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Il locus CYYR1 identificato e clonato sul cromosoma 21 umano è stato caratterizzato dal punto di vista molecolare come un sistema multitrascritto, esclusivo dei vertebrati che ad oggi è orfano di una funzione specifica. Dati presenti in lettura e rintracciati mostrano una possibile relazione tra il gene CYYR1 e il pathway di Sonic Hedgehog (SHH). In questo progetto di tesi è stato utilizzato il modello animale Danio rerio per indagare il ruolo funzionale dell’ortologo (cyyr1), attraverso esperimenti di gain e loss of function che hanno permesso di dimostrare un suo coinvolgimento nello sviluppo del sistema nervoso centrale, del cuore e del tessuto muscolare. Lo studio dell’ortologo in zebrafish è stato associato all’utilizzo di linee cellulari di rabdomiosarcoma umano. I risultati ottenuti dall’induzione al differenziamento miogenico di queste linee, insieme ai dati ottenuti in Danio rerio, confermano il possibile coinvolgimento del gene CYYR1 nella miogenesi. Lo studio delle relazione tra il pathway di SHH e l’espressione del gene CYYR1 è stato condotto in entrambi i modelli con l’utilizzo di differenti inibitori della via di segnalazione. I risultati ottenuti mostrano che sistemi inibitori agenti direttamente sul recettore SMO riducono l’espressione del gene. Un dato inaspettato in Danio rerio ottenuto durante questi esperimenti di inibizione, ha aperto una nuova linea di ricerca in collaborazione con l’Università di Warwick tesa a verificare la relazione tra il gene cyyr1 e il gene lefty1. Gli esperimenti condotti presso il laboratorio della Prof.ssa Sampath hanno dimostrato la localizzazione del prodotto proteico cyyr1 in Danio rerio e indagato co-localizzazioni con la proteina lefty1. Infine, in collaborazione con Dr. Deflorian e della Prof.ssa Pistocchi, è stato generato un mutante di Danio rerio deleto per il gene cyyr1 con la tecnica CRISPR/Cas9. La caratterizzazione del mutante cyyr1 -/- ha confermato alcuni dei dati ottenuti attraverso esperimenti di loss of function., CYYR1 (Cysteine/tyrosine-rich 1) cloned on HC21 defines a new family of highly conserved vertebrate-specific genes. The human locus is characterized by a multitranscript-system including alternative spliced isoforms and one ncRNA gene overlapped in antisense orientation. To date, the function of the CYYR1 product is still unknown even if original results suggest the need of further investigations in order to verify a putative role of CYYR1 in the tumorigenic process of rhabdomyosarcoma, caused by dysfunction of cell differentiation. We perform a full characterization of cyyr1 expression and function in zebrafish model; the gene is present in single copy and the predicted protein maintains almost 58% of identity with human protein. A broad expression in central nervous system (CNS), somites and muscles is demonstred by WISH approach during development up to 24-48 hpf. The cyyr1 knock-down with two different MOs targetting the ATG and the first splice-site of the transcript, affected both CNS and muscle development with a significant rescue in embryo co-injected with cyyr1 mRNA. Defects were also evident in ciliated cells of neuromast of the lateral line. Morphologically the cyyr1-MOs injected embryos display features of embryos inhibited for the Hh pathway through injection of the lefty mRNA, moreover cyyr1 expression was significantly inhibited following Hh inhibition. Interestingly, the injection of cyyr1 mRNA was able to partially rescue Hh-defective phenotype in embryos at 24 hpf. Results from immunofluorescent staining, qPCR and western blotting, support a role for cyyr1 in primary myogenesis probably downstream of Hh pathway. The analysis of CYYR1 expression in rhabdomyosarcoma cell lines seems to indicate an inverse correlation between CYYR1 expression and the range of differentiating capabilities of these cells. These data have encouraged us to better investigate the possible interplay between cyyr1 and Hh-mediated myogenesis even in cyyr1 editing conditions and, as well in rhabdomyosarcoma cell model.

