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

Permanent URI for this collectionhttps://lisa.unical.it/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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    La catestatina migliora la risposta Frank-Starling in cuori di ratto normotesi e ipertesi agendo come attivatore fisiologico del pathway trasduzionale ossido nitrico-dipendente
    (16-12-2015) Cantafio, Patrizia; Canonaco, Marcello; Angelone, Tommaso
    The myocardial response to mechanical stretch (Frank-Starling law) is an important physiological cardiac determinant. Modulated by many endogenous substances, it is impaired in the presence of cardiovascular pathologies and during senescence. Catestatin (CST: hCgA352-372), a 21-amino-acid derivate of Chromogranin A (CgA), displays hypotensive/vasodilatory properties and counteracts excessive systemic and/or intra-cardiac excitatory stimuli (e.g., catecholamines and endothelin-1). CST, produced also by the myocardium, affects the heart by modulating inotropy, lusitropy and the coronary tone through a Nitric Oxide (NO)-dependent mechanism. This study evaluated the putative influence elicited by CST on the Frank-Starling response of normotensive Wistar Kyoto (WKY) and hypertensive (SHR) hearts by using isolated and Langendorff perfused cardiac preparations. Functional changes were evaluated on aged (18-month-old) WKY rats and SHR which mimic human chronic heart failure (HF). Comparison to WKY rats, SHR showed a reduced Frank-Starling response. In both rat strains, CST administration improved myocardial mechanical response to increased end-diastolic pressures. This effect was mediated by EE/IP3K/NOS/NO/cGMP/PKG, as revealed by specific inhibitors. CST-dependent positive Frank-Starling response is paralleled by an increment in protein S-Nitrosylation, AKT/eNOS/nNOS and PLN phosphorylations. Our data suggested CST as a NO dependent physiological modulator of the stretch-induced intrinsic regulation of the heart. This may be of particular importance in the aged hypertrophic heart, whose function is impaired because of a reduced systolic performance accompanied by delayed relaxation and increased diastolic stiffness.
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    Sistemi vegetali e patterns di risposta fattori abiotici
    (2006) Pangaro, Tiziano; nnocenti, Anna Maria; Cozza, Radiana
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    Aspetti citotassonomici, embriologici e morfologici del genere taraxacum Wigg. (Asteraceae) in Calabria
    (2006) Aquaro, Gabriella; Innocenti, Anna Maria; Cesca, Giuliano
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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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    Ligh-regulated and circadian expression of tomato photoreceptors
    (2006) Facella, Paolo; Perrotta, Gaetano; Innocenti, A.M.
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    Filogenesi e processi speciativi in orchidee Mediterranee non nettarifere del genere Serapias L.(Orchidaceae)
    (2006) Bellusci, Francesca; Innocenti, Anna Maria; Musacchio, Aldo
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    Nuclear and Mitochondrial Genetic Risk Factors in Frontotemporal Dementia
    (2006-10-05) Longo, Teresa; Rose, Giuseppina; Bruni, Amalia C.; De Benedictis, Giovanna
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    Heteroplasmy of the mitochondrial DNA control region in families of centenarians
    (2006-10-05) Scornaienchi, Vittorio; Rose, Giuseppina; De Benedictis, Giovanna
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    Study of transcriptional factors which regulate the enhancer activity of a VNTR located within the SIRT3 gene
    (2006-10-05) Covello, Giuseppina; Bellizzi, Giuseppina; De Benedictis, Giovanna
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    Exploring new routes in genetic studies on human aging and longevity
    (2007 ) Montesanto,Alberto; Passarino,Giuseppe; De Benedictis,Giovanna
    The past few decades has witnessed a growing scientific interest in genetic studies on human aging and longevity. This growing interest may be explained by the increasing number of elderly subjects in developed countries over the last 50 years due to the continued improvements in health care. Such a fast increase of these population segments represent a huge problem for the societies in terms of social care and welfare. The difficulty in understanding the biological basis of human aging and longevity is mainly represented by (i) the cohort and the population-specificity of human aging and longevity, (ii) the lack of a clear and objective definition of the phenotype, (iii) the polygenic nature of the human longevity. On the basis of these observations, the studies reported here represent new approaches for the study of human aging and longevity. They were carried out on data obtained from the Calabrian population, characterized by high genetic homogeneity and a scarce level of immigration due to geographical, historical and social reasons. The first approach we used was to analyze the spatial distribution of long-lived individuals in Calabria. Using this approach, we verified that the spatial distribution of such individuals in this region is not uniform. In addition, by using surname data, we verified a significant correlation between population inbreeding and male longevity in a particular area of this region. The second approach is based on the application of a Cluster Analysis with well established geriatric parameters to identify aging phenotypes in the Calabrian population. The results obtained show that, the aging phenotypes recognized by Cluster Analysis are consistent from a geriatric point of view and have a clear genetic component.Finally, using a multilocus approach, we analyzed the influence of genetic variability of candidate genes on survival at old age in good health. On the basis of a synthetic survival curve built using historic mortality data from the Italian population and using multiple regression models, we found that genetic factors influence survival at advanced ages in good health in a sex and age specific way. The results reported here show that the application of these new approaches may be useful in human aging and longevity studies. Furthermore, they demonstrate that a multidisciplinary approach is necessary to analyze human aging and longevity.
