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.Canonaco, MarcelloStart date: 2010

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    Implementazione di sistemi innovativi per la qualità e tracciabilità di “Healthy Food”
    (2017-07-03) Macchione, Claudia; De Cindio, Bruno; Canonaco, Marcello
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    Innovativi multiclassificatori con applicazione ai sistemi di supporto alle decisioni cliniche
    (2017-07-03) Groccia, Maria Carmela; Conforti, Domenico; Canonaco, Marcello
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    Processi di trattamento di reflui e materiali di scarto
    (2017-12-07) Albo, Laura; Canonaco, Marcello; Calabrò, Vincenza
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    Cateslytin and Chromofungin, two CgA derived peptides: actors of the immune and cardiac systems
    (2017-05-03) Scavello, Francesco; Canonaco, Marcello; Angelone, Tommaso; Schneider, Francis; Metz-Boutigue, Marie-Hélène
    Chromogranin A (CgA) belongs to the granin family of uniquely acidic secretory that are ubiquitous in secretory cells of the nervous, endocrine, immune system. Numerous cleavage products of the granins have been identified, some of these peptides showed biological activities and are costored in secretory granules of different cells. Chromofungin (Chr: CgA47-66) and Cateslytin (Ctl: CgA344–358) are peptides that display antimicrobial activities and activate neutrophils, with important implications in inflammation and innate immunity. Staphylococcus aureus is an opportunistic pathogen and the leading cause of a wide range of severe clinical infections and one of the most important cause of hospital-acquired infections, in fact infections caused by this bacterium have classically an important impact in morbidity and mortality in the nosocomial and community scene. Furthermore, this pathogen is the primary cause of surgical site infections and the most frequently isolated pathogen in Gram-positive sepsis. In the specific field of cardiovascular disease S. aureus leading infective cause of destruction of endocardial tissue after implantation of prosthetic heart valve. This pathogen is also notorious for its ability to resist the available antibiotics and dissemination of various multidrug-resistant S. aureus clones that limit therapeutic options for a S. aureus infection. Aslam et al. in 2013 shown that Ctl is resistant to the degradation of S. aureus protease and is the most antibacterial CgA derived peptide against this bacterium. The aim of study was to evaluate the: 1) Effects of Chr on isolated and Langendorff perfused rat hearts in basal and pathological conditions; 2) In vitro antibacterial activity of a synthetic Cateslytin-derived peptide to cover artificial heart valves and prevent infection by S. aureus; 3) In vivo antibacterial activity of Ctl in rat infected with S. aureus. The first part of the study was performed by using the isolated and Langendorff perfused rat hearts, Elisa assay and real-time PCR. We found that, under basal conditions, increasing doses (11–165 nM) of Chr induced negative inotropic effects without changing coronary pressure. The AKT/eNOS/cGMP/PKG pathway mediated this action. We also found that Chr acted as a postconditioning (PostC) agent against ischemia/reperfusion (I/R) damages, reducing infarct size and LDH level. Cardioprotection involved PI3K, RISK pathway, MitoKATPand miRNA-21. Therefore, we suggest that Chr directly affects heart performance, protects against I/R myocardial injuries through the activation of prosurvival kinases. Results may propose Chr as a new physiological neuroendocrinemodulator able to prevent heart dysfunctions, also encouraging the clarification of its clinical potential. In the second part of the study, two new synthetic peptides containing Ctl (RSMRLSFRARGYGFR) were designed: D*T*Ctl (DOPA-K-DOPA-K-DOPATLRGGE- RSMRLSFRARGYGFR), T*Ctl (TLRGGE-RSMRLSFRARGYGFR) with D*: DOPA-K-DOPA-K-DOPA and T*: TLRGGE. This study is based on the observation of the adhesive properties of the DOPA-K-DOPA-K-DOPA sequence and on the ability of S. aureus endoprotease Glu-C to cleave the TLRGGE sequence. Firstly, using techniques of biochemistry, proteomics (sequencing, mass spectrometry) and microbiology we shown that the digestion by the Glu-C protease of T*Ctl and D*T*Ctl is able to release active Ctl. The prediction analisys of the secondary structure suggested the presence of an alpha helix domain in the case of D*T*Ctl with respect to T*Ctl. The D* group stabilized the secondary structure and facilitated the cleavage by Glu-C to the release of the active peptide Ctl. Subsequently, the effect of the oxidation by NaIO4 of D*T*Ctl on the release of Ctl and the antibacterial activity was analized. Proteomic analysis showed the formation of polymers inhibiting the action of Glu-C and the release of Ctl. We also shown that D*T*Ctl had a MIC value around 75 μM against different strains of S. aureus. This data shown that D*T*Ctl had a direct action against the bacteria without Glu-C cleavage. However, in oxidizing conditions the formation of aggregates of D*T*Ctl reduced the antibacterial action of this synthetic peptide. In the last part of this thesis, we evaluated the in vivo antibacterial activity of Ctl and whether and to which extent Ctl elicit cardioprotection in rat infected with S. aureus, as a model of infection with this bacterium. Identification of specific molecular targets of tissue and systemic inflammation and damage were analysed by Western blotting, ELISA and microbiological analysis in cardiac homogenates and plasma. A strong reduction of plasma bacterial growth, TNF-α, IL-1β and LDH plasma levels was observed in infected rat treated with Ctl. Western blotting analysis of cardiac extracts showed that Ctl treatment is accompanied by reduction of expression of pro-inflammatory markers, such as iNOS and COX-2. These preliminary data suggest that in vivo Ctl treatment is able to counteract the deleterious effects of S. aureus, and elicits myocardial protection.
