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Modulation at transcriptional and post-translational level to develop therapeutic approaches for coagulation factor deficiencies
No full textNegli ultimi anni, sono stati fatti molti sforzi per lo sviluppo di approcci terapeutici per le malattie geniche. Questi approcci sono di grande interesse per i pazienti affetti da coagulopatie, in quanto possono trarre giovamento anche da un piccolo aumento di proteina funzionale.
Nel Capitolo 1 si descrive l’utilizzo di composti che mimano chaperoni molecolari come strategia terapeutica per i pazienti affetti da Emofilia B severa. Nell’Emofilia B la maggior parte delle mutazioni causanti malattia sono missenso, e possono essere motivo di cambiamenti strutturali della proteina. Le proteine mal ripiegate, possono aggregare in compartimenti intracellulari o essere degradati dal proteasoma, riducendone l’espressione. Tenendo conto del ruolo naturale dei chaperoni, i ricercatori hanno sviluppato diverse molecole chiamate chaperoni chimici e farmacologici. Queste molecole sono in grado di modulare il folding proteico e di ristabilire la sintesi delle proteine in presenza di mutazioni missenso.
Per questi motivi, abbiamo selezionato sei mutazioni missenso che causano Emofilia B di tipo I e abbiamo confermato che il nostro modello d’espressione fosse in grado di mimare il fenotipo clinico. Si è andati ad effettuare saggi di immunofluorescenza per valutare il trafficking proteico per confermare la ritenzione delle varianti all’interno del reticolo endoplasmatico.
Lo screening con i composti ha rivelato solo due candidati in grado di incrementare la secrezione proteica in maniera dose dipendente. Inoltre, da studi sull’attività abbiamo individuato una sola variante, la R294Q, avente un’attività specifica tale da poter ristabilire i livelli simili a quelli di un fenotipo clinico moderato in presenza del trattamento col composto Na-PBA.
Nel Capitolo II si descrive un approccio mediato da fattori trascrizionali ingegnerizzati fusi alla proteina Cas9 disattivata come strategia terapeutica per il deficit da FVII e l’Emofilia A. Inoltre, si è voluto comparare questo sistema con un altro, già usato precedentemente per il deficit da FVII, che utilizza proteine TALE (trascription activation-like effector) fuse ad attivatori trascrizionali.
Le mutazioni del promotore che inficiano la trascrizione rappresentano una piccola seppur considerevole causa dei difetti di fattori della coagulazione e di tutti i disordini genetici. In uno studio precedente, si è voluto studiare la capacità di proteine TALE di ristabilire la trascrizione in presenza di mutazioni del promotore. Per questo motivo, si è deciso di esplorare un nuovo sistema personalizzabile in grado di indurre la trascrizione, il sistema CRISPR activation. Per questo studio si è deciso di utilizzare il modello utilizzato per il precedente studio con le proteine TALE in modo da poter effettuare un confronto. I modelli dove sono stati testati i due sistemi sono il promotore wild-type del FVII e una mutazione severa del promotore (-61T>G), che ricade nel sito naturale di legame del fattore di trascrizione epato-specifico HFN-4. Tramite l’utilizzo vettori d’espressione contenenti geni reporter, abbiamo confrontato la proteina TALE risultata efficace dal precedente studio con un gRNA che abbiamo disegnato in modo da riconoscere la stessa sequenza sul promotore. I dati ottenuti dal confronto hanno mostrato una maggior efficienza del sistema CRISPRa nei saggi con il gene reporter, e, in maniera interessante, anche un aumento sulla proteina endogena FVII con questo sistema in confronto con la TALE.
Grazie alla versatilità del sistema CRISPRa abbiamo deciso di applicarlo anche al promotore del F8. Abbiamo disegnato sei diversi gRNA per esplorare una porzione di promotore. È stato interessante notare che in cellule trattate con una combinazione di due gRNA l’incremento dei livelli di reporter avviene in maniera additiva, dimostrando che la coppia di gRNA 1+2 sono capaci di raggiungere un incremento di 40 volte.In the last decades, enormous efforts have been pushed toward the development of therapeutic approaches for human genetic diseases. These approaches are of great interest for patients with coagulation disorders, since they would benefit from even a small increase in functional protein levels.
