Lorena Zentilin

International Centre for Genetic Engineering and Biotechnology, Trst, Friuli Venezia Giulia, Italy

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Publications (104)651.29 Total impact

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    ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
    No preview · Article · Dec 2015 · Proceedings of the National Academy of Sciences

  • No preview · Article · Dec 2015 · Journal of Tissue Engineering
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    ABSTRACT: Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.
    No preview · Article · Aug 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at around three weeks after injection and persisting for almost one year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified mAb 14F7.Gene Therapy accepted article preview online, 16 July 2015. doi:10.1038/gt.2015.71.
    Full-text · Article · Jul 2015 · Gene therapy
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    ABSTRACT: Vascular endothelial growth factor (VEGF)-B activates cytoprotective/antiapoptotic and minimally angiogenic mechanisms via VEGF receptors. Therefore, VEGF-B might be an ideal candidate for the treatment of dilated cardiomyopathy, which displays modest microvascular rarefaction and increased rate of apoptosis. This study evaluated VEGF-B gene therapy in a canine model of tachypacing-induced dilated cardiomyopathy. Chronically instrumented dogs underwent cardiac tachypacing for 28 days. Adeno-associated virus serotype 9 viral vectors carrying VEGF-B167 genes were infused intracoronarily at the beginning of the pacing protocol or during compensated heart failure. Moreover, we tested a novel VEGF-B167 transgene controlled by the atrial natriuretic factor promoter. Compared with control subjects, VEGF-B167 markedly preserved diastolic and contractile function and attenuated ventricular chamber remodeling, halting the progression from compensated to decompensated heart failure. Atrial natriuretic factor-VEGF-B167 expression was low in normally functioning hearts and stimulated by cardiac pacing; it thus functioned as an ideal therapeutic transgene, active only under pathological conditions. Our results, obtained with a standard technique of interventional cardiology in a clinically relevant animal model, support VEGF-B167 gene transfer as an affordable and effective new therapy for nonischemic heart failure. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Journal of the American College of Cardiology
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    ABSTRACT: Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.
    Full-text · Article · Jun 2015 · Nature Communications
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    ABSTRACT: Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRed (loxP/loxP) Cripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury.
    Full-text · Article · May 2015 · Frontiers in Cell and Developmental Biology
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    ABSTRACT: The Neuregulin/ErbB system plays an important role in the peripheral nervous system both in normal and pathological conditions. We previously demonstrated that expression of soluble ecto-ErbB4, the released extracellular fragment of the ErbB4 receptor, stimulated glial cell migration in vitro. In this study we examined the possibility to manipulate this system in vivo in order to improve injured peripheral nerve regeneration. Transected rat median nerves of adult female Wistar rats were repaired with a 10-mm-long graft made by muscle-in-vein combined nerve guide previously transduced with either the adeno-associated viral (AAV) vector AAV2-LacZ or AAV2-ecto-ErbB4. Autologous nerve grafts were used as control. Both stereological and functional analysis were performed to assess nerve regeneration. Data show that delivery of soluble ecto-ErbB4 by gene transfer in the muscle-in-vein combined nerve guide has a positive effect on fiber maturation, suggesting that it could represent a potential tool to improve peripheral nerve regeneration.Gene Therapy accepted article preview online, 04 May 2015. doi:10.1038/gt.2015.46.
    No preview · Article · May 2015 · Gene therapy
  • Lorena Zentilin · Rudy Ippodrino · Miguel Mano · Mauro Giacca

