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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Aspetti neurocomportamentali e possibili approcci terapeutici del Disturbo dello Spettro Autistico nel modello roditore BTBR T+Itpr3tf/J (BTBR)
(Università della Calabria, 2024-04-29) Olivito, Ilaria; Angelone, Tommaso; Facciolo, Rosa Maria
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental syndrome characterized by a diverse range of clinical manifestations, encompassing social deficits and repetitive stereotypical behaviors. These symptoms often coexist with psychiatric and medical comorbidities, adding intricacy to the pathological profile of the disorder. The pervasive prevalence and enduring symptoms across the lifespan underscore the significant public health impact of ASD. However, the underlying pathophysiological mechanisms remain poorly understood, and there is a current lack of specific targeted medications. Given the intricate and heterogeneous nature of the symptomatology associated with this syndrome, the adoption of a multifaceted approach emerges as a valuable strategy to deepen the comprehension of its pathophysiology and advance more effective therapeutic interventions. Within these strategic considerations, inducing a ketogenic metabolic state presents a promising avenue, capable of mitigating abnormal behaviors while concurrently ameliorating psychological and sociological aspects across various neurological conditions. This approach operates through diverse molecular pathways, including the modulation of the gut microbiota, identified as a pivotal center for brain regulation. In the context of addressing these challenges, the current doctoral project aims to significantly contribute to the comprehension of ASD's pathophysiology. This is achieved through a comprehensive exploration of behavioral aspects, molecular dynamics, the association with the gut microbiota, and potential therapeutic strategies. Noteworthy is the adopted methodological approach, which involves analyzing the effects of ketosis in the BTBR rodent model of autism, encompassing both in vivo and in vitro assessments. This analysis includes evaluations of behavioral performance, key molecular mechanisms, and integration with the assessment of the gut microbiota. Results from an initial in vivo study demonstrated that the adoption of a ketogenic diet (KD) notably mitigated social deficit (p=0.002), repetitive behaviors (p<0.001), and memory impairments (p=0.001) in BTBR mice. These behavioral enhancements were associated with reduced levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 in the plasma (p=0.007; p<0.001, and p=0.023, respectively), prefrontal cortex (PFC; p=0.006; p=0.04, and p=0.03), and hippocampus (HIP; p=0.02; p=0.09, and p=0.03). Furthermore, the KD mitigated oxidative stress, evidenced by modifications in lipid peroxidation levels (TBARS) and superoxide dismutase (SOD) activity in the brain areas of BTBR mice. Of particular interest, the KD positively influenced beneficial microbiota (Akkermansia and Blautia) and normalized aberrant levels of Lactobacillus in the feces of BTBR mice, underlining its neurophysiological impact through the modulation of the gut microbiota. The correlation between gut microbiota modulation and behavioral changes is substantiated by a meticulous analysis of the effects of five Lactobacillus strains in a study conducted during the planned abroad period within the doctoral project. This specific investigation unveiled a differential impact of the strains L. salivarius - LS7892, L. gasseri - LG6410, L. plantarum - LP14D, L. reuteri - LR92, and L. camelliae - LC LMG 24277 on social behavior, motor activity, and anxiety in BTBR mice. Particularly noteworthy, LS7892 emerged as a potential probiotic for ASD treatment among the examined strains. Following in vitro investigations revealed alterations in the cytoskeletal and synaptic organization in PFC and HIP neurons of BTBR, linked to fluctuations in Brain-Derived Neurotrophic Factor (BDNF) expression levels. The induction of a ketogenic state exhibited a beneficial modulation of the neural network, stabilizing compromised connections, and significantly increasing BDNF levels (p<0.001). In conclusion, the outcomes of this doctoral project underscore the significant relevance of the ketogenic approach and the modulation of the gut microbiota in the context of ASD. This research contributes to a more profound understanding of the pathophysiological foundations, offering innovative perspectives for the development of personalized and targeted therapeutic approaches.
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.