The Chapter 1 describes the use of chaperone-like compounds as a potential strategy for severe Haemophilia B patients. In Haemophilia B the majority of the disease-causing mutations are missense, and they can cause a structural change of the protein. Misfolded proteins can aggregate in intracellular compartments or be degraded from the proteasome in the unfolded protein response (UPR) pathway resulted in protein reduced expression. Taking into account molecular chaperones’ role, researchers developed different molecules called chemical and pharmacological chaperones. These molecules are able to modulate the protein folding and to restore the biosynthesis of proteins impaired by missense mutations. For all these reasons, we explored a therapeutic approach based on the treatment of cells expressing Factor IX missense mutations with chemical and pharmacological chaperones. We selected six missense mutations causing severe HB and first of all we confirmed that our expression model was able to mimic the clinical phenotype observed in patients (type I Haemophilia B <1% of secreted wild type protein). Were also performed immunofluorescence assays to evaluate the cellular trafficking of the missense variants and to demonstrate the retention into endoplasmic reticulum. Screening with chaperone-like compounds revealed that only two chemical chaperones were effective for our variants and were able to increase the secretion in dose dependent manner. The activity assays revealed that only one variant, the R294Q, has an encouraging activity for this type of approach and can be rescued to a moderate phenotype with Na-BA compound.
The Chapter 2 describes the usage of engineered transcription factors (eTFs) fused with a deactivated Cas9 (dCas9) as a potential strategy for FVII deficiency and for Haemophia A. Moreover, it was compared with another system, that we previously used for FVII deficiency, the transcription activator-like effector protein (TALE) fused to a transcription activator. Transcription impairment by promoter mutations represents a small but considerable cause of severe coagulation factors defects and of all genetic diseases. In a previous study, we explored the Transcription Activation-like Effectors (TALE). For this reason, we decided to explore a new and customizable system able to induce transcription activation, the CRISPR activation system (CRISPRa). We decided to use the FVII deficiency model in order to compare two different transcription activation systems in-vitro. As a model to exploit which of the two systems was more effective in restoring transcription, we chose to use the wild type F7 promoter and one severe mutation the -61 T>G, falling in the HNF-4 hepato-specific transcription activator binding site. Through the expression of gene reporter plasmids, we tested a TALE protein resulted effective in our previous work (TF4) and a gRNA that was designed to bind the same sequence. The data obtained from gene reporter assays showed a great efficacy of the CRISPRa system in comparison with TF4. Interestingly, the effect on the endogen FVII protein increase is higher in the cells treated with gRNAF7.5/dCas9-VPR than in the cells treated with TF4.
Due to the versatility of the CRISPR activation system and the effortlessness of realization we decided to explore this technique to F8 promoter. We designed different gRNAs able to explore a significant sequence near the transcription start site. Interestingly, in cells transfected with a combination of two gRNAs the reporter gene levels increase in an additive manner, reporting that the couple of gRNAs 1+2 were able to achieve an additive increment of about 40-fold
The carboxyl-terminal region of human coagulation factor X as a natural linker for fusion strategies
No full textFusion with human serum albumin (HSA), which represents a well-established technique to extend half-life of therapeutic proteins, commonly exploits intervening peptide linkers as key components. Here, we explored the human coagulation factor X (FX) carboxyl-terminal region, previously demonstrated by us to be dispensable for secretion and coagulant activity, as a natural linker for fusion purposes. To test our hypothesis, we compared direct FX-HSA fusion with the designed FX-HSA fusion proteins mimicking the recombinant activated factor VII (rFVIIa)-HSA or factor IX (FIX)-HSA chimeras, both strongly dependent from artificial linkers. Three constructs were produced by direct tandem fusion (FX-HSA) and through flexible (glycine/serine; FX-GS-HSA, mimicking rFVIIa-HSA) or cleavable (incorporating the FX activation site; FX-CL-HSA, mimicking FIX-HSA) linkers. The FX-HSA was efficiently secreted and displayed prolonged plasma persistence in mice. All chimeras possessed remarkable pro-coagulant activity, comparable to FX for FX-HSA (88.7 ± 6.0%) and FX-CL-HSA (98.0 ± 16.4%) or reduced for FX-GS-HSA (55.8 ± 5.4%). Upon incubation with activators, FX-HSA and FX-CL-HSA displayed a correct activation profile while the FX-GS-HSA activation was slightly defective. In fluorogenic-based assays, FX-HSA showed normal activity over time and a specific amidolytic activity (1.0 ± 0.12) comparable to that of FX. Overall, the FX-HSA features indicate that the FX carboxyl-terminal region represents an intrinsic sequence allowing direct tandem fusion. Our results provide the first experimental evidence for i) a coagulation factor fusion protein with biological properties independent from artificial linkers, ii) the suitability of FX carboxyl-terminal region as a natural linker for fusion purposes
An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes
No full textTailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations
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
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