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1
Elia, Ines. "SNAI1 target genes in myoblasts." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1142998.
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SNAI proteins are zinc finger transcription factors that act as transcriptional repressors through a conserved domain (SNAG domain) located in the N-terminus of the protein. These factors bind to a palindromic sequence of the E-box group (CANNTG) in the regulatory regions of their target genes. The role of SNAI11 and SNAI2 is well known in the epithelial mesenchymal transition, where they act as regulators increasing the capacity of tumor cells to metastasize. Less is known about their role as mediators in tissue homeostasis and differentiation. Recent studies have showed SNAI1 and SNAI2 as repressors of muscle differentiation, with the function of maintaining myoblasts in an undifferentiated state during the proliferative phase.
In this study, we explored the function of SNAI1 and SNAI2 in myogenesis both in vitro and in vivo. In vitro, we analyzed the expression of SNAI1 and SNAI2 in proliferating murine myoblasts, at various time points after inducing their differentiation. To evaluate their expression during myogenesis in vivo, we induced skeletal muscle regeneration by injecting the myotoxic agent Bupivacaine in the tibialis anterior muscles of wild-type and transgenic mice. We demonstrated that SNAI1 and SNAI2 are upregulated in proliferating myoblasts both in vitro and in vivo.
Through the analysis of the transcriptome in C2C12 myoblasts silenced for the expression of SNAI1, we have identified several target genes, among which Fgf21 and Atf3. FGF21 is a growth factor involved in muscle differentiation as well as in glucose and lipid metabolism. In muscle differentiation, FGF21 expression is increased during myogenic differentiation and its knockdown impairs myogenic differentiation in C2C12 cells. ATF3 is a transcription factor that induces endoplasmic reticulum stress (ER-stress), phenomenon behind numerous physiological processes, including muscle differentiation and metabolism regulation. Recent studies have showed that ATF3 is able to regulate chemokine mRNA expression in C2C12 myotubes and it attenuates inflammation of skeletal muscle upon muscle-damaging eccentric exercise.
Herein, we analyzed the direct involvement of SNAI1 in the regulation of Fgf21 and Atf3.
For this purpose, several Fgf21 and Atf3 promoter deletion mutants, cloned in front of the reporter gene for luciferase, were generated in order to progressively exclude the possible binding sites for SNAI1. We used the Dual-Luciferase Reporter Assay System and ChIP-qPCR analysis to demonstrate that SNAI1 directly binds to the promoter region of Fgf21 and Atf3, leading to the activation of Fgf21 and Atf3 expression in mouse C2C12 myoblasts.
Finally, we generated a SNAI1 knockout C2C12 cell line, using the CRISPR-Cas9 genome editing technique and we confirmed that SNAI1 acts as repressor of Fgf21 and Atf3 in proliferating myoblasts.
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Mahabir, Mark Ashford. "Senescence in normal and DMD myoblasts." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ58835.pdf.
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Zhao, Shuai. "Effects of Hypoxic Conditions on Skeletal Myoblasts." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1482831468411105.
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Babić, Nikolina. "Regulation of energy metabolism of heart myoblasts /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/11563.
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Yazid, Muhammad Da'In Bin. "Analysis of cell signalling in dystrophin-deficient myoblasts." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7342/.
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An absence of dystrophin in muscle has a massive impact throughout muscle development, and Duchene Muscular Dystrophy (DMD) is one of the consequences. The disruption of the dystrophin-glycoprotein complex (DGC) is caused by a mutation in the dmd gene, which effects muscle integrity, resulting in progressive muscle degeneration and weakness. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation; however, dfdl3 myoblasts did not achieve terminal differentiation. It has been suggested that Pax7 may play a major role during myogenesis, therefore its expression pattern and transport protein were examined for any impairments. It was established that Pax7 localises in the cytoplasm of dystrophindeficient myoblasts and high expression is retained during differentiation. Colocalisation of Pax7 with subcellular markers analysis indicated that Pax7 is synthesised during the proliferative state. Pax7 was shown to possess a nuclear location signal and KPNA2 was suggested as escort protein for Pax7 translocation into the nucleus. The PTEN-PI3K/Akt signalling pathway was investigated and protein synthesis regulation and Fox03 was found to be impaired. Autophagy related genes were found to be highly expressed; however, LC3 lipidation and autophagy flux showed a reduction upon differentiation, indicating defective autophagy. The contribution of PTEN overexpression was assessed in relation to endoplasmic reticulum (ER) stress and activation of the unfolding protein response (UPR). It was established that a reduction in ER stress and changes to UPR activation lead to apoptosis. Finally, minidystrophintransfection of both types of myoblasts was utilised to examine the effect, especially in dystrophin-deficient myoblasts. Minidystrophin improved protein synthesis activation and increased autophagy (increased LC3 lipidation), suggesting that minidystrophin ameliorates dystrophic events at the level of autophagosome formation. To conclude, destabilisation of the plasma membrane owing to a dystrophin mutation causes cell signalling alterations which minidystrophin restoration can partly improve.
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Akohene-Mensah, Paul. "Examining the Role of L-Type Amino Acid Transporter 1 (SLC7A5) in Myoblasts." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41036.
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Skeletal muscles represent the largest tissue mass within the body and are primarily involved in the generation of force for voluntary movement. Skeletal muscles have a remarkable capacity to repair, due primarily to the actions of muscle stem cells (MuSCs). MuSCs are normally quiescent in adult skeletal muscle; however, in response to myotrauma (trauma to muscle tissue) from muscle injury or exercise, MuSCs become activated, either undergo self-renewal to replenish the quiescent population or commit to the myogenic lineage as myoblasts, proliferate, and differentiate into myotubes in vitro or fuse to existing myofibers in vivo. This process of generating new myofibers from quiescent MuSCs is termed myogenesis and a full understanding of how myogenesis is regulated remains to be understood. Mounting evidence suggests that amino acids, particularly the essential amino acid leucine, play a role in MuSC regulation. Leucine is specifically translocated and sensed by the L-type amino acid transporter 1 (LAT1); which facilitates leucine uptake in mature myofibers. Inside the cell, leucine activates mammalian or mechanistic target of rapamycin complex 1 (mTORC1) to stimulate cell growth, proliferation, and protein synthesis. Whether leucine has direct effects on myoblast function via LAT1 is unknown. Thus, our overall objective was to begin to characterize the role of LAT1 in myogenesis. Our results indicate that myoblasts differentially expressed LAT1 throughout myogenesis with peak protein content occurring during differentiation (p<0.05 vs. early proliferation). Further, our results indicate thatpharmacological LAT1 inhibition reduced myoblast expansion and differentiation in vitro (both p<0.05 vs. control). Interestingly, myoblast LAT1 protein content did not change in response to leucine supplementation in vitro; however, was lower under in vitro atrophic conditions (p<0.05 vs. control). Based on these findings, we conclude that LAT1 plays an important role in regulating myogenesis. As such, we uncover a novel role for LAT1 in regulating muscle mass via contributing to the control of MuSC function.
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Gersbach, Charles Alan. "Runx2-Genetically Engineered Skeletal Myoblasts for Bone Tissue Engineering." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/11600.
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Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.
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Mazzuca, Delfina Maria. "Regulation and function of glucose transporters in rat myoblasts." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ30668.pdf.
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Ren, Huiping. "MBD2bdemethylase is involved in the myogenesis of C2C12 myoblasts." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80861.
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Initiating the muscle differentiation pathway typically results in activation of muscle-specific genes previously maintained in a silenced state. One of the processes controlling changes in gene expression during differentiation is a global demethylation event. The mechanisms involved in this global hypomethylation are not fully understood.Promoter methylation is one of the normal mechanisms inactivating gene expression. A single CpG site in the 5' flanking region of myogenin is reported to undergo demethylation during C2C12 differentiation. Considering the demethylase feature of MBD2b and its expression profile during C2C12 differentiation, I propose the hypothesis that MBD2b/demethylase is involved in C2C12 differentiation by demethylating the promoter of the myogenin gene as well as other genes involved in myogenic differentiation.
To test this hypothesis, I determined the consequences of up-regulation and down-regulation of MBD2b/demethylase in C2C12 cells.
The state of the myogenin promoter was also altered as determined using a probe recognizing a single HpaII site, which was previously reported to become demethylated during C2C12 differentiation. (Abstract shortened by UMI.)
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Lund, Dane. "It's a Jungle Out There| Myoblasts, Matrix, and MMPs." Thesis, University of Missouri - Columbia, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10182609.
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Razvadauskaite, Giedre. "Survival and differentiation of implanted skeletal myoblasts in the native and in the cryoinjured myocardium." Link to electronic thesis, 2003. http://www.wpi.edu/Pubs/ETD/Available/etd-0106103-155714.
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Thesis (M.S.)--Worcester Polytechnic Institute.Keywords: myoblasts; dexamethasone; infarction; cryoinjury; desmin; myosin heavy chain; differentiation. Includes bibliographical references (p. 54-59).