Identification of epigenetic mechanisms involved in seed coat development
(Università della Calabria, 2024-04-29) Talarico, Emanuela; Angelone, Tommaso; Bruno, Leonardo
The evolution of seeds is a fascinating aspect of evolutionary history and plant biology. Seeds have evolved over millions of years and are considered a significant adaptation that has contributed to the success of land plants. Seeds provide protection and a means of dispersal, enabling plants to reproduce successfully in a wide range of conditions. This evolutionary history has led to the rich diversity of plant species we see on Earth today. Seeds show remarkable adaptations to survive long journeys, including different shapes, sizes and mechanisms for dispersal. But none of these features would have been possible without the evolution of the ovule, within which sexual reproduction occurs. Indeed, ovule is the structure in which take place the formation of female gametophyte, fertilisation, embryogenesis and seed development upon fertilisation. In this scenario, the aim of this Ph. D. project was to identify, at evolutionary level, the molecular and epigenetic mechanisms involved in the ovule-to-seed switch in Ginkgo biloba plants. In particular, the focus was only on the pollination event, which in such a system is separated from fertilisation by a long time interval (i.e. four/five months). Indeed, Ginkgo biloba, a member of the gymnosperms and the only extant species of the order Ginkgoales, was used as experimental model because it is considered a living fossil due to its very ancient origins, dating back to the Permian period, the last period of the Palaeozoic, when an integument developed for the first time to cover the megasporangium. An interesting characteristic of Ginkgo, which makes it suitable for this purpose, is the production of fleshy fruit-like structures that are attractive to animals. Indeed, already after pollination, the integument takes on a consistency similar to that of mesocarp of fleshy fruit, leading to the hypothesis that it may represent a precursor to the fruit, although it cannot be identified as such because gymnosperms lack an ovary. Various approaches, including RNA sequencing, in situ gene expression, hormones localization and chromatin immunoprecipitation following by sequencing (ChIP-seq), on ovules at different stages were performed in order to identify the key pathways and the epigenetically regulated genes involved in ovule-seed switch. In order to identify the main pathways modulated by the crucial pollination event, three developmental stages of the Ginkgo ovule, collected immediately after the time frame in which pollination drop emission occurs, were used. In this context, pollination drop emission is an interesting aspect because, in many gymnosperms, it identifies the time point of possible pollen reception. Therefore, samples were collected from two different experimental fields, the first characterised by the presence of both male and female plants, and the second where only female plants are present. The two experimental fields are geographically distant from each other, which means that the plants in the second field are unable to receive pollen, so they are useful for understanding how ovule development proceeds in the absence of the pollination event. Moreover, this Ph. D. thesis was part of a larger project, which involved collaboration with the research groups coordinated by Professor Barbara Baldan, University of Padua, and Professor Lucia Colombo, University of Milan, helping to produce a large amount of data on Ginkgo, but also on Arabidopsis, which has always been the model species in plant biology. With the contribution also of the results we produced, it was possible to compare the two species and describe some of the key genes involved in ovule development in Ginkgo. Finally, most of the bioinformatic analyses related to the ChIP-seq experiment reported in this thesis were performed in collaboration with Professor Ernesto Picardi of the University of Bari Aldo Moro and Dr. Antonella Muto, post-doc in my research group at the University of Calabria.
