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Altered mRNA processing and FVIII biosynthesis/function as determinants of phenotype variability in the frequent Arg2016Trp Haemophilia A patients.
No full textThe relationship between the molecular defect, the coagulation and the clinical phenotype in Haemophilia is often unclear. While the association of the so-called “null mutations” with severe forms is well-established, that of missense mutations, frequent in Haemophilia A (HA) and largely predominant in Haemophilia B (HB), with the phenotype is elusive. Missense mutations are candidate to affect protein biosynthesis/function but might also impair pre-mRNA splicing, an emerging mechanism whose pathophysiological impact is still poorly investigated. The interplay between these mechanisms might concur to factor VIII/IX levels and to HA/HB severity.
As an ideal model to investigate this issue, we propose the frequent F8 c.6046C>T mutation that has been found in severe and moderate HA patients with different degrees of bleeding tendency. The mutation is candidate to affect FVIII protein biology, and preliminary data suggest it alters F8 pre-mRNA splicing.
Aim
To elucidate the pathogenic mechanisms of the frequent F8 p.Arg2016Trp (c.6046C>T) mutation and define the underlying residual factor VIII levels, thus identifying determinants of phenotypic variability of Haemophilia A.
Expected results
By investigation at the protein and mRNA level in several p.Arg2016Trp patients and with recombinant FVIII variants we expect to define the contribution of impaired mRNA splicing and/or protein biosynthesis/function to FVIII expression. This will provide insights into a poorly known, and largely underestimated feature of missense mutations, which are frequent cause of Haemophilia and of the other rare coagulation factor disorders, and could boost the investigation of mRNA splicing patterns in patients with missense mutations to help interpretation of the related coagulation and clinical phenotypes.
We believe that our findings on the frequent F8 c.6046C>T mutation could help clinicians in the patient’s diagnosis and treatment
Molecular basis and therapeutic strategies to rescue pre-mRNA splicing defects in Haemophilia A, Familial Dysautonomia and Spinal Muscular Atrophy
No full textLo splicing dei pre-mRNA è un punto essenziale della regolazione dell’espressione genica il quale, controllato da complesse interazioni tra elementi in cis- ed in trans- che variano con le specie, tipi cellulari e durante lo sviluppo, può comportare la produzione di un grandissima varietà di isoforme proteiche a partire da un unico trascritto. Mutazioni o variazioni naturali all’interno delle sequenze codificanti e non-codificanti dei trascritti possono compromettere questo processo cellulare, con implicazione patologiche. Exon specific U1 snRNAs (ExSpeU1s) sono molecole di U1 snRNAs modificate per riconoscere in modo specifico le sequenze introniche che si trovano a valle di esoni negativamente influenzati da mutazioni. Gli ExSpeU1s sono stati recentemente proposti come molecole in grado di modificare lo splicing aberrante, correggendo i fenotipi patologici [1]. In questa tesi, per dimostrare l’applicabilità degli ExSpeU1s partendo da una caratterizzazione molecolare in-vitro alla valutazione della loro efficacia in-vivo, ho studiato lo splicing aberrante in: emofilia A, Disautonomia familiare ed atrofia muscolare spinale. L’emofilia A (HA) è una patologia della coagulazione causato da una deficienza del fattore della coagulazione VIII (FVIII). Tra le oltre 1300 mutazioni identificate in emofilia A, il cambio c.6046C>T (p.R2016W) rappresenta la seconda mutazione più frequente nel Nord Italia. Attraverso un sistema di minigeni ed una piattaforma lentivirale per l’espressione di FVIII ricombinante, ho dimostrato che la mutazione c.6046C>T ha un effetto sia a livello del processamento dell’RNA che su quello proteico del FVIII, rispettivamente inducendo un salto dell’esone 19 (≈30%) e riducendo sia la secrezione (11.0±0.4%) che l’attività (6.0±2.9%) del FVIII. Qui, ho caratterizzato un elemento regolatorio di splicing esonico (ESRE) all’interno dell’esone 19 del F8, identificando tre mutazioni missenso (c.6037G>A, p.G2013R; c.6053A>G, p.E2018G; c.6113A>G, p.N2038S) con un diverso impatto sulla secrezione/attività del FVIII e con un severo effetto sullo splicing dell’esone 19, inducendo un salto dell’esone superiore al 60%. Una combinazione di ridotta secrezione ed attività proteica con un’alterazione dello splicing ha prodotto un gradiente di deficit del FVIII, da una forma moderata ad una severa di emofilia A, anche per mutazioni raggruppate nell’esone 19. Interessatemene, ho dimostrato che lo splicing aberrante dell’esone 19 causato da queste mutazioni missenso può essere corretto da uno U1 snRNA modificato, stimolando l’inclusione dell’esone ed aumentando la quantità di trascritti completi. La Disautonomia familiare (FD) è una patologia caratterizzata da un difetto del sistema nervoso autonomo causato nella maggior parte dei casi (>99%) da una mutazione intronica (IVS20+6T>C) nel gene IKBKAP che induce un salto dell’esone 20 attraverso una riduzione dell’affinità tra lo U1 snRNA cellulare ed il sito donatore. Mediante l’uso di sistemi di minigeni, ho creato diversi ExSpeU1s che, riconoscendo la regione intronica a valle dell’esone 20, hanno efficientemente corretto lo splicing aberrante di IKBKAP in-vitro. Qui, ho inoltre dimostrato che questi ExSpeU1s promuovono una inclusione dell’esone 20 in modo attivo e non attraverso meccanismi antisenso. In aggiunta, sfruttando un sistema lentivirale per introdurre la molecola all’interno dei fibroblasti di pazienti FD, ho dimostrato l’efficacia di un ExSpeU1 in exvivo, fornendo una nuova opportunità