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Cowan, Joanne L. "Translational control during cellular stress and differentiation of C2C12 myoblasts." Thesis, University of Sussex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436824.
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Swailes, Nathan. "Actin and non-muscle myosin II in pre-fusion myoblasts." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416842.
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Briggs, D. "An investigation into sub-populations of satellite cells and myoblasts." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1386193/.
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One potential therapy for muscular dystrophy is myoblast transplantation. This will require cell expansion before transplantation and donor cells must therefore retain their stem cell capacity after propagation. I used a mouse model with the aim of sub-populating freshly isolated satellite cells and cultured satellite cell-derived myoblasts to obtain those that retain their ability to contribute to muscle regeneration upon grafting into dystrophin-deficient mdx-nude mouse muscles. Using flow cytometric cell sorting, satellite cells or myoblasts were separated on the basis of proliferative state and the level of expression of reactive oxygen species (ROS). DNA dyes that separated cells in G0 from those proliferating in G1 were useful for analysis but were toxic, limiting further comparisons. A donor transgenic mouse, which expresses GFP only in activated satellite cells, was used to separate quiescent GFP– from activated GFP+ satellite cells. In culture, the sorted GFP– satellite cells became activated, turned on GFP and showed enhanced proliferation over GFP+ satellite cells. When engrafted into mdx-nude mice, GFP– satellite cells regenerated more muscle than GFP+ satellite cells. Freshly isolated satellite cells showed low levels of ROS, but upon expansion in culture, ROS levels increased. Myoblasts were sorted into ROSlow and ROShigh populations and compared. ROSlow cells had a higher proliferative capacity in vitro than ROShigh cells and this corresponded with an increased ability to regenerate muscle in vivo. Expansion in low oxygen improved myoblast growth. Furthermore, myoblasts that had been grown in 5% O2 contributed to more muscle fibres of donor origin than the same number of myoblasts that had been grown in 20% O2. Transgenic mice expressing GFP or β-gal, under ubiquitous promoters, were compared, to find the most reliable marker for donor-cell transplantation, with the bactinGFP mouse selected for use. In summary, selecting for cell populations that have a greater proliferative ability increases the capacity of transplanted myoblasts to regenerate muscle fibres in vivo.
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Pouliot, Yannick 1963. "Study of L6 myoblast cell-cell adhesion." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61797.
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Butler, David Christopher. "The role of Id2 phosphorylation at serine 5 in C2C12 myoblasts." Morgantown, W. Va. : [West Virginia University Libraries], 2008. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5669.
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Thesis (Ph. D.)--West Virginia University, 2008.Title from document title page. Document formatted into pages; contains v, 42 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
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Bray, Jonathan Alexander. "Comparing insulin and insulin-like growth factor-1 signalling in myoblasts." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596876.
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In this study a chimeric receptor system was employed in which the extracellular domain of the TrkC receptor was fused to the intracellular portion of either the insulin (TIR) or IGF-1 (TIGFR) receptor. These chimeric receptors were expressed in separate populations of the skeletal muscle cell line L6. Initial analysis of individual downstream signalling components and assessment of cell proliferation, induced by TIR or TIGFR stimulation revealed little difference between the two chimeras. To more comprehensively screen for potential differences, microarray analysis was used to compare regulation of gene expression by the two chimeric receptors. This led to the identification of several differentially regulated genes. Whilst it was initially hypothesised that skeletal muscle cells might yield several selectively insulin-sensitive genes, the majority of genes selectively regulated by one receptor were preferentially IGF-1 responsive, consistent with previous studies in other cell types. This perhaps reflects the more mitogenic effect of this ligand in vivo, manifest as an increased ability to regulate transcription per se. Of the differentially regulated genes, that encoding Fit-1m was found to be preferentially induced through activation of the TIGFR rather than the TIR. Further characterisation using real-time PCR established that induction of Fit-1 expression required an intact MAPK signalling pathway. Similar effects were observed when the regulation of Fit-1 expression by insulin and IGF-1 was examined. Subsequent work attempted to establish regions of promoter responsible for the preferential induction of Fit-1m expression by IGF-1. Despite defining promoter and putative enhancer regions which are important for Fit-1m transcription, no region was found which confers a response to stimulation with various ligands, including IGF-1. Rather, a high level of constitutive expression was driven by these DNA sequences, suggesting that an IGF-1 response inhibitory factor may control expression of this gene, binding outside the regions examined. Fit-1 joins an increasing list of genes preferentially regulated by the IGF-1R over the IR and provides and end point with which to analyse potential inherent differences in the signalling capacity of these two highly homologous receptors.
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Hou, Yuguo. "Roles of cholesterol in the proliferation and differentiation of bovine myoblasts." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/87466.
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The objective of this study was to assess the potential role of extracellular, cytosolic, and membrane cholesterol in the proliferation and differentiation of bovine myoblasts. In the first experiment, myoblasts isolated from Angus or Angus crossbred steers were cultured with 2% lipoprotein deficient fetal calf serum (LPDS) or normal fetal calf serum. Culturing with LPDS did not alter the cytosolic or membrane cholesterol content, or myoblast differentiation, but inhibited myoblast proliferation, compared to culturing with normal fetal calf serum. In the second experiment, myoblasts were cultured with or without lovastatin, a selective inhibitor of cholesterol synthesis. Culturing with 5 μM lovastatin did not affect medium concentration of cholesterol, but reduced cytosolic and membrane cholesterol contents, compared to culturing with vehicle control. Culturing with 5 μM lovastatin inhibited both myoblast proliferation and differentiation. In the third experiment, myoblasts were cultured with or without methyl-βcyclodextrin (MβCD), a chemical that depletes cholesterol from cell membranes. Treating myoblasts with 10 mM MβCD for 30 minutes reduced membrane and cytosolic cholesterol contents while increasing medium cholesterol concentration. Treating with MβCD inhibited both myoblast proliferation and differentiation compared to treating with vehicle control. Overall, this study showed that lovastatin- or MβCD-induced reductions in cytosolic and membrane cholesterol contents were associated with reduced proliferation and differentiation in bovine myoblasts. These associations suggest that cytosolic cholesterol, membrane cholesterol, or both may play a role in bovine myoblast proliferation and differentiation.Master of Science
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Evans, Darrell John Rhys. "The behaviour and commitment of myoblasts during mammalian skeletal muscle formation." Thesis, University of Aberdeen, 1994. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU603173.
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During mammalian skeletal muscle development, muscle fibres form in a biphasic manner from the fusion of myoblasts. Primary fibres form first, which subsequently provide a surface for later secondary fibres to form on. The purpose of the present study was to successfully develop new and existing techniques and to employ them in order to study the commitment and behaviour of myoblasts during muscle development in mice. Following part 1; a general introduction into the development of skeletal muscle, the thesis is divided into two subsequent parts giving details of the investigations performed. In the main section (part 2) of this thesis, I investigated the commitment of myoblasts during the foetal development. It has been suggested, that separate populations of myoblasts are present, each committed to producing the different fibre types seen during development. The aim of this study was to see if different populations produced primary and secondary fibres, by seeing if clones of related cells were restricted to fusing with a single type of fibre. Following the injection of replication deficient retroviruses into the hindlimbs of Embryonic day (E)15 and El 7 foetal mice, cells became marked with the lac Z gene encoding for the enzyme [Special character omitted]-galactosidase. The infected cells, their descendants and the fibres they fused with could then be demonstrated histochemically. 83% of the clusters of marked fibres obtained following processing were found to contain both primary and secondary fibres as identified by electron microscopy. The clusters were assumed to be the result of the fusion of a single clone of cells. It was concluded that at these ages, a single population of cells contributes to primary and secondary fibres. Part 3 of the thesis describes a second, shorter study whereby the in vitro behaviour of El 7, El 9 and E21 myoblasts was investigated on artificial grooved substrata. Most cells on grooves with depths of 250nm-6um were found to align parallel with the direction of the grooves. Cells on the shallower grooves (40-140nm) either aligned parallel or perpendicular to the grooves. E21 cells however, orientated randomly on these groove sizes. It was generally concluded however, that myoblasts at the ages studied do align in grooves similar to those formed in vivo by adjacent primary and secondary fibres. It is suggested that grooves such as the ones mentioned may be a possible site for secondary myogenesis. The results of both my studies contribute to the current work being carried out on skeletal muscle development, and may also, in addition, provide useful information towards the development of myoblast transfer therapy, a possible treatment for Duchenne Muscular Dystrophy sufferers.
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Peduto, Giovanni. "Long-term accommodation to encapsulated xenogeneic myoblasts engineered to secrete erythropoietin /." [S.l.] : [s.n.], 1999. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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El, Haddad Marina. "Approche pharmacologique dans la thérapie cellulaire : rôle de l'acide rétinoïque dans la survie et la différenciation des myoblastes humains." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT003.