Il ruolo della Cromogranina A e dei suoi peptidi derivati, Serpinina e Catestatina, nella fisiopatologia cardiaca
(2012-11-24) Gentile, Stefano; Cerra, Maria Carmela; Angelone, Tommaso; Canonaco, Marcello
Il ruolo cardiovascolare della CgA e dei suoi peptidi bioattivi è stato ampiamente documentato. I livelli plasmatici di CgA (range fisiologico da 0.5nM a 5nM), inizialmente utilizzati nella pratica clinica come biomarker di tumori neuroendocrini (O’Connor and Bernstein, 1984; Stridsberg and Husebye, 1997), rappresentano anche un importante marker per le disfunzioni del sistema cardiocircolatorio come ad esempio l’ipertensione essenziale, le cardiomiopatie ipertrofico/dilatative e l’insufficienza cardiaca (Ceconi et al., 2002). Recentemente Jansson et al. (2009), e Rosjo et al. (2010), hanno dimostrato che i livelli di CgA nella sindrome coronarica acuta forniscono informazioni prognostiche indipendentemente dagli altri markers di rischio convenzionali. L’importanza della CgA nella biologia cardiaca è inoltre supportata dall’osservazione che la delezione del gene per la CgA nei topi provoca lo sviluppo di ipertensione, che può essere riportata a livelli fisiologici trattando gli animali con CST o reintroducendo il gene per la CgA in topi con background Chga-/- (Mahapatra et al., 2005). I livelli plasmatici di CgA aumentano in condizioni di eccessiva stimolazione del sistema simpatico, particolarmente evidente nell’insufficienza cardiaca. Ceconi et al. (2002) e Pieroni et al. (2007), hanno dimostrato che in pazienti affetti da insufficienza cardiaca le concentrazioni plasmatiche di CgA sono aumentate (10-20 nM; 500-1000ng/ml), e strettamente correlate alla severità della patologia. Al momento non sono però disponibili informazioni sugli effetti diretti della CgA intera sul cuore, e sui fattori che regolano la sua produzione e processamento a livello miocardico.Il presente lavoro ha quindi lo scopo di chiarire se, e in che misura, la CgA intera induce effetti diretti sulla performance cardiaca, e la possibilità di un processamento proteolitico intracardiaco stimolo-dipendente della proteina. Utilizzando ratti normotesi (WKY) e ipertesi (SHR), abbiamo valutato i) gli effetti miocardici e coronarici della CgA intera nel cuore di ratto isolato e perfuso secondo metodica Langendorff; ii) il pathway trasduzionale (Akt/NOS/NO/cGMP/PKG) coinvolto nel suo meccanismo d’azione; iii) il processamento intracardiaco della CgA in seguito a stimolazione -adrenergica con ISO. Dal processamento della CgA deriva una serie di peptidi bioattivi; fra questi i peptidi N-terminali VS-1/2, (VS-1 CgA1-76; VS-2 CgA1-113), e il peptide C-terminale CST (CgA352-372), hanno effetti cardioattivi. La CST promuove l’angiogenesi (Theurl et al., 2010), abbassa la pressione sanguigna (Mahapatra et al., 2005; Fung et al., 2010; Gaede and Pilowsky, 2012), riduce la contrattilità cardiaca (Angelone et al., 2008; Mazza et al., 2008; Imbrogno et al., 2010), e incrementa la sensibilità dei barocettori (Gayen et al., 2009a; Gaede and Pilowsky, 2010). Recentemente è stato scoperto un nuovo frammento derivato dalla CgA. A livello della regione C-terminale, altamente conservata, il clivaggio proteico ad opera delle pro-ormone convertasi (PC1/2/3), genera un frammento di 2.9 kDa, la “serpinina” (Ala26Leu), il quale può subire una modificazione all’estremità N-terminale per formare un residuo di piro-glutammato (pGlu23Leu o pGlu-serpinina) (Koshimizu et al., 2011a,b). La presenza di entrambe le forme di serpinina è stata rilevata in colture di cellule di ghiandola pituitaria (AtT20). Questi peptidi sono in grado di inibire la morte cellulare indotta da stress ossidativo (Koshimizu et al., 2011a), e di promuovere la biogenesi dei granuli secretori nelle cellule endocrine regolando l’espressione di un inibitore delle proteasi, la proteasi nexina-1 (PN-1), che previene la degradazione delle proteine dei granuli nell’apparato di Golgi. È stato osservato che i peptidi della serpinina agiscono attraverso il pathway AC/cAMP/PKA (Koshimizu et al., 2011b), suggerendo che il meccanismo eccitatorio indotto dalla serpinina potrebbe controbilanciare