    No preview · Conference Paper · May 2015
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    ABSTRACT: Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.
    Full-text · Article · Mar 2015 · Oncotarget
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    ABSTRACT: Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca(2+)], which control aerobic metabolism, cell death, and survival pathways. We investigated in vivo the effects of mitochondrial Ca(2+) homeostasis in skeletal muscle function and trophism by overexpressing or silencing the mitochondrial calcium uniporter (MCU). The results demonstrate that in both developing and adult muscles, MCU-dependent mitochondrial Ca(2+) uptake has a marked trophic effect that does not depend on aerobic control but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-Akt/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca(2+)-dependent organelle-to-nucleus signaling route that links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Cell Reports
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    ABSTRACT: Although in the last decades the molecular underpinnings of the cell cycle have been unraveled, the acquired knowledge has been rarely translated into practical applications. Here, we investigate the feasibility and safety of triggering proliferation in vivo by temporary suppression of the cyclin-dependent kinase inhibitor, p21. AAV-mediated, acute knockdown of p21 in intact skeletal muscles elicited proliferation of multiple, otherwise quiescent cell types, notably including satellite cells. Compared with controls, p21-suppressed muscles exhibited a striking two-threefold expansion in cellularity and increased fiber numbers by 10 days posttransduction, with no detectable inflammation. These changes partially persisted for at least 60 days, indicating that the muscles had undergone lasting modifications. Furthermore, morphological hyperplasia was accompanied by 20% increases in maximum strength and resistance to fatigue. To assess the safety of transiently suppressing p21, cells subjected to p21 knockdown in vitro were analyzed for γ-H2AX accumulation, DNA fragmentation, cytogenetic abnormalities, ploidy, and mutations. Moreover, the differentiation competence of p21-suppressed myoblasts was investigated. These assays confirmed that transient suppression of p21 causes no genetic damage and does not impair differentiation. Our results establish the basis for further exploring the manipulation of the cell cycle as a strategy in regenerative medicine.Molecular Therapy (2015); doi:10.1038/mt.2015.27.
    No preview · Article · Feb 2015 · Molecular Therapy
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    ABSTRACT: Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. Production of the rAAV8RSM was carried out using transient transfection and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to sixteen laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50x1011 pt/ml; CI, 4.26x1011 to 6.75x1011 pt/ml), vector genomes (mean, 5.75x1011 vg/ml; CI, 3.05x1011 to 1.09x1012 vg/ml), and infectious units (mean, 1.26x109 IU/ml; CI, 6.46x108 to 2.51x109 IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time where the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
    Full-text · Article · Oct 2014 · Human Gene Therapy
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    ABSTRACT: Notch signalling plays an important role in synaptic plasticity, learning and memory functions in both Drosophila and rodents. In this paper, we report that this feature is not restricted to hippocampal networks but also involves the olfactory bulb (OB). Odour discrimination and olfactory learning in rodents are essential for survival. Notch1 expression is enriched in mitral cells of the mouse OB. These principal neurons are responsive to specific input odorants and relay the signal to the olfactory cortex. Olfactory stimulation activates a subset of mitral cells, which show an increase in Notch activity. In Notch1cKOKln mice, the loss of Notch1 in mitral cells affects the magnitude of the neuronal response to olfactory stimuli. In addition, Notch1cKOKln mice display reduced olfactory aversion to propionic acid as compared to wildtype controls. This indicates, for the first time, that Notch1 is involved in olfactory processing and may contribute to olfactory behaviour.
    No preview · Article · Sep 2014 · European Journal of Neuroscience
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    ABSTRACT: Rationale: The Notch pathway plays a key role in stimulating mammalian cardiomyocyte proliferation during development and in the early postnatal life; in adult zebrafish, reactivation of this pathway is also essential to drive cardiac regeneration after injury. Objective: We wanted to assess efficacy of Notch pathway stimulation in neonatal and adult hearts as a means to induce cardiac regeneration after myocardial infarction in mice. Methods and results: In early postnatal life, cardiomyocyte exit from the cell cycle was paralleled by decreased Notch signaling and the establishment of a repressive chromatin environment at Notch-responsive genes, characterized by recruitment of the polycomb group enhancer of zeste homolog 2 methyltransferase and the acquisition of the histone 3 Lysine 27 trimethylation histone mark, as detected by chromatin immunoprecipitation. Forced Notch pathway activation by adenoassociated virus gene transfer of activated Notch1 or its ligand Jagged1 expanded the proliferative capacity of neonatal cardiomyocytes; this correlated with increased transcription of Notch target genes and maintenance of an open chromatin conformation at their promoters. The same adenoassociated virus vectors, however, were largely ineffective in stimulating cardiac repair after myocardial infarction in adult mice, despite optimal and long-lasting transgene expression. Analysis of Notch-responsive promoters in adult cardiomyocytes showed marks of repressed chromatin and irreversible CpG DNA methylation. Induction of adult cardiomyocyte re-entry into the cell cycle with microRNAs was independent from Notch pathway reactivation. Conclusions: Notch pathway activation is crucial in regulating cardiomyocyte proliferation during the early postnatal life, but it is largely ineffective in driving cardiac regeneration in adults, because of permanent epigenetic modification at Notch-responsive promoters.