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Les cellules satellites sont considérées comme de véritables cellules souches du muscle squelettique. Une fois transplantées dans un muscle hôte, les myoblastes, cellules filles des cellules satellites, sont capables de fusionner avec les fibres musculaires existantes, permettant ainsi de modifier de façon permanente le muscle receveur. La greffe des cellules satellites est donc une des thérapies dans la lutte contre les maladies musculaires dégénératives ou myopathies. Malheureusement les premiers essais cliniques sont décevants compte tenu en partie d’une mortalité massive au sein des myoblastes implantés. Plusieurs approches ont été développées pour réduire la mortalité des myoblastes. Notre approche a été de sélectionner et de purifier une population de cellules plus aptes à résister au stress cytotoxique. Les aldéhydes déhydrogenases (ALDH) sont une famille d’enzymes capables de détoxiquer efficacement les résidus aldéhydes générés par les espèces réactives de l’oxygène. Nous avons montré récemment que l’activité ALDH est élevée dans une majorité des myoblastes humains. Cette activité est associée à une augmentation de la survie cellulaire, ex vivo, suite à un stress oxydant induit au peroxyde d’hydrogène (H2O2) et, in vivo, lorsque les myoblastes ALDHhigh sont implantés dans des souris immunodéprimées scid. De plus, nous avons montré que la protéine Aldh1a1 est responsable de la totalité de l’activité ALDH dans les myoblastes humains. La protéine Aldh1a1 fait partie d’un sous groupe des ALDH appelées rétinaldehydes. Ces enzymes catalysent l’oxydation de la vitamine A en acide rétinoïque qui se lie et active les récepteurs nucléaires de l’acide rétinoïque. Dans ce projet, je vais chercher à savoir si l’activité anti-apoptotique de l’ALDH dans les myoblastes humains est dépendante de la synthèse d’acide rétinoïque. Au cours de mon stage M2R dans le laboratoire, j’ai montré que les myoblastes humains exposés à un stress oxydatif perdent leur intégrité cellulaire. Le traitement par l'acide rétinoïque protège les myoblastes humains de ce stress cytotoxique. L'analyse des transcriptomes des myoblastes traités à l’acide rétinoïque a révélé que la glutathione péroxydase 3 et la superoxyde dismutase 2, gènes codant pour des enzymes antioxydantes, sont des gènes cibles potentiels de l’acide rétinoïque. Dans ce projet, objectif 1, je propose d'étendre ces résultats aux myoblastes provenant de patients atteints de dystrophie Facioscapulohumeral (FSHD), une maladie musculaire dégénérative. Puisque l’équipe de Winokur et notre équipe ont démontré la présence d’une susceptibilité au stress oxydant dans ces myoblastes FSHD, nous postulons que la voie de signalisation des rétinoïdes pourrait stabiliser ce stress oxydatif et protègerait les myoblastes FSHD durant le processus de transplantation. Dans l’objectif 2, je vais inactiver l’expression de GPx3 et de SOD-2 en utilisant des shRNA afin de déterminer si GPX3 et SOD-2 sont responsables des effets anti-apoptotiques de l'acide rétinoïque. L’objectif 3 aura pour but de déterminer si l'acide rétinoïque améliore la survie des myoblastes sains et FSHD dans des essais de transplantation chez des souris immunodéficientes. Enfin, dans un projet à plus long terme, objectif 4, je testerais l'hypothèse que le statut en vitamine A (le précurseur de l'acide rétinoïque) est important pour la survie des cellules satellites et leur expansion chez l’adulte. Par conséquent, améliorer la survie des cellules souches musculaires afin d'augmenter la masse musculaire pourrait s'avérer être une stratégie thérapeutique importante pour contrecarrer l’évolution des dystrophies musculairesMouse and human satellite cells have been shown to be functional muscle stem cells. Since myoblasts, the progeny of satellite cells can be transplanted and fuse with endogenous muscle fibers to form hybrid cells, myoblast transplantation represents a potential approach for the treatment of muscle diseases. Although other limitations, such as immune rejection or limited spread into the host tissue are also important, failure of myoblast transfer in the initial clinical trials was at least partly related to poor survival rate of transplanted myoblasts. Several approaches have been developed to reduce early loss of injected myoblasts. The approach in the laboratory was to select and purify a pool of myoblasts characterized by an improved survival response. Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Their findings indicate that high ALDH activity is present in a majority of human myoblasts. This activity is correlated ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when ALDHhigh myoblasts were transplanted into host muscle of immune deficient scid mice. They demonstrated that Aldh1a1 protein contributes to most if not all ALDH activity in human myoblasts. Aldh1a1 catalyzes the irreversible oxidation of vitamin A (retinol) to retinoic acid (RA) which binds and activates nuclear retinoic acid receptor (RAR)/Retinoic X receptor (RXR) heterodimers. Since high ALDH activity is correlated to improved cell viability, we will ask whether part of this biological activity is mediated by retinoic acid synthesis. In this project, we propose to determine whether retinoids (vitamin A and retinoic acid) protect human muscle precursor cells from cytotoxic damages and improved cell survival in transplantation assays. During my M2R training, I showed that human myoblasts exposed to an oxidative stress lost their integrity. Treatment with retinoic acid impaired these cytotoxic damages ex vivo. Microarray analysis of retinoic acid treated myoblasts revealed glutathione peroxidase 3 and superoxide dismutase 2, genes encoding antioxidant enzymes, as a potential RA target genes. In this project, aim 1, I propose to extend these results to myoblasts derived from patients with Facioscapulohumeral dystrophy (FSHD), a muscle degenerative disease. Since the team of Winokur and our team found that FSHD myoblasts were highly susceptible to an induced oxidative stress, we postulate that retinoid signalling pathway may stabilise this oxidative stress and protect FSHD myoblasts during the process of transplantation. In aim2, I will inactivate Gpx-3 and SOD2 using shRNA to determine whether SOD-2 and GPx3 mediate the anti-apoptotic effects of retinoic acid. In the aim 3, I will determine whether retinoic acid improves myoblast survival in transplantation assays in animals. Finally, in a more long-term project, aim 4, I will test the hypothesis that vitamin A status (the precursor of retinoic acid) is important for satellite cell survival and expansion in the offspring. Therefore, manipulating cell survival in order to increase the mass of muscle produced from a pool of muscle precursor cells could be an important therapeutic strategy to counteract the course of muscular dystrophy
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Sudarsan, Vikram. "Coordinating cell fate signalling during Drosophila development." Thesis, University of Sheffield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247190.
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Lim, Sean. "The Relationship Between Metabolic Circumstance and Epigenetic Acetylation in Myoblast Fate and Function." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42659.
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Muscle tissue is grown and maintained by muscle stem cells termed satellite cells. Activated satellite cells become myoblasts, which must proliferate then differentiate into functional muscle. This process, known as myogenesis, is controlled by a cascade of epigenetic regulatory events. One facet of this regulation is histone acetylation, which can be influenced by the availability of metabolites within a cell. In this study, the ability of glucose, pyruvate, or glutamine to change histone acetylation levels in cultured myoblasts was investigated. Changing concentrations of glucose or pyruvate had no effect but decreasing the availability of glutamine in cell culture from 2mM to 0.2mM resulted in proliferating myoblasts accruing a hyperacetylated histone phenotype. However, when the same concentration of glutamine was used on differentiating myoblasts the hyperacetylated phenotype was lost and no change to differentiation was observed. This study demonstrates the potentials and limitations of altering epigenetic acetylation with metabolic circumstance. -- Le développement du tissu musculaire est soutenu par les cellules souches musculaires, communément appelées cellules satellites. Les cellules satellites activées se transforment en myoblastes qui doivent ensuite proliférer et se différencier en muscle fonctionnel. Ce processus, connu comme myogenèse, est contrôlé par une cascade de régulation épigénétique. Un aspect de ce processus est l’acétylation d’histones, qui peut être influencée par la disponibilité de métabolites dans la cellule. Dans cette étude de cas, la capacité du glucose, pyruvate, ou glutamine à changer les niveaux d’acétylation d’histones a été examinée. Le changement des concentrations de glucose ou de pyruvate n’a généré aucun effet, mais la diminution de la disponibilité de la glutamine dans la culture cellulaire de 2mM à 0.2mM a eu pour résultat une prolifération de myoblastes présentant un phénotype d’histones hyper-acétylées. Pourtant, quand la même concentration de glutamine a été utilisée pour différencier les myoblastes, le phénotype hyper-acétylé n’a pas été observé et aucun changement de différenciation n’a pu être détecté. Cette étude démontre le potentiel et les limites des modifications de l’acétylation épigénétique selon les circonstances métaboliques.
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Joshi, Shilpy. "Specific and redundant roles of the Tead family of transcription factors in myogenic differentiation of C2C12 cells and primary myoblasts in vitro." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ093/document.