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    Analysis of C538T polymorphims of the SSADH gene in humans: functional and evolutionary aspects
    (2007) Leone,Ofelia; Passarino,Giuseppe; De Benedictis,Giovanna
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    Identificazione e caratterizzazione di FoxP nel sistema nervoso centrale di Octopus vulgaris (Mollusca, Cephalopoda)
    (2007) 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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    L’Angiotensina II modula il calcio intracellulare attivando AT1R, tramite la via di trasduzione dell’IP3 e i canali per il calcio di Tipo T (TCC).
    (2008) Martini, Aurela; Martino, Guglielmo; Canonaco, Marcello
    L’ Angiotensina II è il maggiore effettore del sistema renina-angiotensina. L’Angiotensina I, formata in seguito all’attività enzimatica della renina, interagisce con l’ACE plasmatico e con quello dell’endotelio polmonare convertendosi in Ang II.1 Successivamente l’Ang II è veicolata dal circolo ai suoi organi bersaglio regolando la pressione sanguigna, il bilancio idrico e il tono muscolare. Gli effetti indotti dal peptide sono mediate principalmente da due tipi di recettori di membrana, AT1 e AT2.2 I recettori AT1 stimolano un elevato numero di sistemi di trasduzione del segnale all’interno della cellula, quali fosfolipasi A (PLA), fosfolipasi D (PLD), fosfolipasi C (PLC), le MAP kinasi e la mobilizzazione del calcio intracellulare; infatti è ben nota la modulazione della concentrazione intracellulare di questo ione dall’idrolisi di fosfatidilinositolo-4,5-difosfato. Un’ ulteriore quantità di Ca2+ entra nella cellula anche dall’esterno grazie all’apertura dei canali del calcio. Recenti studi hanno dimostrato l’esistenza dei canali per il calcio di tipo T a livello delle cellule endoteliali, ma il loro vero ruolo non è ancora del tutto chiaro.3 Scopo dello studio: Esaminare il coinvolgimento dell’Ang II nell’incrementare la [Ca2+]i anche attraverso l’attivazione dei canali del calcio di tipo T nelle cellule HUVEC e determinare quale dei due recettori, AT1 o AT2, è coinvolto della attivazione di questi canali. La prima tappa di questo studio è stata la messa a punto del protocollo per la determinazione della vitalità cellulare, mediante l’ Arancio di Acridina, della concentrazione del Calcio, NO e ROS utilizzando rispettivamente le sonde: Fluo-3AM, DAF-2DA e HDCFH-DA. Metodo: Le HUVEC utilizzate al terzo passaggio, sono state mantenute e cresciute in coltura mediante mezzo specifico per cellule endoteliali EGM® Bullet Kit (Lonza) contenente 10% FBS. Le cellule sono state trattate con Ang II alle concentrazioni: 10-9 M o 10-7 M o 10-6 M in presenza o meno degli antagonisti dei recettori AT1 o AT2 per la messa a punto del protocollo. Nel secondo studio le cellule sono state trattate con Ang II alle suddette concentrazioni in presenza degli antagonisti e in presenza o meno dell’ inibitore della via IP3 o del TCC. Le cellule trattate con le sonde sono state osservate dopo 3, 6 e 9 ore. Le immagini sono state catturate col microscopio Olympus utilizzando ProImagePlus 4.0 ed analizzate col programma NIH ImageJ. Risultati: La valutazione dell’effetto dell’Ang II sulla vitalità e sulla modulazione della concentrazione del Calcio, NO e ROS a livello delle cellule endoteliali, ha evidenziato che la concentrazione ottimale per la valutazione degli effetti intracellulare di questo octapeptide nelle cellule endoteliali è 10-7 M; le cellule HUVUC rappresentano un buon modello per valutare l’azione dell’Ang II sull’ endotelio capillare. Il secondo studio ha dimostrato che l’ Ang II induce un alterazione del calcio intracellulare attraverso l’interazione col recettore AT1 stimolando la via IP3 per rapidi effetti fisiologi mentre attiva i canali del calcio di tipo T per tempi maggiori o uguali a 9 ore. Tali risultati suggeriscono che i T-Type Calcium channels regolano direttamente la permeabilità al calcio delle membrane plasmatiche di cellule HUVEC, mediante canali selettivi e non solo tramite la via IP3. Questo risultato mette in risalto il ruolo dei TCC nella regolazione metabolica e strutturale diretta delle cellule endoteliali, accanto a quella delle cellule muscolari lisce dei capillari.
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