Browsing by Author "Fiorito, Graziano"

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    Arm growth and regeneration in octopus (Octopus vulgaris and Eledone moschata)
    (Università della Calabria, 2020-02-12) Baldascino, Elena; Cerra, Maria Carmela; Fiorito, Graziano
    Here I evaluated the timing and rate of arm regeneration in octopus, a cephalopod mollusc, and contributed to the understanding of the molecular machinery involved in arm growth and regeneration in two octopus’ species: Octopus vulgaris and Eledone moschata. This work stands and revisit in part the first experimental study on arm regeneration in cephalopod molluscs, including the two species carried out by M. Lange (1920). An arm of octopus may fully regenerate and regrowth in few months after amputation. Lange recognized three stages: wound healing, tissue degeneration and tissue renewal. I applied the 3Rs principle to this study utilizing samples and data from a previous study, thus limiting the number or live animals humanely killed for the purpose of this PhD project. During the analysis of data, I found the same three stages in O. vulgaris arm regeneration to occur. I was not able to observe similar series of events in E. moschata, probably due to limited time and conditions of the animals. I found that O. vulgaris increased significantly body weight in 21 weeks. The growth appeared to be affected by repeated anesthesia, thus encouraging further studies on the physiological responses to stress in octopus. Data I analyzed confirm the view that O. vulgaris is characterized by rapid non-asymptotic growth, with high individual variability. Octopuses elongate their arms continuing to grow over time with no significant difference in the relative rate of elongation between anterior and posterior arms. The arms that underwent to regeneration, appear to have a faster growth when compared with the contralateral arms (but repeated anesthesia influenced the rate of growth of regenerating arms). Data analyzed extent the study of Lange, by providing morphometric information on the relative growth rate of the arms (regenerating vs contralateral), for the first time. A significant change in the relative expression profile of the tip vs the proximal part of the arm in O. vulgaris was observed using different approaches, confirming and extending the view that the tip the arm represents an exceptional biological system where continuous growth is achieved through massive cellular differentiation and interesting molecular events. I identified 39 genes in O. vulgaris and seven in E. moschata and studied their involvement in arm growth and/or regeneration events in octopus arms. These studies are novel.
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    Arm growth and regeneration in octopus (Octopus vulgaris and Eledone moschata)
    (Università della Calabria, 2020-02-07) Baldascino, Elena; Cerra, Carmela; Fiorito, Graziano
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    Distribuzione e analisi funzionale dei neuromodulatori in Octopus vulgaris
    (2017-03-13) Ponte, Giovanna; Cerra, Maria Carmela; Fiorito, Graziano
    ments the pattern of distribution of biogenic amines in the brain of the cephalopod mollusc Octopus vulgaris. The data show that dopamine, noradrenaline and octopamine are not ubiquitary in the brain, as considered before, and that a division of labour between visual and tactile processing system exists. These data largely revise previous available knowledge and for the firt time depict the distribution of octopamine in the brain of the octopus. This despite this molecule has been for the first time discovered in the salivary gland of this animal more than 100 years ago.
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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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    Learning and memory in Octopus vulgaris: search of the underlying biological machinery
    (Università della Calabria, 2021-03-01) Manzo, Paolo; Cerra, Maria Carmela; Fiorito, Graziano
    The cephalopod mollusc Octopus vulgaris is known for the richness of behavioral repertoire, neural and behavioral plasticity, and complex cognition rivaling higher Vertebrates. Animals are known to learn over a variety of tasks, equipped with different sensory-motor systems, i.e. visual and tactile, and able to recall the outcomes of their experience for long term (e.g., ‘one month’ Sutherland, 1957; ‘some months’ Sanders, 1970). In the present study, a fear conditioning training protocol was adopted to evaluate O. vulgaris behavioral responses to an artificial stimulus to be avoided. Behavioral outcomes have been tested for the effect of a protein synthesis inhibitor on memory acquisition and retention, and for changes in the pattern of expression of genes potentially involved in memory formation. Applying the 3Rs principle, I used samples from a previous study, thus limiting the number or live animals humanely killed for the aims of this PhD project. My experiments and analysis allowed to: i. Identify that cycloheximide-induced protein synthesis inhibition did not alter the octopus ability to acquire an avoidance learning task. However, octopuses ability to retain and recall the memory was impaired; ii. Data available to me did not allow to rule out a state-dependent effect of cycloheximide injection that somehow affects memory recall and octopuses ability to learn; iii. Identify 24 target genes, nine memory-related genes and 15 epigenetic modifiers, from O. vulgaris transcriptome, and studied their gene expression profile in relation with learning and memory consolidation.
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