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La famille Tead de facteurs de transcription reconnaît l'élément MCAT trouvé dans le promoteur de gènes spécifiques au muscle. L'analyse génétique de leur fonction dans la différenciation musculaire a révélé difficile en raison de la redondance susceptible parmi les membres de la famille. Dans cette étude, nous avons utilisé le silencing siRNA médiation pour aborder le rôle des facteurs TEAD dans la différenciation des myoblastes primaire.Contrairement aux cellules C2C12 où Tead4 joue un rôle essentiel, son silence dans les myoblastes primaires a eu peu d'effet sur leur différenciation. Silence de facteurs individuels TEAD n'a eu aucun effet significatif sur la différenciation des myoblastes primaires, alorsque le silençage combinatoire a conduit à l'inhibition de leur différenciation indiquant laredondance parmi ces facteurs. Dans les cellules C2C12 aussi, combinatoire silençageTead eu des effets beaucoup plus puissants que de faire taire Tead4 seule indiquant une contribution des autres Teads dans ce processus. En intégrant Tead1 et les données Tead4ChIP-Seq avec les données d'ARN-Seq suivante combinatoire Tead1 / 4 silencieux, nous identifions ensembles distincts, mais qui se chevauchent de gènes Tead réglementés dansles deux cellules C2C12 myoblastes et primaires. Nous avons également intégré les / 4 données Tead1 ChIP-seq avec des ensembles de données publiques sur Myog et MYOD1ChIP-Seq et chromatine modifications à identifier une série d'éléments de régulation actifsliés par des facteurs TEAD seul ou avec Myog et MYOD1. Ces données disséquer les fonctions spécifiques et combinatoires de ces facteurs de transcription dans les réseaux derégulation de le differentiation musculaireThe Tead family of transcription factors recognise the MCAT element found in thepromoters of muscle-specific genes. Genetic analysis of their function in muscledifferentiation has proved elusive likely due to redundancy amongst the family members.We previously used shRNA-mediated silencing to show that loss of Tead4 function resultedin abnormal differentiation characterised by the formation of shortened myotubes. ChIP-chipcoupled to RNA-seq data identified a set of potential target genes that are either activatedor repressed by Tead4 during differentiation. In this study, we have used siRNA-mediatedsilencing to address the role of the Tead factors in primary myoblast differentiation. Incontrast to C2C12 cells where Tead4 plays a critical role, its silencing in primary myoblastshad little effect on their differentiation. Silencing of individual Tead factors had no significanteffect on primary myoblast differentiation, whereas combinatorial silencing led to inhibitionof their differentiation indicating redundancy amongst these factors. In C2C12 cells also,combinatorial Tead silencing had much more potent effects than silencing of Tead4 aloneindicating a contribution of other Teads in this process. By integrating Tead1 and Tead4ChIP-seq data with RNA-seq data following combinatorial Tead1/4 silencing, we identifydistinct but overlapping sets of Tead regulated genes in both C2C12 cells and primarymyoblasts. We also integrated the Tead1/4 ChIP-seq data with public data sets on Myogand Myod1 ChIP-seq and chromatin modifications to identify a series of active regulatoryelements bound by Tead factors alone or together with Myog and Myod1. These datadissect the specific and combinatorial functions of these transcription factors in muscledifferentiation regulatory networks
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Beauchamp, Pascal. "The functional role of the RNA-binding protein HuR in the regulation of muscle cell differentiation /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111586.
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Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.
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Li, Hongmei. "An Investigarion of PAX3 Isoforms (PAX3c, e AND g) in C2c12 Myoblasts." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492771.
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Rapa, Elizabeth. "Characterisation of the differences in gene expression between rhabdomyosarcoma cells and myoblasts." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510209.
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Alsharidah, Mansour. "Behaviour of human myoblasts in vitro : role of ageing and inflammatory cytokines." Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/behaviour-of-human-myoblasts-in-vitro-role-of-ageing-and-inflammatory-cytokines(f544ecce-946d-40f1-91a4-fd750aacee11).html.
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Ageing is associated with a loss of muscle mass, a processes known as “sarcopenia”. It has been postulated that one of the reasons underlying this muscle loss is a decreased ability to repair itself in response to damage. Repair of muscle is facilitated by a specific population of progenitor adult stem cells known as satellite cells in situ and myoblasts ex vivo. The work in this thesis has used a cell culture approach to study the behaviour of human myoblasts and compare the inherent effects of age (by studying myoblasts taken from young and old people) and using an in vitro model of ageing, (i.e. proliferative senescence), and investigating the contribution of environmental factors by culturing the cells in human serum from young and old participants and treating them with recombinant cytokines. Several parameters were studied, but the main focus was on ability of myoblasts to proliferate and differentiate. Markers of proliferating muscle cells were desmin, NCAM and Ki67, and markers of differentiating muscle cells were myogenin and myosin heavy chain. Additional parameters observed were DNA damage and analysis of specific cytokines in the cell secretome. No differences in any parameter measured were found between cells of young and old people. However senescent myoblasts differed significantly in all parameters from early passage cells. Differentiation was studied over a seven day period. There was a delay of two-four days in the onset of markers expression between senescent and early passage cells, as well as a decrease in the expression levels of myogenin (50 ± 3% in young, 49 ± 3% in old and 6 ± 1% in senescent) after three days of differentiation and myosin heavy chain (MHC; 71 ± 2% in young, 70 ± 1.4% in old and 15 ± 1% in senescent) after five days of differentiation. In addition, increased DNA damage (7 ± 1% in young, 8 ± 1% in old and 90 ± 4% in senescent), increased TGF-β secretion (111 ± 13pg/ml in young, 115 ± 19 pg/ml in old and 268 ± 11pg/ml in senescent), and decreased myotube area (151574 ± 22968μm2 in young, 132531 ± 25106μm2 in old and 47765 ± 763μm2 in senescent) were observed in senescent cells compared to early passage cells. For influence of environmental factors, freshly isolated cells were cultured in human sera or medium with or without cytokines. No differences were observed in the myoblasts cultured in sera from young and elderly individuals (Ki67 expression was 85 ± 2% in young and 84 ± 2% in old, and desmin expression was 81 ± 2% in young and 83 ± 3% in old at three days after isolation). There was, however, a significant decrease in desmin and myogenin expression when cells were exposed to TGF-β1 (1 ng/ml), TNF-α (1 ng/ml) or IL-1β (1 ng/ml). Committed myoblasts were also cultured in the presence or absence of TGF-β1, TNF-α, or IL-1β. Myogenin but not desmin expression was significantly inhibited in the presence of cytokines. These findings support the observation of many studies in vivo in both human and animal models which show that satellite cells from old muscle can contribute to muscle repair and regeneration. This suggests the satellite cells per se may not be critically involved in the mechanism responsible for sarcopenia. However, their sensitivity to inflammatory cytokines suggests that if these were present in vivo they would affect the myogenic behaviour of the cells.
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Parolini, D. "CALCIUM HANDLING IN MYOGENIC PROGENITORS AND SKELETAL MYOBLASTS: THE ROLE OF CD20." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217445.
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The calcium ion plays an essential role in the physiology of all living cells. Accordingly, multiple mechanisms contribute to the precise control of its intracellular concentration ([Ca2+]i). Particularly in skeletal muscle, the efficient regulation of cytosolic Ca2+ is crucial for tissue functionality and impairment of Ca2+ homeostasis has been shown to contribute to the etiology of muscular disorders such as Duchenne muscular dystrophy (DMD). Although the impairment of Ca2+ homeostasis affecting dystrophic muscular cells has been extensively reported, the pathways involved in calcium-release and the role of store-operated Ca2+ channels in dystrophic myogenic progenitors were not investigated before. Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133+ cells displaying myogenic properties. Interestingly, expression of the B-cell marker CD20 was observed in blood-derived CD133+ stem cells. Among the scarce available data about the biological role of the membrane protein CD20, there are some evidences of its involvement in the regulation of intracellular calcium concentration ([Ca2+]i). Here, we show that a CD20-related pathway leading to an increase of cytosolic calcium is differently activated in normal and dystrophic blood-derived CD133+ stem cells, supporting the assumption of a CD20-related calcium impairment affecting dystrophic cells. Although CD20 can modulate cytosolic calcium through a specific signaling pathway, other studies demonstrated its association with lipid raft domains of the plasma membrane, where it probably functions directly as a store-operated Ca2+ channel. Recent works indicated that store-operated Ca2+ entry (SOCE) plays a central role in skeletal muscle physiology and development, but there remain a number of unresolved issues relating to SOCE modulation in this tissue. That being so, and considering that blood and muscle share common mesodermic origins, we were prompted to investigate whether CD20 contributes to calcium handling in committed muscular cells. Expression of CD20 was observed in skeletal muscle, displaying a membrane localization in myoblasts and adult muscle fibers. Additionally, we showed that inhibition of CD20 resulted in specific impairment of SOCE in C2C12 myoblasts. Together, reported findings contributed to identify deregulated pathways affecting dystrophic stem cells and potentially involved in DMD pathology. Moreover, our results suggested that functional CD20 is required for SOCE to consistently occur in C2C12 myoblasts, providing a novel insight to improve the understanding of store-operated Ca2+ entry regulation in skeletal muscle.
