structural-functional dichotomy of some motor-controlling brain regions. Indeed during the day, ORX-A might be very likely promoting the awakening state via low-affinity ORXR sites at hypothalamic (HTH) and mesencephalic (MES) levels so inducing the promotion of STF and FS. On the other hand, during the night ORX-A might result in a promotion of R via activation of mostly telencephalic (TEL) high-affinity sites. As far as GABAAR influence is concerned, MUS was capable of inducing a moderate (p<0.05) increase in STF and this tends to not only underlie a highly conserved GABA stimulus on feeding but also that such an activity relies on the involvement of ORXR in most brain regions of both fish models. Moreover, it was also possible to observe that such a GABAAR agonist elicits a neuroprotection via ORXR as suggested by both the reduction of BIC-dependent mRNA down-regulations and the very low argyrophilic reactions in brain regions of MUS+BIC-treated fish. When the increase of STF was next checked to see whether it was linked to an enhancement of food consumption, I observed that ORX-A and GABAAR  drugs (MUS and BIC) did not modify this activity. On the other hand, GABAAR  subunits appeared to not only influence STF maneuvers but also food consumption as demonstrated by ZOL and DZP evoking very great increases in STF coupled with great and very great amounts of food consumption, respectively. Interestingly, also in 8 this case ZOL- and DZP-dependent hyperphagia seemed to be correlated to ORXR expression variations in mostly HTH and MES, brain areas that are involved in energy homeostasis and motor activities. At this point it was interesting to investigate if ORX- and GABAAR-induced STF effects were mediated through rewarding pathways like that reported for mammals. With such an aim, T-maze and Conditioned Place Preference (CPP) paradigms were performed. For this part, ORX-A accounted for a moderate improvement of T-maze performances that became great when associated to color visual stimuli, suggesting that ORX-A might be operating on memory processes also in fish. Moreover from CPP analysis in which ORX-A acted as a main agent responsible for great increase of color-dependent change in CPP, it seems that this neuropeptide and not GABAAR drugs, might be potentiating reward pathways in goldfish. Also in this case, behavioral performances are consistent with ORXR up-regulations in some TEL regions and in the nucleus of the posterior tuberculum, homologous of the amygdala plus hippocampus and ventral tegmental area that in mammals control learning and reward processes. It is worthy to note that DZP only accounted for a moderate increase in CPP change while BIC completely blocked ORX-A-induced change in CPP and this tends to point to a dual neurosignaling mechanism between ORX circuit and GABAAR / subunits on motivational-reward behaviors. Overall these results highlight, for the first time, not only an ORX neuronal driving force operating on feeding behaviors in goldfish, but above all its participation in the control of goal-oriented behaviors like in mammals.
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    Ruolo del cross-talking GABA-glutammato nello sviluppo morfo-funzionale dei neuroni ippocampali di Mesocricetus auratus
    (2014-03-24) Di Vito, Anna; Cerra, Maria Carmela; Canonaco, Marcello
    The hippocampus (HIP) is recognized as a major telencephalic area processing learning and episodic memory events through the accumulation of neuronal signals deriving from the different subregions. In particular, hippocampal neurons have received particular attention due to some of their functional properties being regulated by GABAergic and Glutamat- (Glu)-ergic neuronal signals during both postnatal and adult stages. For the present study, growth of hamster hippocampal neurons on biohybrid membrane substrates allowed us to show for the first time that the two major GABAA α receptor subunits (α2,5) promote early neuronal shaping plus expression differences of the main neuronal cytoskeletal factors (GAP-43, MAP2, Syn, the neurotrophin BDNF) along with Gluergic subtypes. In a first case, the selective GABAAR agonist diazepam (DZP; α2,5) caused very great (p<0.001) increases of dendritic sprouting and branching processes mainly at DIV3, while its effects continued to account even for great (p<0.01) axonal length during the entire culture period. In addition, DZP also accounted for great (p<0.01) up-regulation of neuritic NR1, GluR2 and MAP2 while it moderately (p<0.05) increased synaptophysin (Syn) at DIV7. Such effects were abolished by its highly selective antagonist flumazenil. The application of the inverse α5 agonist (RY-080) tended to strongly suggest its specific role on the dendritic component via the inhibition of BDNF, as shown by a reduction of dendritic length at DIV7 concomitantly to very low levels of the neurotrophin. Conversely, the effects of the preferentially specific agonist for hippocampal α2 subunit (flunitrazepam) were, instead, directed at the formation of growth cones at DIV3 in the presence of greatly (p<0.01) diminished GAP-43 levels as displayed by strongly reduced axonal sprouting. It is interesting to note that contextually to these morphological variations, the transcription of some Gluergic receptor subtypes resulted to be altered. Indeed, the α2 agonist was responsible for a distinctly rising expression pattern of axonal NR1 mRNA levels from DIV3 (p<0.01) until DIV7 (p<0.001), along with a very great up-regulation of Syn at DIV7. In the case of RY-080, it evoked a very great (p<0.001) downregulation of dendritic GluR2 at only DIV7. Together, our results demonstrate that GABAA α2,5 receptor-containing subunits are considered key modulating neuronal elements of HIP morphological features by regulating the precise synchronization of cytoskeletal factors. Moreover, the notable NR1 and GluR2 transcription differences promoted by these GABAA α subunits tend to favorably corroborate the early role of α2 + α5 on HIP neuronal networks in hibernating rodents through the recruitment and activation of silent neurons. We are still at the beginning but further studies are required to establish the nature of molecular signals controlling responses of the different hippocampal subregions, which may have therapeutic bearings for neurological disorders.
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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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    Carabid beetles as natural antagonists of the olive fly Bactrocera oleae Rossi 1790
    (2014-03-20) Odoguardi, Rosalba; Canonaco, Marcello; Zetto, Tullia; Bonacci, Teresa