1,720,982 research outputs found
Back to basics: optimization of DNA and RNA transfer in muscle cells using recent transfection reagents
No full textPublication Experimental Cell Research
Optimization of DNA and RNA transfer in muscle cells using recent transfection reagent
Understanding the maintenance of neuromuscular integrity: role of CtBP1 and EZH2 in skeletal muscle response to denervation
No full textLoss of functional innervation of skeletal muscles is key in several pathologies, including sarcopenia. Neuromuscular junctions (NMJs) maintenance involves local expression of synaptic genes restricted to sub-synaptic nuclei, and a proper response to spontaneous denervation to re-innervate and preserve muscle functions. We hypothesized that the Akt/mTORC1 signaling pathway acts as a regulatory platform coordinating muscle responses to denervation. During my PhD, I characterized the expression pattern of two candidate targets, C-terminal binding protein 1 (CtBP1) and enhancer of zeste homolog 2 (EZH2) in both innervated and denervated conditions. I also studied the consequences of knocking down and/or overexpressing CtBP1 or EZH2 on synaptic gene expression, NMJ maintenance, muscle histology and metabolism. Lastly, I evaluated the role of Akt/mTORC1 in the regulation of CtBP1 and EZH2 in skeletal muscle, by comparing their expression pattern in control and TSCmKO mice, characterized by Akt/mTORC1 deregulation and used as a model for sarcopenia
Investigating the pathomechanisms underlying the selective involvement of skeletal muscle in X-linked Myopathy with Excessive Autophagy (XMEA)
No full textX-linked myopathy with excessive autophagy (XMEA) is a rare neuromuscular disorder characterized by muscle atrophy and weakness. Mutations in the gene VMA21 have been identified as the genetic cause of the disease in 2013. Since then, the number of XMEA patients with VMA21 mutations has increased, along with varying ages of onset and disease severity. Intriguingly, mutations in VMA21 have also been recently linked to a liver disease. VMA21 is a crucial chaperone protein that regulates the v-ATPase proton pump, which ensures the acidification of lysosomes and other organelles. Proper acidification is required for processes like autophagy, which are vital for muscle cell homeostasis. While autophagy defects have been described in cells from XMEA patients, the molecular mechanisms underlying muscle pathology in XMEA and the distinct clinical manifestations associated with VMA21 mutations remain largely unknown.
During my PhD, I explored the expression pattern and pathophysiological roles of VMA21. In a first study, I uncovered that VMA21 is expressed as two main isoforms in humans and mice: the previously known, ubiquitous VMA21-101 isoform and a yet unknown isoform, VMA21-120. I demonstrated that VMA21-120 is expressed specifically in skeletal muscle cells after their differentiation. Notably, both isoforms were absent in cells from two XMEA patients, opening new insights into the potent pathogenic role of VMA21-120 in XMEA-associated muscle alterations. In a second study, I characterized the consequences of VMA21 depletion in skeletal muscle using a mouse model newly generated in the lab. While 50% reduction in the expression of VMA21 did not perturb muscle homeostasis, the loss of the two VMA21 isoforms in skeletal muscle (Vma21mKO mice) resulted in early lethality and profound muscle degeneration. Importantly, VMA21 depletion caused a blockage in the autophagic flux at the degradation step, prior muscle degeneration. In parallel, RNA sequencing analysis revealed perturbations in several key pathways, such as endocytosis, as well as processes previously associated with VMA21- related disorders, including ER stress and lipid metabolism. Consistently, there was a dramatic extrusion of vesicles resembling autophagic vesicles and/or multivesicular bodies in Vma21mKO muscles before their degeneration. The combined disruption of lysosomal, autophagic and endocytic pathways may precipitate muscle fibres to death and trigger the extremely severe phenotype observed in Vma21mKO mice.