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Bensimon, Maharaj Victoria. "Novel Cell Cycle Regulation of the Transcription Factor p53 and the Kinase c-Abl in Skeletal Myoblast Cultured in Differentiation Media and the Fusion of Myoblasts." Cleveland State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=csu1600275796611256.
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Lee, Antonio Seung Jin, and n/a. "Myogenic mononucleated cell populations in the developing vertebrate limb in vivo." University of Otago. Department of Anatomy & Structural Biology, 2007. http://adt.otago.ac.nz./public/adt-NZDU20070321.143922.
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Skeletal muscles of the limb are derived from somites and their precursors migrate to the limb prior to muscle formation. Upon migration, a limited number of stem cells multiply and differentiate to give rise to fusion-competent muscle cells, which fuse to form the multinucleated myotubes. During the course of myogenesis there is thus a period of few days when cells at different developmental stages such as migrating, proliferating, differentiating and fully differentiated co-reside within the developing limb bud. Current understanding on how these cells interact and behave during early and later myogenesis in vivo is lacking. The aim of this project was to identify and further classify the mononucleated myogenic cells present within the developing limb muscle and examine their behaviours at different stages of myogenesis.
The lack of an appropriate method to extract and visualise cellular constituents of developing muscles has been a major limitation hindering such investigations in vivo. In this project, we first developed a unique cell isolation method to extract mononucleated cells from developing muscles, allowing examination of mononucleated cells in vivo using immunocytochemistry. As Pax3, Pax7 and Myogenic Regulatory Factors (MRFs) are the key players for the muscle formation, they were used to mark the different myogenic sub-populations.
The results from chicken and rats clearly demonstrate that three myogenic cell pools, namely Pax3, Pax7 and MRFs positive cells, and 4 sub-populations formed by their overlap, co-exist in specific proportions within the developing limb muscle, and that their proportions undergo dynamic changes during the course of myogenesis. The most striking observation was that the sizes of Pax3 and MRF compartments remain constant while that of Pax7 compartment increases dramatically during myogenesis. Thus each myogenic cell compartment in the developing muscle has different cell kinetics during primary and secondary myogenesis. The dynamic changes in the proportions of these myogenic sub-populations may constitute a dynamically maintained cellular niche, within which the muscle stem cells reside. Our study suggests that the concept of community effect - the interaction between a group of cells and their surrounding cells, originally from invertebrate muscle system, may be conserved in mammalian systems. Furthermore, this study for the first time, reports that the earliest fully differentiate muscle cells in the rat hindlimb are highly elongated mononucleated cells which express Pax3, MyoD, myogenin and myosin but not Myf-5 protein.
In summary, this study provides quantitative data to demonstrate dynamic changes in various mononucleated myogenic cell populations during skeletal muscle formation and reveals that Pax7(+ve) population becomes significantly upregulated during secondary myogenesis.
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Lu, Lin, and 鹿琳. "Mechanisms involved in the release of ATP from skeletal myoblasts at low pH." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B47323772.
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Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR.
Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6.
Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel.
We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux.
The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current.
These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release.published_or_final_version
Physiology
Master
Master of Philosophy
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Abbas, Hesham Magdi. "Estrogenic Compounds Protect Rat Cardiac Myoblasts (H9c2 Cells) Against Doxorubicin-Induced Cell Death." VCU Scholars Compass, 2006. http://scholarscompass.vcu.edu/etd_retro/131.
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The antineoplastic drug doxorubicin is widely used in the treatment of various types of cancers including breast, colon and lung cancer. However, doxorubicin has adverse effects on the heart and prolonged doxorubicin administration results in cardiomyopathy and congestive heart failure. In the present study we have established that treatment of rat cardiac myoblasts (H9c2 cells) for 24 hours with doxorubicin resulted in concentration and time dependent cell death as determined by proliferation assay. Almost 50-55% cell death was attained at 24 hours treatment of H9c2 cells with 5 μM doxorubicin. We have selected about 50% cell injury as an optimum doxorubicin-induced cell injury because once this threshold is reached, cells became irreversibly injured and are unable to respond to protective treatment. We have observed that another potent antineoplastic drug, cyclophosphamide, had no cardiotoxic effects even with exposure at 35 μM concentrations for a treatment time of up to 72 hours. Pretreatment of H9c2 cells for 24 hours with 100 nM 17β-estradiol protects about 30% cell death against subsequent treatment for 24 hours with 5 μM doxorubicin. Interestingly 500 nM quecertin and 20 μM resveratrol pretreatment provide about 30% and 40% protection, respectively, to the H9c2 cells against subsequent doxorubicin treatment. However, diethylstilbestrol (DES), bisphenol A, and estrone exhibit no protective effects. It is concluded that 17β-estradiol, resveratrol and quercetin considerably protect H9c2 cells against doxorubicin-induced cell death.
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LIBETTI, DEBORA. "ROLE OF NF-YA ISOFORMS IN MOUSE EMBRYONIC STEM CELLS AND MYOBLASTS DIFFERENTIATION." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/732914.
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The Nuclear Factor Y (NF-Y) is a Transcription Factor (TF) composed by three different subunits, NF-YA, NF-YB and NF-YC, all necessary to recognize and bind the CCAAT box, a DNA- regulatory region highly enriched in active enhancers in human and mouse Embryonic Stem cells (ESCs).
NF-YB and NF-YC subunits have a histone-like domain (related to core histones H2B and H2A respectively) that associate forming a dimer required to interact with the NF-YA subunit. In particular, NF-YA has an evolutionary conserved domain in the C-terminal part responsible for NF-YB/NF-YC interaction and DNA binding, while the N-terminal domain is characterized by a glutamine rich (Q- rich) trans-activation domain. NF-YA comes in two isoforms, the long (NF- YAl) and the short (NF-YAs), differing for 28 amino acids coded by exon 3. Mouse ESCs preferentially express the short isoform, but after differentiation by Embryoid bodies (EBs) formation, a complete switch from the short to the long isoform is observed. Moreover, the progenitor muscle C2C12 cells express the long isoform, but after complete differentiation into muscle tissue, NF-YA protein is not detected.
The first project of my PhD thesis aims to shed light on the function of NF-YAs and NF-YAl in controlling cell differentiation by generating mES and C2C12 cell lines with a genomic deletion of exon 3 through the CRISPR/Cas9 Nickase system. Two isolated homozygous clones for each cell line were obtained and they expressed only the NF-YA short isoform (NF-YAl-KO), as expected.
Both cell lines, after exon 3 deletion, maintained the same morphology of wild type cells, but after differentiation stimuli, NF-YAl-KO clones and wild type cells showed different responses. In mESCs, NF-YAl-KO clones maintained the typical stem cell morphology with high levels of stemness genes and low levels of differentiation markers, compared to the wild type cells. In C2C12, after differentiation induction, wild type cells originated the classical myotubes, while the NF-YAl-KO clones maintained the myoblast identity. These results were supported by a low expression levels of muscle-associated genes in NF-YAl-KO clones, compared to wild type cells. These data confirm a different role of the two NF-YA isoforms in stemness maintenance and in particular suggest that the long one has a pivotal role during the differentiation process. The second project has been inspired by the study of NF-YA protein 3D structure that highlighted the presence of two features typical of CPPs in the evolutionary conserveddomain. Previous studies have shown that the GST-TAT-NF-YA short fusion protein stimulated Hematopoietic Stem cells (HSCs) growth enhancing cell proliferation. Moreover, mESCs transfected with the GST-TAT-NF-YA short fusion protein maintained their pluripotency identity even after Leukemia Inhibitory Factor (LIF) withdrawal, which is necessary for mESCs to maintain stemness identity in culture media. The work presented in this thesis demonstrated the NF-YA capability to enter cells and to translocate into nuclei in a TAT- independent manner. Moreover, the differentiation induction of myoblast C2C12 cells after NF-YA short recombinant protein transfection inhibited myotubes formation demonstrating the functionality of the transduced recombinant protein.
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Wang, Kun. "Development of new fibrin-based biomaterials." Electronic Thesis or Diss., Sorbonne université, 2021. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2021SORUS430.pdf.