CYYR1 (Cysteine/tyrosine-rich 1) cloned on human chromosome 21 defines a new family of highly conserved vertebrate-specific genes1,2. The analysis of the human locus revealed the presence of a multitranscript-system that includes alternative spliced isoforms and one ncRNA gene overlapping CYYR1 in antisense orientation3. Original results suggest the need of further investigations in order to verify a putative role of CYYR1 in the tumorigenic process, caused by dysfunction of cell differentiation and possibly related to the Hh pathway4; to date, the specific function of the CYYR1 product is still unknown. The zebrafish cyyr1 is present in single copy and maintains almost 58% of identity with human protein therefore, we decided to perform a full characterization of cyyr1 expression and function using zebrafish as model system. WISH approach defined a broad expression in central nervous system (CNS), somites and muscles during somitogenesis and at 24-48 hpf. The cyyr1 knock-down with two different MOs targetting the ATG and the first splice-site of the transcript, affected both CNS and muscle development with a significant rescue in embryo co-injected with cyyr1 mRNA. Defects were also evident in ciliated cells of neuromast of the lateral line. Morphologically, the cyyr1-MOs injected embryos display some features of embryos inhibited for the Hh pathway through injection of the lefty mRNA and cyyr1 expression was significantly inhibited following Hh inhibition. Interestingly, the injection of cyyr1 mRNA was able to partially rescue Hh-defective phenotype in embryos at 24 hpf. Results obtained through immunofluorescent staining, qPCR and western blotting, support a role for cyyr1 in primary myogenesis probably downstream of Hh pathway. 1. Vitale L et al. Gene 2002, 290:141-51. 2. Casadei R et al. Gene Expr Patterns 2011, 11: 271-6. 3. Casadei R et al. Mol Biol Rep 2014, 41:6025-38. 4. Xu J et al. Genetics 2006, 174:735-52.

OBJECTIVESWe sought to determine whether the clinical effects of early angiotensin-converting enzyme (ACE) inhibitor (ACEi) treatment for acute myocardial infarction (MI) are influenced by the concomitant use of aspirin (ASA).BACKGROUNDAspirin and ACEi both reduce mortality when given early after MI. Aspirin inhibits the synthesis of vasodilating prostaglandins, and, in principle, this inhibition might antagonize some of the effects of ACEi. But it is uncertain whether, in practice, this influences the effects of ACEi on mortality and major morbidity after MI.METHODSThis overview sought individual patient data from all trials involving more than 1,000 patients randomly allocated to receive ACEi or control starting in the acute phase of MI (0–36 h from onset) and continuing for four to six weeks. Data on concomitant ASA use were available for 96,712 of 98,496 patients in four eligible trials (and for none of 1,556 patients in the one other eligible trial).RESULTSOverall 30-day mortality was 7.1% among patients allocated to ACEi and 7.6% among those allocated to control, corresponding to a 7% (standard deviation [SD], 2%) proportional reduction (95% confidence interval 2% to 11%, p = 0.004). Angiotensin-converting enzyme inhibitor was associated with similar proportional reductions in 30-day mortality among the 86,484 patients who were taking ASA (6% [SD, 3%] reduction) and among the 10,228 patients who were not (10% [SD, 5%] reduction: chi-squared test of heterogeneity between these reductions = 0.4; p = 0.5). Angiotensin-converting enzyme inhibitor produced definite increases in the incidence of persistent hypotension (17.9% ACEi vs. 9.4% control) and of renal dysfunction (1.3% ACEi vs. 0.6% control), but there was no good evidence that these effects were different in the presence or absence of ASA (chi-squared for heterogeneity = 0.4 and 0.0, respectively; both not significant). Nor was there good evidence that the effects of ACEi on other clinical outcomes were changed by concomitant ASA use.CONCLUSIONSBoth ASA and ACEi are beneficial in acute MI. The present results support the early use of ACEi in acute MI, irrespective of whether or not ASA is being given.

Parkinson’s disease (PD) is one of the most common progressive neurodegenerative diseases. Clinical and epidemiological studies indicate that sex differences, as well as genetic components and ageing, can influence the prevalence, age at onset and symptomatology of PD. This study undertook a systematic meta-analysis of substantia nigra microarray data using the Transcriptome Mapper (TRAM) software to integrate and normalize a total of 10 suitable datasets from multiple sources. Four different analyses were performed according to default parameters, to better define the segments differentially expressed between PD patients and healthy controls, when comparing men and women data sets. The results suggest a possible regulation of specific sex-biased systems in PD susceptibility. TRAM software allowed us to highlight the different activation of some genomic regions and loci involved in molecular pathways related to neurodegeneration and neuroinflammatory mechanisms.