Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

Gesù continuatore di Giovanni. Studio storico-esegetico sulla relazione tra Gesù di Nazaret e Giovanni il Battista

L’enigma della relazione tra Gesù e Giovanni il Battista ha da sempre stimolato l’immaginazione storica degli studiosi, suscitando una varietà di ipotesi e valutazioni, spesso assai diverse. Ciò nonostante, tutti concordano su un punto: che, almeno nella sua fase maggiore in Galilea, il ministero di Gesù fosse una realtà essenzialmente autonoma, diversa, originale e irriducibile rispetto alla missione di Giovanni. In controtendenza con questa “impostazione predefinita”, il presente studio sostiene la tesi secondo cui Gesù portò avanti la sua missione come intenzionale e programmatica prosecuzione dell’opera prematuramente interrotta di Giovanni. Nella prima parte, si esamina approfonditamente quale memoria della relazione sia conservata nelle fonti più antiche, cioè Q (qui analizzata con particolare attenzione) e Marco – a cui si aggiunge Matteo, che, in ragione dello stretto legame storico-sociologico con Q, offre un esempio illuminante di rinarrazione della memoria altamente originale eppure profondamente fedele. La conclusione è che la memoria più antica della relazione Gesù-Giovanni è profondamente segnata da aspetti di accordo, conformità e allineamento. Nella seconda parte si esaminano una serie di tradizioni che attestano che Gesù era percepito pubblicamente in relazione al Battista e che egli stesso condivideva e alimentava tale percezione riallacciandosi a lui in polemica con i suoi avversari, e dipingendolo come una figura di capitale importanza nella predicazione e nell’insegnamento a seguaci e discepoli. Infine, si argomenta l’esistenza di ampie e sostanziali aree di accordo tra i due a livello di escatologia, istruzioni etiche e programma sociale, missione penitenziale verso i peccatori e attività battesimale. L’ipotesi che Gesù portasse avanti l’attività riformatrice di Giovanni, in termini di una campagna purificatoria “penitenziale-battesimale-esorcistica” in preparazione dell’avvento di Dio, consente infine di armonizzare in modo soddisfacente i due aspetti più caratteristici dell’attività di Gesù (normalmente giustapposti, quando non contrapposti): escatologia e miracoli, il Gesù profeta e il Gesù taumaturgo.

Proposta di traduzione italiano-spagnolo di alcuni capitoli tratti dal libro “Ricordi di scuola” di Giovanni Mosca

"Ricordi di scuola", il più celebre e personale tra i romanzi del giornalista, scrittore, umorista, nonché vignettista Giovanni Mosca, è il diario di un giovane maestro delle scuole elementari nella Roma fascista degli anni ’30. Il libro presenta uno stile semplice, fresco e lineare e racconta gli episodi di un maestro elementare e dei suoi alunni. La figura prevalente è sicuramente quella del maestro, un uomo “speciale” dalla personalità gentile, disponibile e per nulla severo, come ci si potrebbe aspettare, data l’epoca in cui il romanzo è ambientato, dimostrandosi comprensivo ed indulgente con i propri alunni senza però dimenticare la sua funzione educativa. Dal punto di vista della didattica il maestro Mosca si mostra diverso, quasi “rivoluzionario”. E’ un maestro che non dà voti e non boccia, che critica il nozionismo, i problemi astrusi e le poesie a memoria. Il racconto si compone di ventuno episodi, nei quali l’autore non nasconde la sua simpatia nei confronti degli alunni per la loro fantasia e la loro candida immaginazione e la sua ironia e la sua disapprovazione nei confronti degli adulti, di quei “grandi” che pensano di possedere la saggezza e la verità, ma che spesso si rivelano personaggi più immaturi e fragili dei loro stessi alunni. L’elaborato si compone di: introduzione al libro, traduzione di alcuni capitoli in lingua spagnola e analisi delle difficoltá traduttive incontrate durante la stesura.

Lo spazio del chiostro, progetto di restauro del chiostro di San Giovanni Battista a Faenza

Tesi in Restauro Architettonico del chiostro di San Giovanni Battista a Faenza. L'edificio è situato in Vicolo San Giovanni Battista nel centro storico di Faenza. L'intervento prevede un restauro scientifico delle murature esistenti, e la progettazione di un nuovo volume in aggiunta per risolvere i problemi di accessibilità. La fase progettuale si sviluppa su tre campi: il volume in aggiunta in cui collocare elementi di collegamento, la rampa interna al chiostro per superare le differenze di quota e l’intervento al piano superiore in cui sviluppare uno spazio funzionale ad un attività.