Overall, my research projects highlight the crucial physiological role of VMA21 in maintaining muscle cell homeostasis. My work confirmed that VMA21 deficiency leads to autophagy impairment in muscle fibres, and unveiled alterations in other processes, such as the endocytic pathway, expanding our understanding of XMEA pathogenesis. These discoveries open new avenues for future translational investigations on VMA21-related disorders and the development of therapeutic strategies. </p
Optimization and application of a new <i>in situ</i> method for metabolic profiling of adult muscle stem cells in normal and pathological conditions
No full textMuscle stem cells (MuSCs) are essential cells allowing muscle regeneration following injury. The fate of MuSCs is finely regulated by transcriptional modifications allowing metabolic flexibility to finally establish a balance between quiescence and activation. In pathologies such as Duchenne muscular dystrophy, there is a disturbance of the MuSC niche, which may impact the regenerative capacities of MuSCs. So far, the metabolism of MuSCs has been studied only after isolation of MuSCs from their niche, even though the latter seems to be crucial for efficient muscle repair. In order to obtain a picture as close as possible to physiology, I used the technique developed by my supervisor A. Prola to study the enzymatic metabolism of MuSCs within tissues of dystrophic and wild-type mice.Les cellules souches musculaires (MuSCs) sont des cellules essentielles à la régénération musculaire suite à des lésions. Les MuSCs sont finement régulées par des modifications transcriptionnelles permettant une flexibilité métabolique pour finalement établir un équilibre entre quiescence et activation. Dans des pathologies telles que la dystrophie musculaire de Duchenne, une perturbation de la niche des MuSCs pourrait affecter les capacités régénératrices des cellules souches musculaires. Jusqu’à présent, le métabolisme des MuSCs a été étudié uniquement suite à leur isolement hors de la niche, même si cette dernière semble être déterminante pour permettre le fonctionnement correct des cellules souches. Afin d'obtenir une image aussi proche que possible de la physiologie, j'ai utilisé la technique développée par mon superviseur A. Prola pour étudier le métabolisme enzymatique des MuSCs au sein de tissus sains et dystrophiques
Deciphering the roles of VMA21 in skeletal muscle to understand the pathogenesis of a rare Autophagic Vacuolar Myopathy
No full textMutations in the VMA21 gene, encoding a V-ATPase chaperone protein, cause a X-linked myopathy with excessive autophagy and a congenital disorder of glycosylation altering muscle and liver, respectively. It remains unclear why mutations in VMA21 result in different diseases. To understand the clinical spectrum associated with VMA21 mutations, we investigated the consequences of the modulation of VMA21 in vitro and in vivo. First, we used lentiviral infection to generate stable cell lines of primary myoblasts, either 1) knock-out (KO) for Vma21, or 2) overexpressing (OE) the ubiquitous (VMA21-101) or muscle-specific (VMA21-120) isoforms of VMA21. Our results showed that overexpression of VMA21-120 perturbs the autophagic flux and the growth of primary myoblasts. In parallel, we failed to obtain Vma21KO and VMA21-101-OE cell lines, probably due to cell lethality. Second, we further characterized Vma21mKO mice recently developed in the lab to understand their premature lethality. We showed that skeletal muscle starts degenerating at 26 days of age in Vma21mKO mice, while autophagic lesions are already observed at 23 days. These results provide important information on the physiological role of VMA21 isoforms and XMEA pathogenesis. Des mutations dans le gène VMA21, codant pour une protéine chaperonne de la V-ATPase, causent une myopathie liée à l'X avec autophagie excessive et un trouble congénital de la glycosylation altérant le muscle et le foie, respectivement. La raison pour laquelle ces mutations entraînent des maladies distinctes reste floue. Pour mieux comprendre le spectre clinique associé aux mutations de VMA21, nous avons étudié les conséquences de sa modulation in vitro et in vivo. Tout d’abord, nous avons utilisé des lentivirus pour générer des lignées stables de myoblastes primaires, soit 1) délétées pour Vma21 (KO), 2) surexprimant (OE) l’isoforme ubiquitaire (VMA21-101) ou muscle-spécifique (VMA21-120) de VMA21. Nos résultats montrent que la surexpression de VMA21-120 perturbe le flux autophagique et la dynamique des myoblastes primaires. En revanche, les lignées Vma21KO et VMA21-101-OE n’ont pas pu être générées, probablement à cause de la létalité consécutive des cellules. En parallèle, nous avons caractérisé les souris Vma21mKO, récemment générées au laboratoire, afin de comprendre la raison de leur mort prématurée. Nous avons montré que les muscles squelettiques dégénèrent dès l'âge de 26 jours chez les souris mutantes, alors que les défauts autophagiques sont déjà détectables à 23 jours. Ces résultats fournissent des informations cruciales sur le rôle physiologique des isoformes de VMA21 et la pathogenèse de l'XME
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
No full text
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
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