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    Structure/function relationships of the human heterodimeric amino acids transporter 4F2hc/LAT1
    (2017-06-09) Napolitano, Lara; Canonaco, Marcello; Indiveri, Cesare; Scalise, Mariafrancesca
    Amino acids transport in mammalian cells is mediated by different amino acid transporters whose activity allow the flow of an important source for metabolic need of cells. Moreover, some amino acids such as Gln, Arg and Leu work as signalling molecules and their availability and concentration represent key factors in the regulation of intracellular signalling pathways responsible of cellular growth. Thus, amino acids flow, which is important under physiological condition, becomes particularly relevant under pathological conditions such as in tumours cells to satisfy their unique metabolic and proliferative needs. Therefore, since in tumours upregulation of amino acids transporters is an important step to satisfy the increased demand for these nutrients, the same transporters are potential drug targets for cancer therapy. However, the certainty that a specific transporter could be a target in human therapy requires its functional characterization and the knowledge of the enchanting structure/function relationships. In this context, an important transporter that became of particular interest for its overexpression in many tumours is LAT1, and the aim of this work has been that to shed light on still unclear aspects of its function hLAT1 belongs to SLC7 family and into the plasma membrane forms heterodimers with the glycoprotein 4F2hc (also known as CD98 in mice), member of SLC3 family. Studies conducted in intact cells showed that 4F2hc/LAT1 complex catalyses amino acids transport; however, in this experimental model it was not possible to clarify whether one or both subunits are competent for transport activity and substrate recognition. Thus, aimed to unravel the dark side of 4F2hc/LAT1 mediated transport, different experimental strategies were adopted allowing to demonstrate that LAT1 is the sole transport competent unit of the heterodimer. Indeed, using western blot analyses and transport assays in liposomes reconstituted with proteins extracted from SiHa cells and in liposomes reconstituted with recombinant LAT1, it has been demonstrated that neither the covalent interaction nor the association of 4F2hc with LAT1 influence transport and specificity of LAT1. Moreover, the suitability of proteoliposome model used for reconstitution of recombinant LAT1, allowed to identify a functional asymmetry of this transporter which, on a physiological point of view, exhaustively elucidates the reciprocal correlation between the transport activity of LAT1 and that of another important amino acids transporter overexpressed in tumours cells, ASCT2. To the same extent, proteoliposome tool together with bioinformatics and site-directed mutagenesis have been useful to probe critical residues of the substrate binding site of LAT1. These results laid the groundwork for deciphering molecular mechanism of LAT1 function and for setting up studies aimed to identify new potent and specific inhibitors great for human health.
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    Investigating how movement affects prey camouflage using an insect predator
    (2019-06-09) Umeton, Diana; Canonaco, Marcello; Brandmayr, Pietro
    Patterns that help prey camouflage themselves whilst stationary prove to be ineffective once prey move. Given that motion breaks camouflage, can a moving prey ever be effectively concealed? Recent studies have found that certain patterns might help prey deceive their predators whilst moving, as in the case of ‘motion dazzle’. However, research with moving prey has been conducted using only humans or birds as predator models, and consequently, it is now known how other predator species might behave. In addition, it is important to know not just how motion affects camouflage, but also how the speed of motion can affect the efficacy of different defensive patterns. This thesis aims to address these current gaps in the field. First, I explore the visual acuity in a group of insect predators, the praying mantids, to explore if different species vary in their visual acuity, which could impact on what they can perceive and which selective pressure they could exert on prey defensive patterns. Second, using praying mantids tracking computer-generated stimuli, I empirically investigate how cryptic and conspicuous patterns might enhance the survival of moving prey. In particular, I specifically investigate if high contrast striped prey could reduce predation risk through the visual phenomenon known as “flicker fusion effect”. I found that when prey were slow moving, all patterns were equally detectable by the mantids. However, once prey moved at faster speed, a cryptic pattern was more likely to be tracked than a more conspicuous black-and-white striped pattern suggesting that the latter was successful in inducing flicker fusion effect in praying mantids’ eyes. This thesis starts to disentangle how pattern and speed could combine to help camouflage an animal when moving through its environment. The outcome of the study are discussed in the wider context of how animals coloration and behaviour evolved together to confer them survival advantages.