terapeutica per il trattamento di questa patologia alla quale tuttora non esistono alternative. L’atrofia muscolare spinale (SMA) è una patologia che colpisce principalmente gli α-motoneuroni ed è causata da mutazioni nel gene di sopravvivenza dei motoneuroni 1 (SMN1) che codifica per la proteina SMN. Nello stesso cromosoma si trovano una o più copie del gene parologo (SMN2) che, tuttavia, contiene una mutazione sinonima (c.840C>T) che induce il salto dell’esone 7 di SMN2, riducendo l’espressione della proteina. In questa tesi, ho valutato l’efficacia in-vivo di due ExSpeU1s già caratterizzati per questa patologia [2]. Attraverso un virus adeno-associato 9, ho somministrato queste molecole per via intraperitoneale in modelli animali di SMA con fenotipo lieve e severo. Nel modello di topo SMA lieve, questi ExSpeU1s hanno efficientemente corretto lo splicing aberrante di SMN2, migliorando il fenotipo attraverso il recupero della lunghezza della coda. Nel modello di topo SMA severo, che normalmente muore dopo 10-12 giorni, ho dimostrato che il trattamento con ExSpeU1 estende significativamente la sopravvivenza con un 40% degli animali ancora vivi dopo 250 giorni. Complessivamente, questi risultati ottenuti in-vitro, ex-vivo ed in-vivo forniscono nuove conoscenze riguardanti le potenzialità terapeutiche basate su molecole di U1 snRNA modificate e forniscono le basi per ulteriori studi volti allo sviluppo di nuove terapie per patologie genetiche.Pre-mRNA splicing is an essential step of gene expression regulation, supervised by complex interactions between cis- and trans-acting factors that vary with species, celltypes and during development, which might dictate the synthesis of a multiplicity of different protein isoforms. Mutations or natural variations within both coding and noncoding sequences can affect this process with pathophysiological implications. Exon Specific U1 snRNAs (ExSpeU1s), modified U1 snRNAs that specifically bind to intronic sequences downstream affected exons, have been recently described as splicing-switching molecules able to revert pathological phenotypes [1]. In this thesis, in order to demonstrate the applicability of ExSpeU1s from the molecular characterization in-vitro to the evaluation of the efficacy in-vivo, I studied the misregulation of splicing in Hemophilia A, in Familial Dysautonomia, and in Spinal Muscular Atrophy. Hemophilia A (HA) is a bleeding disorder due to the deficiency of the blood coagulation factor VIII (FVIII). Among the over 1300 point mutations found in HA, the c.6046C>T (p.R2016W) substitution represents the second most frequent mutation in Northern Italy. Through a hybrid minigene system and a lentiviral platform to express recombinant FVIII variants, I demonstrated that the c.6046C>T mutation impacts both the RNA and protein FVIII biology , respectively promoting skipping of exon
(≈30%) and reducing both the FVIII secretion (11.0±0.4%) and activity (6.0±2.9%). I characterized the presence of an exonic splicing regulatory element (ESRE) within the F8 exon 19, identifying three missense mutations (c.6037G>A, p.G2013R; c.6053A>G, p.E2018G; c.6113A>G, p.N2038S) with differential impact on FVIII secretion/function and a severe impact on F8 exon 19 splicing, inducing >60% of exon skipping. Combination of reduced protein secretion and activity with splicing alteration produced a gradient of FVIII deficiency, from mild to severe HA, even for mutations clustered in
exon 19. Interestingly, I demonstrated that aberrant F8 exon 19 splicing caused by missense mutations can be improved by a modified U1 snRNA, promoting exon inclusion and increasing the amount of full length transcripts. Familial Dysautonomia (FD), characterized by impairment of the autonomous nervous system, is mainly caused (>99% of cases) by the intronic mutation IVS20+6T>C in the IKBKAP gene that promotes skipping of exon 20 through a reduced affinity between the wild type U1 snRNA and the donor site. With minigene assays I developed several ExSpeU1s that, targeting the intronic region downstream of the exon 20 donor site, efficiently correct the aberrant IKBKAP splicing in-vitro. Here, I also demonstrated that ExSpeU1s promote exon 20 inclusion in an active manner and not through antisense mechanisms. Moreover, taking advantage of a lentiviral delivery in FD patients’ fibroblasts, I demonstrated the ex-vivo efficacy of one ExSpeU1, providing a novel therapeutic opportunity to treat a disease for which there are no currently alternatives. Spinal Muscular Atrophy (SMA) primarily affects α-motor neurons and is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene encoding the SMN protein. Interestingly on the same chromosome is located one or more copies of its paralog (SMN2) that however carries a synonymous mutation (c.840C>T) promoting skipping of SMN2 exon 7, which reduces the SMN expression. In this thesis, I challenged the in-vivo efficacy of two already characterized ExSpeU1s for this disease [2]. Through an Adeno-associated virus 9, I intraperitoneally delivered these molecules in SMA animal models with a mild and a severe phenotype. In the mild SMA mouse the ExSpeU1s efficiently corrected the aberrant SMN2 splicing and improved the phenotype that consist of a recover of the tail lenght. In the severe SMA mouse, that normally die at 10-12 days, I demonstrated that treatment with ExSpeU1 significantly extend the survival (40% of animals alive after 250 days). Altogether these in-vitro, ex-vivo and in-vivo data provide novel insights into the potential of the RNA therapeutics based on modified U1 snRNAs and lay the foundation for further studies aimed at developing novel therapies for genetic disorders
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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