1,720,964 research outputs found
La Displasia Fibrosa in modelli in vitro e in vivo
No full textLa displasia fibrosa (DF) è una malattia genetica dell’osso e del midollo osseo causata da mutazioni missenso attivanti nel gene codificante per la subunità α della proteina G stimolatoria, Gs (Gsα). Fratture patologiche, deformità e dolori ossei rappresentano comunemente l’espressione clinica della malattia, correlata a sostituzione di osso normale e midollo osseo con tessuto abnorme, a carente mineralizzazione ed instabilità dell’osso, a midollo osseo fibrotico e non ematopoietico. Tali anomalie dipendono dalla disfunzione delle cellule che formano l’osso (osteoblasti), causata dalla presenza della mutazione nelle cellule stesse e nei loro progenitori, le cellule stromali del midollo osseo (BMSC).
Ad oggi, non sono note le modificazioni molecolari generate dalla mutazione, né quale sia il contributo dei diversi tipi cellulari al fenotipo malattia, né è disponibile una cura efficace per la DF. Per definire esaustivamente a livello molecolare, cellulare ed organismico gli eventi fisiopatologici della DF, e per identificare nuove strategie terapeutiche, abbiamo generato e studiato modelli di DF in vitro e in vivo.
Per analizzare la modulazione trascrizionale indotta dalla mutazione attivante di Gsα (R201C), abbiamo esaminato con i microarray il profilo di espressione di BMSC umane, ingegnerizzate per esprimere stabilmente la mutazione GsαR201C. L’analisi interpretativa dei dati di microarray ha evidenziato la modulazione di geni che sottendono i fondamentali cambiamenti tissutali osservati nella DF, tra cui MGP (artefice della sotto-mineralizzazione dell’osso) e RANKL (responsabile dell’eccessivo riassorbimento osseo), entrambi possibili bersagli terapeutici.
D’altra parte, per valutare il contributo di specifiche popolazioni cellulari al fenotipo malattia abbiamo generato modelli murini che consentono l’espressione tessuto-specifica di GsαR201C. In particolare, abbiamo prodotto topi con l’espressione della GsαR201C confinata alle cellule murali, ossia i progenitori scheletrici intesi come cellule microvascolari. L’analisi ai raggi X di questi animali ha messo in luce un’alterazione radiografica delle ossa femorali dei topi, suggerendo che l’espressione della Gsα mutata nelle cellule murali causi anormalità del tessuto scheletrico. Inoltre, per ottenere nuovi modelli murini tessuto-specifici, abbiamo prodotto topi condizionali per l’espressione tessuto-specifica della GsαR201C (Lox-Stop-Lox-GsαR201C). Lo studio radiografico di questi animali ha confermato l’assenza di anomalie ossee. Questi animali potranno essere incrociati con topi che esprimano la ricombinasi Cre nei diversi tessuti di interesse, per ottenere un’ ampia gamma di topi GsαR201C tessuto-specifici.
Nel suo insieme, questo lavoro è stato importante per l’identificazione di possibili bersagli terapeutici, ha contribuito a definire l’istopatogenesi molecolare della DF, e, in particolare, dall’uso/analisi di topi GsαR201C tessuto-specifici, potrà derivare un’ulteriore caratterizzazione della patologia
High-throughput transcriptional analysis of gene therapy viral vectors effects on brain cells
No full textThe gene therapy approach using viral vectors currently represents one of the best hopes for treating numerous genetic and acquired brain disorders. Different viral vector platforms have been extensively studied and utilised in clinical trials on the Central Nervous System (CNS), by taking advantage of the specific viral features. However, the improvement of viral systems to mediate safe and long-lasting expression of therapeutic transgenes in brain is particularly challenging due to the post-mitotic nature of nervous cells, the high level of compartmentalisation of the CNS, the potential toxicity and the alteration of the neuronal physiology triggered by the virus. Although many studies have proved the efficacy of the viral sources in transducing the brain in vivo, little is known on neuronal cells perturbations following the vector interaction. To address this issue, we have analysed the global transcriptome of differentiated midbrain-derived human neuronal progenitor cells transduced in vitro with HIV-1-, AAV9-, Helper Dependent human adenoviral (HD hAd)- and Helper Dependent canine adenoviral (HD CAV-2)- vectors, at early and late time points. In particular, canine adenovectors have proved to be an interesting alternative to the human Ad, because of their efficiency in transducing human cells and the absence of CAV-specific neutralizing antibodies in human serum, that inhibit the vector effect. This study intends to provide insights in vector development for CNS, consisting in the ability to predict the neuronal functions altered by the vectors and the possibility to act on these with tools aimed at improving the efficacy and reducing the toxicity
Canine adenovirus (CAV-2) vectors induce an innate immune response and the modulation of cell cycle genes in dopaminergic differentiated human midbrain neuronal precursors
No full textCAV-2 vectors circumvent the ubiquitous human anti-human Adenovirus (hAd) memory immune response, are capable of long term neuron-specific expression (>1year in rat brains), do not induce the maturation of dendritic cells and have been proposed for the treatment of neurodegenerative diseases. In the prospect of clinical applications to brain diseases in humans, we investigated the toxicogenomic profile of helper-dependent (HD) CAV-2 vectors in human midbrain precursors differentiated into dopaminergic neurons. We transduced the cultures with HD CAV-2 and, for comparison, with third generation SIN lentiviral vectors (LV) and HD hAd. We evaluated gene modulation by Affymetrix gene chip at 2h and 5 days post transduction. Our analyses of the chip-contained 47,000 transcripts showed that, at the moi of 1000 genomes per cell, HD CAV-2 exhibited a specific modulation profile. It induced genes belonging to the cell cycle, DNA recombination and repair pathways, including p53, RAD51, BIRC5, FANCD2 and MAD2L1. It also up regulated genes involved in the immune response and in inflammation, including TL3 and 4, HAS3 and CD44, and genes involved in neuron projection morphogenesis. HD hAd was less efficient in transduction than HD CAV-2 and its effect on the trascriptome was milder. However, at 2h, it did have an impact on neuron remodeling and trafficking genes. LV transduced very efficiently, had a strong effect on the trascriptome, which overlapped with that of HD CAV-2 for TLR activation, and diverged in specific aspects of the immune and DNA repair gene groups. Single gene and pathway modulation data emerged from our analysis constitute useful information for toxicity prediction, vector comparison and evolution and virus-host interaction studies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Soma-to-germline transmission of RNA in mice xenografted with human tumour cells: possible transport by exosomes.
No full textMendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe 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
High-throughput analysis of downstream effects of activating Gsalpha mutations in skeletal progenitor/stem cells
No full textFibrous dysplasia (FD) of bone is a genetic disease caused by mutations of the α subunit of the stimulatory G protein, Gs (Gsα). The mutation results in enhanced cAMP production, and in a subversion of the structure of bone/bone marrow, mediated by the effects on skeletal stem cells (SSC). To investigate the downstream effects of the mutation in stem cells, we transduced phenotype-purified SSC with a LV vector encoding one of the two mutations causing the disease in humans (R201C). De novo transduction of normal SSC (as opposed to isolation of SSC from bone lesions) and the use of the parent untransduced cell strain as control, allowed to approach a high-throughput analysis of transcriptome changes downstream of mutated Gsα, with a statistical power which would not be permitted by natural variability of clinical material and rarity of the disease. This revealed the up- or down regulation of multiple genes specifically involved in excess bone formation, bone resorption, angiogenesis, hematopoietic control, and adipogenesis. Among these, individual genes were identified which could directly explain the emergence of specific histopathological changes
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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
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