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La fibrine est largement utilisée en clinique mais les hydrogels formés à partir de cette protéine souffrent généralement d’une faible tenue mécanique et d’une biodégradation très rapide. Pour palier ces limites, nous avons développé trois stratégies. La première consiste à former des matériaux hybrides en ajoutant différentes sources de silice. Une étude en conditions diluées a montré que certains silanes pouvaient favoriser la fibrillogénèse et conduire à une stabilisation des gels. A plus forte concentration de silane ou en présence de silice condensée, l’auto-assemblage de la fibrine est perturbé. La deuxième approche implique l’ajout de nanocristaux de cellulose. Des études en conditions diluées ont mis en évidence l’adsorption du fibrinogène sur les nanocristaux, qui facilite leur alignement. En conditions plus concentrées, un renfort mécanique notable a pu être constaté. De plus un ralentissement de la cinétique de gélification a conduit à de nouvelles formulations injectables. La troisième approche repose sur l’association avec du collagène de type I. En utilisant un procédé de gélification du fibrinogène en conditions acides, il a été possible d’obtenir des gels mixtes mais les propriétés rhéologiques ont été peu modifiées. Pour les trois stratégies adoptées, les matériaux ont été évalués pour leur capacité à promouvoir la prolifération de cellules myoblastes et leur différentiation. Des résultats intéressants ont été obtenus en présence de nanocelullose. Ce travail a donc permis d’élucider les interactions de la fibrine avec différents partenaires inorganiques ou bio-organiques et ouvre de nouvelles perspectives pour son application dans le domaine biomédicalFibrin is widely used in clinic, but hydrogels formed by this protein generally suffer from poor mechanical property and very rapid biodegradation. To overcome these limitations, we have developed three strategies. The first was to form hybrid materials by adding different sources of silanes. The certain silanes at low concentration could promote fibrillogenesis and lead to stabilization of the gels. At a higher concentration of silane or in the presence of condensed silica, the self-assembly of fibrin is disturbed. The second approach involved the addition of cellulose nanocrystals. Studies under dilute condition shown the adsorption of fibrinogen to the nanocrystals, which facilitates their alignment. Composite hydrogels from high concentrated fibrinogen and nanocrystals showed a notable mechanical reinforcement. In addition, a slowing down of the gelation kinetics have led to new injectable formulations. The third approach was to form the interpenetrate network with type I collagen. Through the process of gelation of fibrinogen under acidic conditions, it was possible to obtain mixed gels but the rheological properties of which were little modified. For the three strategies adopted, the materials have been shown to have the ability to promote proliferation and differentiation of myoblast cells. Interesting results have been obtained in the presence of cellulose nanocrystals. This work has therefore made it possible to elucidate the interactions of fibrin with various inorganic or bio-organic fillers and opens up new perspectives for its application in the biomedical field
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Harrison, Adrian Paul. "Regulation of porcine skeletal muscle growth and differentiation." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360936.
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Ge, Xiaomei. "Roles of Growth Hormone, Insulin-Like Growth Factor I, and Sh3 and Cysteine Rich Domain 3 in Skeletal Muscle Growth." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77332.
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Three studies were conducted to achieve the following respective objectives: 1) to determine the cellular mechanism by which growth hormone (GH) stimulates skeletal muscle growth; 2) to identify the signaling pathways that mediate the different effects of insulin-like growth factor I (IGF-I) on skeletal muscle growth; and 3) to determine the role of a functionally unknown gene named SH3 and cysteine rich domain 3 (STAC3) in myogenesis. In the first study, the myogenic precursor cells, satellite cells, were isolated from cattle and allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. GH increased protein synthesis without affecting protein degradation in myotubes; GH had no effect on proliferation of myoblasts; GH had no effect on IGF-I mRNA expression in either myoblasts or myotubes. These data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle. In the second study, the signaling pathways mediating the effects of IGF-I on proliferation of bovine myoblasts and protein synthesis and degradation in bovine myotubes were identified by adding to the culture medium rapamycin, LY294002, and PD98059, which are specific inhibitors of the signaling molecules mTOR, AKT, and ERK, respectively. The effectiveness of these inhibitors was confirmed by Western blotting. Proliferation of bovine myoblasts was stimulated by IGF-I, and this stimulation was partially blocked by PD98059 and completely blocked by rapamycin or LY294002. Protein degradation in myotubes was inhibited by IGF-I and this inhibition was completely relieved by LY294002, but not by rapamycin or PD98059. Protein synthesis in myotubes was increased by IGF-I, and this increase was completely blocked by rapamycin, LY294002, or PD98059. These data demonstrate that IGF-I stimulates proliferation of bovine myoblasts and protein synthesis in bovine myotubes through both the PI3K/AKT and the MAPK signaling pathways and that IGF-I inhibits protein degradation in bovine myotubes through the PI3K/AKT pathway only. In the third study, the potential roles of STAC3 in myoblast proliferation, differentiation, and fusion were investigated. Overexpression of STAC3 inhibited differentiation of C2C12 cells (a murine myoblast cell line) and fusion of these cells into myotubes, whereas knockdown of STAC3 had the opposite effects. Either STAC3 overexpression or STAC3 knockdown had no effect on proliferation of C2C12 cells. Myoblasts from STAC3-deficient mouse embryos had a greater ability to fuse into myotubes than control myoblasts; the former cells also expressed more mRNAs for the myogenic regulators MyoD and myogenin and the adult myosin heavy chain protein MyHC1 than the latter. These results suggest that STAC3 inhibits myoblast differentiation and fusion.Ph. D.
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Miller, Mathew Gordon. "Integrin-linked kinase 1 (ILK1) is necessary for myogenic differentiation in rat L6 myoblasts." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0018/MQ54079.pdf.
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Whitney, Marsha L. "Molecular control of skeletal myoblast proliferation for cardiac repair /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8008.
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Talarico, Alexander Phillip. "Myf5 Does Not Induce Apoptosis In Skeletal Myoblasts But Is Regulated By Oncogenic Ras Expression." Cleveland State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=csu1234402667.
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Cogan, John G. "DNA-binding proteins regulating vascular smooth muscle alpha-actin gene expression in myoblasts and fibroblasts /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487863429091115.
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Engelbrecht, Lize. "Grape seed extract affects adhesion competence and maturation of primary isolated rat myoblasts after contusion injury." Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/80380.
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Thesis (MSc)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, activating a series of cellular events. Satellite cells (SC), the key role players in muscle regeneration, are activated to proliferate and develop into mature myoblasts, which could fuse to form new myotubes or to repair damaged fibres. Evidence suggests that anti-oxidants, such as those found in grape seed extract (GSE), enhance repair, but their effect on SCs is still unclear. This study aimed to harvest and culture primary rat myoblasts to investigate the effect of chronic in vivo GSE supplementation on SCs following a standardised crush injury. Using a modified pre-plate technique, myoblasts were harvested from rat muscle and then compared with the immortal C2C12 cell line for proliferation and differentiation competence. Several media options were compared: i) DMEM with or without L-glutamine, ii) Ham‘s F10 or iii) DMEM with L-glutamine and Ham‘s F10 combined. Primary myoblasts proliferated and differentiated at a much slower rate than C2C12 cells. The combined media was selected for further use. To investigate the effects of GSE on the recovery, rats were supplemented daily with GSE or placebo 14 days prior to a standardised mass-drop crush injury to the gastrocnemius. SCs were isolated and cultured from uninjured (NI, baseline) and from injured rats 4 hours (4h), 3 days (3d) or 14 days (14d) post-injury. Expression of myogenic proteins Pax7, M-cadherin, MyoD, CD56, desmin and CD34 was determined by flow cytometry. Myoblasts were sorted according to their CD56 and CD34 expression and three sub-sets were collected and re-cultured, namely CD56+/CD34-, CD56-/CD34+ and CD56+/CD34+. After 24 hours, sorted cells were stained for desmin expression. Pax7, M-cadherin and MyoD were present in 100% of isolated cells from all groups confirming their myogenic SC identity. For all groups, desmin was expressed only in ~80% of SCs. Lower adhesion competency in GSE supplemented groups resulted in lower yield obtained for culturing. Expression of CD56 increased significantly 3d post-injury in the placebo group. In contrast, with GSE, CD56 already increased 4h post-injury and decreased again 3d post-injury. Although CD34 expression did not differ dramatically, expression pattern resembled that of CD56. Immunocytochemistry revealed a range in morphology and desmin expression of sorted myoblasts. More myoblasts with high desmin expression were observed in the two CD56+ sub-sets (irrespective of CD34 expression), indicating that CD56 is still expressed in more mature myoblasts. Flow cytometry revealed a population of myoblasts expressing particularly high levels of desmin, primarily in the non-injured baseline GSE group. We hypothesise that this result is an indication of preparedness of myoblasts to respond earlier to injury, enabling quicker repair. This cell population with high desmin content is restored in skeletal muscle after repair (14d), only when supplemented with GSE. In conclusion, GSE attenuated adhesion competence of primary myoblasts in culture, but resulted in earlier maturation of SCs, possibly due to baseline preparedness of myoblasts in uninjured muscle for a quick response. Both reduced adhesion competence and early progression of myoblasts could enhance wound healing in skeletal muscle.