Symbolic Dynamics Analysis: a new methodology for foetal heart rate variability analysis

Cardiotocography (CTG) is a widespread foetal diagnostic methods. However, it lacks of objectivity and reproducibility since its dependence on observer's expertise. To overcome these limitations, more objective methods for CTG interpretation have been proposed. In particular, many developed techniques aim to assess the foetal heart rate variability (FHRV). Among them, some methodologies from nonlinear systems theory have been applied to the study of FHRV. All the techniques have proved to be helpful in specific cases. Nevertheless, none of them is more reliable than the others. Therefore, an in-depth study is necessary. The aim of this work is to deepen the FHRV analysis through the Symbolic Dynamics Analysis (SDA), a nonlinear technique already successfully employed for HRV analysis. Thanks to its simplicity of interpretation, it could be a useful tool for clinicians. We performed a literature study involving about 200 references on HRV and FHRV analysis; approximately 100 works were focused on non-linear techniques. Then, in order to compare linear and non-linear methods, we carried out a multiparametric study. 580 antepartum recordings of healthy fetuses were examined. Signals were processed using an updated software for CTG analysis and a new developed software for generating simulated CTG traces. Finally, statistical tests and regression analyses were carried out for estimating relationships among extracted indexes and other clinical information. Results confirm that none of the employed techniques is more reliable than the others. Moreover, in agreement with the literature, each analysis should take into account two relevant parameters, the foetal status and the week of gestation. Regarding the SDA, results show its promising capabilities in FHRV analysis. It allows recognizing foetal status, gestation week and global variability of FHR signals, even better than other methods. Nevertheless, further studies, which should involve even pathological cases, are necessary to establish its reliability.

I tre libri di salmi vespertini a otto voci (opp. I, VII e XI) di Giovanni Paolo Colonna

Le tre raccolte di salmi da vespro a otto voci nello stile pieno di Giovanni Paolo Colonna (Bologna 1637-1695), pubblicate rispettivamente nel 1681, nel 1686 e nel 1694 (opp.I, VII e XI), costituiscono un oggetto di studio privilegiato nell’ambito della ricca produzione a stampa dell’autore: in primo luogo, esse ebbero ampia e favorevole recezione, come testimoniano la diffusione degli esemplari, le ristampe, le copie manoscritte ricavate dalle edizioni; in secondo luogo, la fortuna postuma del compositore fu legata in buona parte alla sua musica sacra a doppio coro e, in particolare, al favore riscosso dai suoi libri di salmi; infine, l’analisi delle tre raccolte consente di confrontare le risorse tecniche e stilistiche messe in opera da Colonna in composizioni afferenti a uno stesso genere ma risalenti a periodi diversi. La dissertazione è articolata in tre parti: nella prima sono presi in esame gli ordinamenti liturgici secenteschi relativi alla celebrazione dei vespri, onde illustrare la cornice rituale alla quale i salmi di Colonna erano destinati, e si passano in rassegna alcune definizioni di stile pieno e di contrappunto a otto voci desunte dalla trattatistica coeva. La seconda parte è dedicata alla lettura storico-critica delle opp. I, VII e XI nel contesto generale della produzione dell’autore. La terza parte contiene l’edizione integrale dell’opera VII e XI, nonché una scelta di brani tratti dall’op. I.

Epigenetic role of N-Myc in Neuroblastoma

Childhood neuroblastoma is the most common solid tumour of infancy and highly refractory to therapy. One of the most powerful prognostic indicators for this disease is the N-Myc gene amplification, which occurs in approximately 25% of all neuroblastomas. N-Myc is a member of transcription factors belonging to a subclass of the larger group of proteins sharing Basic-Region/Helix–Loop–Helix/Leucin-Zipper (BR/HLH/LZ) motif. N-Myc oncoproteins may determine activation or repression of several genes thanks to different protein-protein interactions that may modulate its transcriptional regulatory ability and therefore its potential for oncogenicity. Chromatin modifications, including histone methylation, have a crucial role in transcription de-regulation of many cancer-related genes. Here, it was investigated whether N-Myc can functionally and/or physically interact with two different factors involved in methyl histone modification: WDR5 (core member of the MLL/Set1 methyltransferase complex) and the de- methylase LSD1. Co-IP assays have demonstrated the presence of both N-Myc-WDR5 and N-Myc-LSD1 complexes in two neuroblastoma cell lines. Human N-Myc amplified cell lines were used as a model system to investigate on transcription activation and/or repression mechanisms carried out by N-Myc-LSD1 and N-Myc-WDR5 protein complexes. qRT-PCR and immunoblot assays underlined the ability of both complexes to positively (N-Myc-WDR5) and negatively (N-Myc-LSD1) influence transcriptional regulation of crititical neuroblastoma N-Myc-related genes, MDM2, p21 and Clusterin. Ch-IP experiments have revealed the binding of the N-Myc complexes above mentioned to the gene promoters analysed. Finally, pharmacological treatment pointed to abolish N-Myc and LSD1 activity were performed to test cellular alterations, such as cell viability and cell cycle progression. Overall, the results presented in this work suggest that N-Myc can interact with two distinct histone methyl modifiers to positively and negatively affect gene transcription in neuroblastoma.