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    Effetti del daidzein sul comportamento mnemonico attraverso le variazioni del sistema neurotensinergico e di fattori infiammatori in aree limbiche di criceti high fat diet
    (2018-04-11) Fazzari, Gilda; Cerra, Maria Carmela; Canonaco, Marcello
    In the past years, growing indications have pointed to food diets as major factors capable of conditioning physical-social states in humans as suggested by high-fat containing food sources which, despite their extremely "tasteful" properties, are responsible for marked morpho-structural alterations in critical brain areas for the regulation of not only psycho-cognitive behaviors but above all for nutritional parameters. It is already known that food consumption and action mechanisms implicated in energetic homeostasis are part of a complex neuronal circuit that when they are altered tend to account for increased body weight and consequently the development of obesity, regarded a chronic disease leading to different medical co-morbidities such as cardiovascular diseases (especially hypertension), dyslipidemia and diabetes type 2. From these effects plus the avoidance of applying drugs with numerous collateral alterations have directed a greater attention towards the production of "safer" drugs natural deriving vegetable extracts such as daidzein (DZ), a glucosidic isoflavone capable of mimicking estrogenic activities at the brain level, which supplied neuroprotective values against increased body weight, elevated feeding duration and diminished locomotor activity of high fat diet (HFD) hamsters (Mesocricetus auratus). This isoflavone also reduced anxiety-like behaviors in hyperlipidic animals as indicated by a greater time spent (p<0.01) in open arms of elevated plus maze (EPM) along with greater exploration (p<0.001) tendencies toward new objects of the novel object recognition (NOR) test. From a molecular point of view, DZ greatly protected the brain against neurodegeneration events as pointed out by few Amino Cupric Silver Stain granules in the hippocampus (HIP), hypothalamus (HTH) and amygdala (AMY) very likely via a diminished interaction of neurotensin receptor1 (anorexic neuropeptide; NTR1) GABAergic-leptin receptor levels in the latter two areas after the conditioned place preference (CPP) test. Contextually the recovered cognitive performances seemed to be tightly linked with reduced NTR1 expression levels in an ERβ manner for HIP after NOR test. In addition, such effects of DZ also strongly required increased levels of the antineuroinflammatory cytokine IL-10 that by reducing adenosine 5' monophosphate kinase (AMPK) and extracellular receptor kinase (ERK) widely improved cognitive together with exploratory behaviors. Overall, these first results supply important evidences on the neuroprotective values of DZ, which through NTR1 + IL-10 protective and repairing mechanisms of, aside the above limbic areas, also appetite-mediated brain centers known for their feeding, cognitive and endocrine functions, may surely comprise a good start for the introduction of novel therapeutic applications against obesity conditions.
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    <> 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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    <> ruolo del GPER nelle arterie uterine e mesenteriche in gravidanza
    (2015-12-16) Tropea, Teresa; Canonaco, Marcello; Mandalà, Maurizio
    Low uteroplacental blood flow has been implicated as a cause of pregnancy hypertension and intrauterine fetal growth retardation. A successful pregnancy outcome requires a sufficient uteroplacental blood flow, which increases of many fold compared to the non pregnant levels. This significant increase is achieved in part by vasodilation of the uterine vasculature because pregnancy induces a physiological remodelling of the maternal uterine circulation. Estrogen may contribute to this effect although the mechanisms involved remain unclear. It is known that estrogen receptor-α and estrogen receptor-β in the uterine vascular endothelium are both functionally implicated in the regulation of the uterine blood flow but so far nothing is known about the novel G-protein coupled estrogen receptor (GPER) in the uterine vasculature. GPER has been identified in many vascular bed exerting its potential vasorelaxing effect. The aim of this study was to investigate the function of GPER in the regulation of the uterine vascular tone during pregnancy. Experiments were carried out on mesenteric arterioles and uterine radial arteries isolated from both non-pregnant (NP) and age-matched pregnant (P) Sprague Dawley rats to compare reproductive and systemic vasculature effect. Arterial segments were pressurized to 50 mmHg in the pressure myography, preconstricted of 40% with phenylephrine and then incubated with incremental doses (10-12-10-6 M) of the specific GPER agonist, G-1. A dose-response curve was obtained for G-1 in the 1 pM – 1 μM range with a maximum vasodilation of uterine arteries of 97,8 ± 2,5% in P vs 66,5 ± 3,7% in NP, p<0.001. G-1 vasorelaxing effect was similar in pregnant (45,5 ± 6,1%) and non-pregnant (53,6 ± 2,3%) mesenteric arteries. Pregnancy induced a significantly higher G-1 vasodilation only in uterine artery and its protein expression was also increased. G-1 effect was significantly reduced by the GPER antagonist, G-15. The NOS inhibitors, L-NAME+L-NNA or endothelium removal reduced the G-1 induced relaxation of uterine artery, suggesting an endothelium-dependent mechanism, involving cGMP pathway but not BKca channels. Immunohistochemistry revealed the GPER expression in the main uterine artery of both eNOS-/- and wild type pregnant mice. This receptor was functionally activated in a dose response manner with a significant less effect in knock out mice, showing the relevance of the NO in GPER pathway. GPER is also present in human chorionic plate arteries and its activation induces a slight vasodilation. These results demonstrate for the first time that GPER may have a role in regulating vascular tone, placental perfusion and normal fetal development, suggesting a potential therapeutic target in pregnant diseases.