AFRIKAANSE OPSOMMING: Kneuswonde veroorsaak aansienlike skade aan skeletspier, wat ‘n reeks sellulêre prosesse in werking stel. Satellietselle, die hoofrolspelers tydens spierregenerasie, vermenigvuldig en ontwikkel tot volwasse mioblaste, wat saamsmelt om nuwe spiervesels te vorm. Antioksidante, soos die wat in druiwepit-ekstrak voorkom, bespoedig herstel, maar hul uitwerking op satellietselle is steeds onduidelik. Die doel van hierdie studie was om mioblaste uit rotspiere te isoleer en te kweek om die effek van langdurige in vivo aanvulling van druiwepit-ekstrak op satellietselle na ‘n kneusbesering te bepaal. 'n Aangepaste protokol is gebruik om primêre mioblaste te isoleer, wat daarna met C2C12 selle, ten opsigte van hul vermenigvuldigings- en differensiasievermoë vergelyk is. Verskeie groeimedia is gebruik: i) DMEM met of sonder L-glutamien, ii) Ham F10 en iii) ‘n kombinasie van DMEM, L-glutamien en Ham F10. Primêre mioblaste het stadiger vermenigvuldig en gedifferensieer as C2C12 selle. Die gekombineerde medium is vir verdere gebruik gekies. Om die uitwerking van druiwepit-ekstrak op spierherstel te ondersoek, is rotte vir 14 dae onderwerp aan daaglikse aanvullings van druiwepit-ekstrak of placebo voor ‘n gestandardiseerde kneusbesering aan die gastrocnemius. Satellietselle is geïsoleer vanuit onbeseerde spier (basiskontrole) en vanuit beseerde spier 4 ure (4h), 3 dae (3d) en 14 dae (14d) na die besering. Die uitdrukking van spierverwante proteïene Pax7, M-cadherin, MyoD, CD56, desmin en CD34 is vasgestel met 'n vloeisitometer. Mioblaste is daarna gesorteer op grond van hul CD56- en CD34-uitdrukking. Drie sub-groepe is versamel en verder gekweek, nl. CD56+/CD34-, CD56-/CD34+ en CD56+/CD34+. Na 24 uur is gesorteerde selle gekleur om desmin-uitdrukking te bepaal. Pax7, M-cadherin en MyoD is deur 100% satellietselle in alle groepe uitgedruk, wat hul spierverwante identiteit bevestig, alhoewel slegs 80% selle in alle groepe desmin uitgedruk. Druiwepit-ekstrak het die vermoë van selle om aan plate te heg onderdruk, wat gelei het tot ‘n laer opbrengs van mioblaste. Drie dae na die besering in die placebo groep het die CD56-uitdrukking beduidend toegeneem. In teenstelling hiermee het CD56-uitdrukking in die druiwepit-ekstrak groep 4 ure na die besering beduidend toegeneem en weer afgeneem na 3 dae. Hoewel daar nie sulke dramatiese verskille was tussen groepe ten opsigte van CD34-uitdrukking nie, was daar ‘n soortgelyke tendens as vir CD56-uitdrukking. Immunositochemie het ‘n verskeidenheid van morfologieë en variërende desminvlakke in gesorteerde mioblaste blootgestel. In die twee CD56+ groepe is meer mioblaste wat hoë desmin vlakke uitdruk gevind, wat aandui dat CD56 uitgedruk word deur meer volwasse mioblaste, ongeag van CD34-uitdrukking. Tydens vloeisitometrie is ‘n populasie selle wat hoë desminvlakke uitdruk, hoofsaaklik in die onbeseerde en 14d druiwepit-ekstrak groepe gevind. Dit is ‘n aanduiding dat sommige mioblaste voorbereid is om na 'n besering vinniger te reageer. Na die herstelproses word hierdie groep selle hernu in die teenwoordigheid van druiwepit-ekstrak-aanvulling. Die resultate het gevolglik daartoe gelei dat druiwepit-ekstrak die hegtingsvemoë van mioblaste verlaag, maar dat die aanvulling in vivo tot vroeër ontwikkeling van mioblaste lei, waarskynlik deur satellietselle voor te berei vir 'n vinnige respons na ‘n besering. Beide die onderdrukking van aanhegting aan kultuurplate en die vroeë ontwikkeling van mioblaste, kan die herstel van die skeletspier verbeter.
NRF and the Harry Crossley bursary for funding
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Horner, Ellias. "Six1 Is Important for Myoblast Proliferation Through Direct Regulation of Ccnd1." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34186.
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The transcription factor Six1 of the sine oculis homeobox family has been tied to skeletal muscle formation. Work completed thus far has allowed our research team to identify the precise mechanism by which Six1 regulates the expression of MyoD, a key myogenic gene, in muscle stem cells. Furthermore, loss-of-function of this protein, mediated by RNA interference, has implicated Six1 as essential towards normal myogenic differentiation. However, beyond Six1 and its involvement towards myogenesis, our data also suggests the transcription factor as a potential regulator of the cell cycle. Data from our lab shows that loss of Six1 expression significantly impairs primary myoblast proliferation and appears to impair satellite cell activation in response to muscle injury in vivo. Furthermore, loss of Six1 decreases the expression of key cell cycle genes. Combining functional genomics approaches such as ChIP-Seq and Gene Expression Profiling together with Gene Ontology Term Enrichment shows a significant representation for biological processes regarding the cell cycle and its regulation; these biological clusters contain a large subset of genes that are bound and modulated by Six1. In particular, Ccnd1 was found to display a similar expression pattern as Six1 in growing myoblasts and its expression was found to be directly controlled by Six1. Furthermore, Ccnd1 over-expression was sufficient to rescue the Six1-knockdown associated cell cycle phenotype. Together, these data suggest that in response to injury Six1 enhances the expression of the cell cycle gene Ccnd1 thus modulating myoblast proliferation for muscle regeneration.
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El, Khatib Nour. "Identification des mécanismes moléculaires et physiopathologiques impliqués dans la dystrophie facioscapulohumérale." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT039.
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La dystrophie musculaire facioscapulohumérale (FSHD) est une maladie autosomique dominante, caractérisée par une faiblesse et une atrophie progressive de certains muscles squelettiques. La FSHD est liée à une répression inefficace de la région des macrosatellites D4Z4 sur le chromosome 4, entraînant l'expression inappropriée dans le muscle squelettique, d’un gène à double homeobox 4 (DUX4), et la dérégulation des gènes avoisinants. La surexpression de DUX4 est responsable du phénotype atrophié des myotubes FSHD et induit la dérégulation de gènes impliqués dans la réponse au stress oxydant. Malgré les avancés majeures dans la compréhension du locus morbide, les mécanismes exacts impliqués dans la FSHD ne sont pas totalement compris et aucun traitement curatif n’est disponible. Cependant, de nombreuses données montrent le rôle prépondérant du stress oxydant dans la FSHD. Récemment, nous avons caractérisé la présence d’un stress oxydant dans les biopsies musculaires et les prélèvements sanguins des patients atteints de FSHD. Nous avons démontré que ce stress est corrélé à une altération de la fonction musculaire chez ces patients et qu’une supplémentation en antioxydants adaptée améliore la fonction musculaire et réduit les dommages oxydatifs. Par ailleurs, nous avons démontré que les myoblastes dérivés des biopsies FSHD sont plus sensibles à des agents pro-oxydants et présentent des défauts de différenciation. L’objectif de nos travaux est de caractériser les mécanismes moléculaires impliqués dans la FSHD afin de faciliter la mise en place d’approches thérapeutiques. Ce projet de thèse original réunit à la fois une approche fondamentale et clinique.Grâce à la mise en place d’un nouveau modèle in vitro de culture primaire de myoblastes de patients atteints de FSHD, nous avons montré la présence d’un stress oxydant dans ces myoblastes corroborant les observations précédemment obtenues aux niveaux systémiques et musculaires chez ces patients. Par ailleurs, les traitements par des agents pro-oxydants (paraquat et peroxyde d'hydrogène) ont un effet différentiel sur l’expression des enzymes antioxydantes par rapport aux contrôles suggérant un défaut dans les mécanismes d'adaptation au stress oxydant chez les patients atteints de FSHD.D’autre part, afin d'améliorer les procédures de réadaptation pour les patients atteints de FSHD, nous avons proposé d'étudier la faisabilité, la sécurité et l'efficacité de l’entraînement de force par électrostimulation neuromusculaire (ESNM) pour contrer la faiblesse musculaire des quadriceps chez ces patients. Cette étude, en cours, semble être une stratégie de réhabilitation prometteuse pour les patients atteints de FSHD et n’a montré aucun effets indésirables jusqu’à présentFacioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease, characterized by progressive weakness and atrophy of specific skeletal muscles. FSHD is linked to an inefficient repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat array on chromosome 4, resulting in the unappropriated expression in skeletal muscle of the double homeobox 4 (DUX4) retrogene. DUX4 overexpression leads to atrophic myotubes phenotype and dysregulation of antioxidant genes. Despite major progress in the understanding of the genetic locus, exact mechanisms that lead to FSHD defects are not completely understood and no curative treatment is available. However, several lines of evidence have proposed oxidative stress and myogenesis defect as the major biological processes affected in FSHD. Recently, we characterized oxidative stress in skeletal muscle biopsies and blood samples from patients with FSHD. We demonstrated that oxidative stress is associated with reduced physical performance in patients with FSHD and that antioxidants adapted strategy was effective to reduce oxidative stress and maintain muscle functions. Furthermore, satellite cell-derived myoblasts from these patients were more susceptible to pro-oxidant agents than control myoblasts and showed a defect in differentiation. The originality of this project relies on creating a synergy between basic and clinical research. The major goal of this work is to identify molecular mechanisms involved in FSHD oxidative stress in order to identify therapeutic approaches.Using in vitro cell model of FSHD, recently developed and optimized in our team, we demonstrate the presence of oxidative stress in FSHD primary myoblast cultures that corroborates previous observations at systemic and muscular levels. Furthermore, treatments with different pro-oxidant agents (paraquat and hydrogen peroxide) have a differential effect on the expression of antioxidant enzymes compared to controls, suggesting a defect in the oxidative stress adaptive response in FSHD myoblasts.Furthermore, in order to improve rehabilitation procedures for patients affected with FSHD, we proposed to investigate the feasibility, safety, and effectiveness of neuromuscular electrostimulation (NMES) strength training to counteract quadriceps muscle weakness in these patients. This ongoing study appears to be a promising rehabilitation strategy and shows no adverse effect for patients with FSHD
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Streppa, Laura. "Characterizing mechanical properties of living C2C12 myoblasts with single cell indentation experiments : application to Duchenne muscular dystrophy." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSEN008/document.