Mitral regurgitation

Mitral regurgitation (MR) involves systolic retrograde flow from the left ventricle into the left atrium. While trivial MR is frequent in healthy subjects, moderate to severe MR constitutes the second most prevalent valve disease after aortic valve stenosis. Major causes of severe MR in Western countries include degenerative valve disease (myxomatous disease, flail leaflet, annular calcification) and ischaemic heart disease, while rheumatic disease remains a major cause of MR in developing countries. Chronic MR typically progresses insidiously over many years. Once established, however, severe MR portends a poor prognosis. The severity of MR can be assessed by various techniques, Doppler echocardiography being the most widely used. Mitral valve surgery is the only treatment of proven efficacy. It alleviates clinical symptoms and prevents ventricular dilatation and heart failure (or, at least, it attenuates further progression of these abnormalities). Valve repair significantly improves clinical outcomes compared with valve replacement, reducing mortality by approximately 70%. Reverse LV remodelling after valve repair occurs in half of patients with functional MR. Percutaneous, catheter-based to mitral valve repair is a novel approach currently under clinical scrutiny, with encouraging preliminary results. This modality may provide a valuable alternative to mitral valve surgery, especially in critically ill patients.

Evaluation of administration of isoflurane at approximately the minimum alveolar concentration on depression of a nociceptive withdrawal reflex evoked by transcutaneous electrical stimulation in ponies

OBJECTIVE: To investigate effects of isoflurane at approximately the minimum alveolar concentration (MAC) on the nociceptive withdrawal reflex (NWR) of the forelimb of ponies as a method for quantifying anesthetic potency. ANIMALS: 7 healthy adult Shetland ponies. PROCEDURE: Individual MAC (iMAC) for isoflurane was determined for each pony. Then, effects of isoflurane administered at 0.85, 0.95, and 1.05 iMAC on the NWR were assessed. At each concentration, the NWR threshold was defined electromyographically for the common digital extensor and deltoid muscles by stimulating the digital nerve; additional electrical stimulations (3, 5, 10, 20, 30, and 40 mA) were delivered, and the evoked activity was recorded and analyzed. After the end of anesthesia, the NWR threshold was assessed in standing ponies. RESULTS: Mean +/- SD MAC of isoflurane was 1.0 +/- 0.2%. The NWR thresholds for both muscles increased significantly in a concentration-dependent manner during anesthesia, whereas they decreased in awake ponies. Significantly higher thresholds were found for the deltoid muscle, compared with thresholds for the common digital extensor muscle, in anesthetized ponies. At each iMAC tested, amplitudes of the reflex responses from both muscles increased as stimulus intensities increased from 3 to 40 mA. A concentration-dependent depression of evoked reflexes with reduction in slopes of the stimulus-response functions was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Anesthetic-induced changes in sensory-motor processing in ponies anesthetized with isoflurane at concentrations of approximately 1.0 MAC can be detected by assessment of NWR. This method will permit comparison of effects of inhaled anesthetics or anesthetic combinations on spinal processing in equids.

Antimicrobial therapy using a local drug delivery system (Arestin) in the treatment of peri-implantitis. I: Microbiological outcomes

OBJECTIVES: To assess the microbiological outcome of local administration of minocycline hydrochloride microspheres 1 mg (Arestin) in cases with peri-implantitis and with a follow-up period of 12 months. MATERIAL AND METHODS: After debridement, and local administration of chlorhexidine gel, peri-implantitis cases were treated with local administration of minocycline microspheres (Arestin). The DNA-DNA checkerboard hybridization method was used to detect bacterial presence during the first 360 days of therapy. RESULTS: At Day 10, lower bacterial loads for 6/40 individual bacteria including Actinomyces gerensceriae (P<0.1), Actinomyces israelii (P<0.01), Actinomyces naeslundi type 1 (P<0.01) and type 2 (P<0.03), Actinomyces odontolyticus (P<0.01), Porphyromonas gingivalis (P<0.01) and Treponema socranskii (P<0.01) were found. At Day 360 only the levels of Actinobacillus actinomycetemcomitans were lower than at baseline (mean difference: 1x10(5); SE difference: 0.34x10(5), 95% CI: 0.2x10(5) to 1.2x10(5); P<0.03). Six implants were lost between Days 90 and 270. The microbiota was successfully controlled in 48%, and with definitive failures (implant loss and major increase in bacterial levels) in 32% of subjects. CONCLUSIONS: At study endpoint, the impact of Arestin on A. actinomycetemcomitans was greater than the impact on other pathogens. Up to Day 180 reductions in levels of Tannerella forsythia, P. gingivalis, and Treponema denticola were also found. Failures in treatment could not be associated with the presence of specific pathogens or by the total bacterial load at baseline. Statistical power analysis suggested that a case control study would require approximately 200 subjects.

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.