gli effetti antiadrenergici e cardioinibitori indotti dalla CST e VS-1. Ad oggi non esistono evidenze sperimentali sul possibile ruolo cardioattivo della serpinina; pertanto nel presente lavoro di tesi è stata valutata la presenza di questo peptide e delle sue forme alternative nel cuore di ratto; è stato inoltre osservato in che modo influenzano la performance miocardica e la vasoattività coronarica. La serpinina e la pGlu-serpinina inducono un effetto positivo dose-dipendente sulla contrazione (inotropismo) del cuore di ratto isolato e perfuso secondo metodica Langendorff e sui muscoli papillari isolati, nonché sul rilassamento (lusitropismo) miocardico. Questi effetti miocardici sono stati accompagnati da una lieve, ma non significativa, vasodilatazione coronarica. Un terzo peptide, la serpinina Ala29Gly, non ha influenzato la performance cardiaca a nessuna delle concentrazioni testate. Sia la serpinina che la pGlu-serpinina sembrano agire attraverso il pathway 1-AR/AC/cAMP/PKA. Questi dati evidenziano le proprietà cardio-circolatorie della serpinina e della pGlu-serpinina, fornendo ulteriori informazioni su come i peptidi CgA derivati possano, controbilanciandosi, regolare finemente l’attività cardiaca in risposta a stimoli -adrenergici.Il quadro clinico dei pazienti affetti da cardiopatie è complicato da un’altra patologia che si accompagna spesso alle cardiopatie, la sindrome metabolica. E’ stato constatato, infatti, che circa il 30% di pazienti con cardiopatia ischemica acuta sono affetti da sindrome metabolica (ad esempio diabete, obesità). Il peptide CgA derivato prancreastatina (CgA250-301, PST) (Tatemoto et al., 1986; O’Connor et al., 2005; Gayen et al., 2009b), svolge diverse funzioni a livello metabolico, in particolare sul metabolismo del glucosio (Tatemoto et al., 1986). Tuttavia non sembra avere effetti a livello cardiovascolare. Studi recenti hanno evidenziato il ruolo della PST e della CST nel regolare la secrezione di insulina mantenendo l’omeostasi fra l’effetto anti-insulina della PST, e gli effetti insulino-sensitizzante della CST. (Gayen et al., 2009b). È stata valutata la possibile azione della CST sul metabolismo lipidico che, come è ben noto, risulta alterato nei soggetti obesi. Le cellule adipose sono regolate dalle catecolamine attraverso quattro tipi di AR: 1, 2, 3 e 2 (Arner, 1999; Arner, 2005). L’attivazione dei recettori -AR, coinvolgento le proteine G stimolatorie, aumenta la produzione di cAMP; questo a sua volta attiva la PKA, la quale fosforila la lipasi ormone sensibile (HSL) causando l’idrolisi dei lipidi. Al contrario, l’attivazione dei recettori 2-AR, accoppiati a proteine G inibitorie, inducono effetti opposti sulla lipolisi (Lafontan et al., 1997; Stich et al., 1999, 2003; Lafontan and Langin, 2009). Pertanto l’azione netta delle catecolamine sulla lipolisi dipende dall’equilibrio fra recettori - e -AR (Arner, 2005). Normalmente l’azione lipolitica indotta dai recettori -AR prevale sull’azione -AR. Una continua stimolazione del sistema nervoso simpatico o un aumento delle catecolamine plasmatiche, è spesso associato alla desensitizzazione dei -AR (Mori et al., 2007). Studi in vivo hanno dimostrato che l’azione lipolitica delle catecolamine è ridotta nei soggetti obesi (Bougneres et al., 1997; Jensen, 1997). Il trattamento ripetuto con epinefrina induce soppressione della lipolisi, basale e indotta da epinefrina, sia in soggetti normopeso che obesi (Townsend et al., 1994). Anche negli studi in vitro la risposta lipolitica indotta dall’epinefrina è diminuita dal pretrattamento con la stessa molecola (Stallknecht et al., 1997). Sulla base di questi dati, è stato ipotizzato che l’aumentata massa adiposa dei topi iperadrenergici Chga-KO (Gayen et al., 2009a) possa rispecchiare la desensitizzazione dovuta all’aumento delle catecolamine circolanti (Mahapatra et al., 2005). I topi Chga-KO mostrano, nonostante gli elevati livelli circolanti di catecolamine e leptina, una notevole adiposità. Le catecolamine inibiscono la secrezione della leptina (Fritsche et al., 1998; Scriba et al., 2000; Couillard et al., 2002); la desensitizzazione del -AR