    Preview · Article · Aug 2014 · Circulation Research
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    ABSTRACT: Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies we applied AAV-liver specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months post-injection. The therapeutic effect was mediated by the presence of transcriptionally active double stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver vs. skeletal muscle transgene expression. We observed that 5%-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dL). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dL), despite 20-30% of hUgt1a1 expression levels, compared to normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.
    Full-text · Article · Jul 2014 · Human Gene Therapy
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    ABSTRACT: Purpose. Familial cardiomyopathies are caused by genetic mutations that induce accumulation of misfolded proteins with consequent cardiomyocyte death and maladaptive cardiac remodelling. Molecular chaperones are a family of proteins devoted to prevent accumulation of misfolded proteins by promoting either their refolding or degradation via the ubiquitin-proteasome or the autophagosome systems and can thus represent a potential mean to treat familial cardiomyopathies. Methods. Melusin is a muscle specific chaperone protein whose overexpression effectively prevents maladaptive cardiac remodeling and heart failure both in pressure overload and myocardial infarct mouse models. In this study we use in vivo gene delivery with cardiotropic adeno associated virus 9 vector to induce to increase melusin expression in the heart of mice carrying H222P Lamin A mutation mimicking human Emery-Dreifuss familial cardiomyopathy. Mutant male mice develop spontaneous dilated cardiomyopathy from 4 months of age and die within 10-12 months of age. Results. Homozygous mutant mice were injected with AAV9-Melusin (I.V. injection of 1012 viral particles/mouse) either at 1 month of age before development of the cardiomyopathy or at 4 months when cardiomyopathy was already present to test for both preventive and therapeutic activity. Cardiac function was monitored by echocardiography for the following months. While untreated mice progressively developed left ventricle dilation and reduced contractility (FS%), mice injected with AAV9-Melusin retained physiological level of contractility and were protected toward LV dilation. Ten months after the treatment, 40% of mutant mice died, while 100% of melusin-treated mice were alive. A second group of mutant male mice was injected with AAV9-melusin at 4 months of age when the cardiomyopathy was already detectable. Interestingly melusin prevented the deterioration of contractility in the following 6 months and significantly reduced mortality. Conclusions These data indicate that melusin chaperone overexpression can effectively delay the onset of Emery-Dreifuss cardiomyopathy as well as arrest the progression of the already established pathology.
    Preview · Article · Jul 2014 · Cardiovascular Research
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    ABSTRACT: Unlabelled: Aberrant DNA replication induced by deregulated or excessive proliferative stimuli evokes a "replicative stress response" leading to cell cycle restriction and/or apoptosis. This robust fail-safe mechanism is eventually bypassed by transformed cells, due to ill-defined epistatic interactions. The COP9 signalosome (CSN) is an evolutionarily conserved regulator of cullin ring ligases (CRLs), the largest family of ubiquitin ligases in metazoans. Conditional inactivation of the CSN in several tissues leads to activation of S- or G2-phase checkpoints resulting in irreversible cell cycle arrest and cell death. Herein we ablated COPS5, the CSNs catalytic subunit, in the liver, to investigate its role in cell cycle reentry by differentiated hepatocytes. Lack of COPS5 in regenerating livers causes substantial replicative stress, which triggers a CDKN2A-dependent genetic program leading to cell cycle arrest, polyploidy, and apoptosis. These outcomes are phenocopied by acute overexpression of c-Myc in COPS5 null hepatocytes of adult mice. Conclusion: We propose that combined control of proto-oncogene product levels and proteins involved in DNA replication origin licensing may explain the deleterious consequences of CSN inactivation in regenerating livers and provide insight into the pathogenic role of the frequently observed overexpression of the CSN in hepatocellular carcinoma.
    Full-text · Article · Jun 2014 · Hepatology
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    Serena Zacchigna · Lorena Zentilin · Mauro Giacca
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    ABSTRACT: The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.
    Preview · Article · May 2014 · Circulation Research
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    Preview · Article · Apr 2014 · Journal of the American College of Cardiology

Publication Stats

3k Citations
651.29 Total Impact Points

Institutions

  • 1992-2015
    • International Centre for Genetic Engineering and Biotechnology
      • Molecular Medicine Laboratory
      Trst, Friuli Venezia Giulia, Italy
  • 2005-2014
    • Università degli Studi di Trieste
      Trst, Friuli Venezia Giulia, Italy
  • 2004
    • Scuola Normale Superiore di Pisa
      Pisa, Tuscany, Italy
  • 1994-1996
    • IRCCS Ospedale Infantile Burlo Garofolo
      • Department of Pediatrics
      Trst, Friuli Venezia Giulia, Italy
    • University of Florence
      • Dipartimento di Scienze Biomediche, Sperimentali e Cliniche
      Florens, Tuscany, Italy