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Cette thèse interdisciplinaire a été dédiée à la caractérisation des propriétés mécaniques de myoblastes (murins et humains) et de myotubes (murins) à l'aide de la microscopie à force atomique (AFM). En modifiant ou en inhibant la dynamique du cytosquelette (CSK) d’actine de ces cellules, nous avons pu montrer que ces propriétés mécaniques variaient. L’enregistrement de courbes de force indentation nous a permis de montrer que la présence de cellules adhérentes introduisait sur les leviers d’AFM un amortissement visqueux supplémentaire à celui d’une paroi solide, et que cet amortissement visqueux dépendait de sa vitesse d’approche et que celui-ci restait non négligeable pour les plus faibles vitesses (1μm/s). Nous avons observé que les propriétés mécaniques des précurseurs de muscles devenaient non linéaires (comportement plastiques) pour des grandes déformations (>1μm) et qu’elles dépendaient de l’état, du type de cellule et de leur environnement. En combinant des expériences d’AFM, des modèles visco-élastiques et des méthodes d'analyse multi-échelle basées sur la transformation en ondelettes, nous avons illustré la variabilité des réponses mécaniques de ces cellules (de visco-élastiques à visco-plastiques). À l'aide de courbes de force-indentation, de l’imagerie morpho-structurale (DIC, microscopie à fluorescence) et de traitements pharmacologiques, nous avons éclairé le rôle essentiel des processus actifs (dépendants de l’ATP) dans la mécanique de myoblastes, en discutant tout particulièrement ceux des moteurs moléculaires (myosine II) couplés aux filaments d’actine. En particulier, nous avons montré que les fibres de stress du cytosquelette d’actine situées autour du noyau pouvaient présenter des évènements de remodelage soudains (ruptures) et que ces ruptures étaient une mesure indirecte de l’aptitude de ces cellules à tendre leur CSK. Nous avons enfin montré qu’il était possible de généraliser cette approche à des cas cliniques humains, en l’occurrence des myoblastes primaires de porteurs sains et de patients atteints de dystrophie musculaire de Duchenne, ouvrant la voie à des études plus larges sur d’autres types cellulaires et pathologiesThis interdisciplinary thesis was dedicated to the atomic force microscopy (AFM) characterization of the mechanical properties of myoblasts (murine and human) and myotubes (murine). We reported that the mechanical properties of these cells were modified when their actin cytoskeleton (CSK) dynamics was inhibited or altered. Recording single AFM force indentation curves, we showed that adherent layers of myoblasts and myotubes introduced on the AFM cantilever an extra hydrodynamic drag as compared to a solid wall. This phenomenon was dependent on the cantilever scan speed and not negligible even at low scan velocities (1μm/s). We observed that the mechanical properties of the muscle precursor cells became non-linear (plastic behaviour) for large local deformations (>1μm) and that they varied depending on the state, type and environment of the cells. Combining AFM experiments, viscoelastic modeling and multi-scale analyzing methods based on the wavelet transform, we illustrated the variability of the mechanical responses of these cells (from viscoelastic to viscoplastic). Through AFM force indentation curves analysis, morpho-structural imaging (DIC, fluorescence microscopy) and pharmacological treatments, we enlightened the important role of active (ATP-dependent) processes in myoblast mechanics, focusing especially on those related to the molecular motors (myosin II) coupled to the actin filaments. In particular, we showed that the perinuclear actin stress fibers could exhibit some abrupt remodelling events (ruptures), which are characteristic of the ability of these cells to tense their CSK. Finally, we showed that this approach can be generalized to some human clinical cases, namely primary human myoblasts from healthy donors and patients affected by Duchenne muscular dystrophy, paving the way for broader studies on different cell types and diseases
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Gu, Jinmo. "An NF-kappaB - EphrinA5 - Dependent Communication between NG2+ Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1458152802.
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Chung, Kevin. "Augmentation of alignment and differentiation in C2C12 skeletal myoblasts through use of nano-to-microscale biochemical patterns." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p1467778.
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Thesis (M.S.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed September 15, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 71-76).
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Powell, Gareth Thomas. "The zebrafish homologues of JAM-B and JAM-C are essential for myoblast fusion." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609399.
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Williams, Drew. "Identification of a protective role for mitochondrial deoxyuridine triphosphate nucleotidohydrolase against oxidative stress-induced apoptosis in mouse myoblasts." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95249.
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Cardiac ischemia results in cardiomyocyte death in part due to apoptosis. Gene therapeutic approaches using the introduction of anti-apoptotic sequences directly into the infarcted heart is being evaluated for the therapeutic value of limiting cell death. In spite of many advances in human genomics, the complete repertoire of cardiac anti-apoptotic genes remains unknown. To increase our understanding of cardiac apoptosis, it is essential that we work towards indentifying and characterizing the repertoire of anti-apoptotic sequences in the human heart. Our laboratory has exploited the observation that many of the processes involved in mediating apoptosis are evolutionary conserved between metazoans and yeast. By screening a human cardiac cDNA library in yeast cells conditionally expressing mammalian pro-apoptotic Bax, our laboratory has identified over 60 clones capable of preventing the deleterious effects of Bax in yeast. The research presented here looks beyond yeast to assess the possible anti-apoptoic effects of these clones in mammalian cells. The protein evaluated here is deoxyuridine triphosphate nucleotidohydrolase (dUTPase). We show that in addition to the anti-apoptotic effects in yeast, dUTPase demonstrates an anti-apoptotic effect in mammalian C2C12 myoblasts subject to oxidative stress-induced apoptosis. These results serve to further exemplify the similarities between the process of anti-apoptosis in yeast and human cells. In addition, dUTPase may have therapeutic value of limiting cell death in the infracted heart.L'ischémie cardiaque provoque la mort des cellules myocardiques en partie en raison de l'apoptose. Des thérapies géniques basées sur l'introduction directe des gènes anti-apoptotiques dans l'infarctus du myocarde sont actuellement en train d'êtres évalués à leur capacité de limiter la mort cellulaire programmée après une crise cardiaque. En dépit du progrès de la génétique humaine, le répertoire complet des gènes cardiaques anti-apoptotiques reste peu connu. Afin d'accroître notre compréhension de l'apoptose cardiaque, il est essentiel d'identifier et caractériser le répertoire des gènes anti-apoptotiques dans le cur humain. En effet, il a été prouvé que le mécanisme moleculaire de l'apoptose est conservé le long de l'évolution entre les métazoaires et la levure. En examinant les effets anti-apoptotiques d'une banque d'ADNc cardiaque dans des cellules de levure exprimant conditionnellement le pro-apoptotique Bax des mammifères, notre laboratoire a identifié plus de 60 clones capables de prévenir les effets délétères de Bax chez la levure. Le présent travail montre également les effets potentiels anti-apoptotiques de ces gènes chez des cellules mammaliennes. Nos resulats ont prouvé aussi que la protéine désoxyuridine triphosphate nucleotidohydrolase (dUTPase) a un effet anti-apoptotique en prévenant le processus apoptotique induite par le stress oxydatif dans les myoblastes mammaliennes C2C12. Ces résultats servent à illustrer les similitudes entre le processus anti-apoptotique chez la levure et les cellules humaines et, indiquent que la dUTPase pourrait avoir un intérêt thérapeutique afin de limiter la mort cellulaire dans les infarctus cardiaques. fr
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Penton, Christopher M., Vasudeo Badarinarayana, Joy Prisco, Elaine Powers, Mark Pincus, Ronald E. Allen, and Paul R. August. "Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion." BIOMED CENTRAL LTD, 2016. http://hdl.handle.net/10150/622727.
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Background: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. Methods: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. Results: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. Conclusions: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.