potrebbe prevenire tale effetto, causando un aumento dei livelli di leptina e della massa adiposa, così come mostrato nei topi Chga-KO e in altri modelli sperimentali di obesità. Come osservato nei topi DIO (diet induced obesity), l’aumento di leptina circolante induce la desensitizzazione dei recettori per la leptina, possibile causa del fenotipo obeso dei topi Chga-KO. Sulla base di queste osservazioni, è stato ipotizzato che la CST possa ridurre l’obesità ripristinando la sensibilità dei recettori adrenergici e dei recettori per la leptina attraverso la normalizzazione dei livelli plasmatici di catecolamine e leptina. È stato infatti osservato che il trattamento cronico con CST induce una significativa riduzione della massa adiposa nei topi Chga-KO. Il trattamento con CST ha inoltre determinato una riduzione del peso corporeo e della massa adiposa anche nei topi DIO, senza alterare l’assunzione di cibo. Sia nei topi DIO che nei topi ob/ob, in cui l’obesità è dovuta all’incapacità di produrre leptina, la CST è in grado di incrementare gli effetti della leptina sul metabolismo e sul signaling del tessuto adiposo. Le nostre osservazioni suggeriscono che la riduzione della massa grassa dopo trattamento cronico con CST è dovuta ad un aumento della lipolisi e della mobilizzazione dei lipidi; inoltre sembra che la CST agisca attraverso i recettori 2-AR e i recettori per la leptina. In linea con tali osservazioni, la CST promuove l’ossidazione degli acidi grassi e il signaling della leptina.
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.
Livelli fisiologici della Cromogranina A esercitano potente effetto cardioprotettivo contro la cardiotossicità indotta da doxorubicina
(Università della Calabria, 2020-02-14) Granieri, Maria Concetta; Pasqua, Teresa; Angelone, Tommaso
The clinical use of doxorubicin (Doxo), a widely used anticancer chemotherapeutic drug, is limited by dose-dependent cardiotoxicity. We have investigated whether chromogranin A (CgA), a cardioregulatory protein released in the blood by the neuroendocrine system and by the heart itself, may contribute to the regulation of cardiotoxic and antitumor activities of Doxo. We evaluated whether and to which extent, the in vivo administration of physiological doses of recombinant full-length CgA may exert cardioprotection in a Doxo-induced cardiotoxicity rat model and modulate the anticancer activity of Doxo in murine models of melanoma, fibrosarcoma, lymphoma, and lung cancer, respectively. The effect of Doxo on circulating levels of CgA was also investigated. At the end of the treatment, hearts were perfused by Langendorff method and Ischemic protocols were used. Western Blot, Immunohistochemistry techniques, and ELISA assay were used for molecular analysis and plasma measurements of TNFα, IL-1β, ROS, LDH, cTnT and CgA. We found that CgA mitigated Doxo-dependent adverse effects, as revealed by the reduction of pro-inflammatory and cardiotoxic markers. Doxo reduced CgA plasma levels. When given together with Doxo, CgA increased the systolic function after ischemia and reduced the infarct size, compared to the Doxo group alone. Molecular analysis indicated that CgA reduced CTGF expression, induced activation of the RISK and SAFE pathways and of AMPK, and reduced inflammatory targets such as iNOS, COX2, NLRP3. CgA reduced intracardiac ROS and the expression of AOX-1 and XO. CgA-dependent cardioprotection was mediated by ARC activation, by the increase of Bcl2 and the reduction of BAX, Caspase3 and the apoptotic nuclei. Furthermore, we observed that Doxo reduces the intracardiac expression and release of CgA (i.e., an important cardioprotective agent) in the blood. On the other hand, CgA did not impair the anticancer activity of Doxo in all the investigated murine models. These data suggest that CgA could mitigate Doxo-induced cardiotoxicity by limiting ischemic injury. The protein can be proposed as a possible biomarker and the administration of exogenous CgA to patients with low levels of the endogenous protein might represent a novel approach to prevent Doxo-induced adverse events without impairing its antitumor effects.
RHEOLOGICAL STUDY OF BITUMINOUS ECOBASES FOR COLD RECYCLING
(Università della Calabria, 2024-06-26) Marchesano, Ylenia Maria; Angelone, Tommaso; Baldino, Noemi; Gabriele, Domenico
The environmental benefits of reduced toxic emissions, energy and economic savings, fewer and faster traffic disruptions, and increased safety for pavement operators make cold recycling pavement technologies far more advantageous than conventional reconstruction strategies. The recycling of asphalt mixes is therefore fundamental to a proper policy of natural resource conservation and "integrated" environmental design. Ecobases are an excellent cold recycling method because they allow the recycling of existing pavements, i.e. RAP, to be combined with the use of bituminous technology formulations, such as bituminous emulsions. Primarily composed of a bituminous and aqueous phase, these materials are undergoing ongoing research and development to enhance their mechanical performance, aiming to surpass that of traditional hot mix pavements. In addition, several studies, including this PhD thesis, are focused on identifying more environmentally friendly surfactants and achieving a higher pH for emulsion stability by using less acid and making the emulsion more stable, as well as on the identification of fillers capable of replacing those currently used, such as cement, which only provide stiffness, with substances capable of providing load-bearing capacity and flexibility at the same time, to resist the loads that road pavements must withstand today To this end, interfacial techniques have been demonstrated to be crucial. Interfacial instruments can measure the surfactant power of substances through interfacial tensions and therefore their ability to create emulsions. The -potential measurements and the observation in cylinders of the emulsions were fundamental for stability monitoring and were consistent with both the interfacial and bulk tests.
Ruolo di GPER sulla funzione cardiovascolare in condizioni di ipertensione: caratterizzazione fisiologica del recettore in Ratti Spontaneamente ipertesi (SHR)
(2013-11-25) De Francesco, Ernestina Marianna; Maggiolini, Marcello; Angelone, Tommaso
Estrogens promote beneficial effects in the cardiovascular system mainly through the estrogen receptor (ER)α and ERβ, which act as ligand-gated transcription factors. Recently, the G protein-coupled estrogen receptor (GPER) has been implicated in the estrogenic signaling in diverse tissues, including the cardiovascular system. In this study, we demonstrate that left ventricles of male Spontaneously Hypertensive Rats (SHR) express higher levels of GPER compared to normotensive Wistar Kyoto (WKY) rats. In addition, we show that the selective GPER agonist G-1 induces negative inotropic and lusitropic effects to a higher extent in isolated and Langendorff perfused hearts of male SHR compared to WKY rats. These cardiotropic effects elicited by G-1 involved the GPER/eNOS transduction signaling, as determined by using the GPER antagonist G-15 and the eNOS inhibitor L-NIO. Similarly, the G-1 induced activation of ERK1/2, AKT, GSK3β, c-Jun and eNOS was abrogated by G-15, while L-NIO prevented only the eNOS phosphorylation. In hypoxic Langendorff perfused WKY rat heart preparations, we also found an increased expression of GPER along with that of the hypoxic mediator HIF-1α and the fibrotic marker CTGF. Interestingly, G-15 and L-NIO prevented the ability of G-1 to down-regulate the expression of both HIF- 1α and CTGF, which were found expressed to a higher extent in SHR compared to WKY rat hearts. Collectively, the present study provides novel data into the potential role played by GPER in hypertensive disease on the basis of its involvement in myocardial inotropism and lusitropism as well as the expression of the apoptotic HIF-1α and fibrotic CTGF factors. Hence, GPER may be considered as a useful target in the treatment of some cardiac dysfunctions associated with stressful conditions like the essential hypertension.
Selenoprotein T as a novel cardiac modulator: from production to expression measurement and pathophysiological implications
(2018-03-02) Rocca, Carmine; Cerra, Maria Carmela; Angelone, Tommaso
The selenoprotein T-mimetic (PSELT) exerts cardiometabolic protection in rat and mouse models of obesity and metabolic syndrone
(Università della Calabria, 2022-01-27) De Bartolo, Anna; Angelone, Tommaso; Giordano, Francesca; Anouar, Youssef
Valorizzazione del Sargasso per la produzione di biometano in Repubblica Dominicana
(Università della Calabria, 2024-05-07) Paletta, Rosy; Angelone, Tommaso; Calabrò, Vincenza; Arcuri, Natale