No description available.

The goals of this dissertation were to explore the role of the Ca2+-sensitive transcription factor NFAT in the regulation of smooth muscle contractility. We have identified a conserved NFAT binding site that overlaps an intronic SRF-binding CArG element that has previously been demonstrated to be essential for expression of smooth muscle α-actin. Transfection of a reporter construct containing the composite CArG/NFAT element, designated SNAP, into SMCs resulted in robust basal reporter activity that was sensitive to the calcineurin/NFAT pathways inhibitors FK506 and CsA. Mutations to either the NFAT or adjacent SRF binding site essentially abolished reporter activity, indicating that both were required. Co-immunoprecipitation assays revealed that NFATc3 and SRF formed a complex in solution, and that the formation of this complex was facilitated by the presence of the SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased α-actin expression in cultured SMCs, suggesting that NFAT plays a role in the expression of smooth muscle contractile proteins such as α-actin. To determine if NFAT transcriptional activity is involved in modulating urinary bladder smooth muscle contractility, we compared the contractile and electrophysiological properties of NFATc3-null mice to wild-type mice. UBSM strips taken from NFATc3-null mice displayed an elevated contractile response to EFS compared to strips from wild-type mice. This increased contractility was due to a decrease in IBTX-sensitive BK current and was supported at the molecular level by reduced expression of mRNA for the pore-forming α-subunit of the BK channel. Single-channel recordings revealed that the β-subunit of the BK channel, which modulates the sensitivity of the BK channel to voltage and Ca2+, was not altered. Interestingly, TEA-sensitive KV currents, and expression of the pore-forming KV2.1 subunit, were increased in the NFATc3-null myocytes. However, the increased contractile response of UBSM strips from NFATc3-null mice indicates that, at least in response to electrical field stimulation, the downregulation of BK current plays a more significant role than does the increase in KV current. Presumably, this is due to the prominent role that BK channels play in shaping the UBSM action potential. Thus, this dissertation provides evidence that NFAT plays a role in modulating smooth muscle contractility via its role in regulating the expression of contractile proteins and ion channels and, furthermore, lays the foundation for future investigations into the specific role of NFAT in the pathological response of the urinary bladder to outlet obstruction.

No description available.

M.Sc. (Physiotherapy), Faculty of Health Sciences, University of the Witwatersrand, 2008. / Paediatric Human Immunodeficiency Virus (HIV) remains a significant challenge to children and caregivers in South Africa. Although the availability of antiretroviral (ARV) therapy has improved, it is not yet universally accessible. Rates of transmission from mother to child thus remain high and the virus widely uncontrolled. One aspect affecting children infected with HIV is that of muscle strength. For children weakness has been inferred by way of developmental studies in young children infected with HIV. Impaired performance in activities such as standing, walking, stair-climbing and jumping have been noted. These gross motor activities require higher muscle outputs and strength against gravity. This study sought to ascertain the feasibility of a full study on muscle strength in children infected with HIV. It analysed the effect of HIV on muscle strength, height and weight of those children receiving and not receiving highly active antiretroviral therapy (HAART). Children were recruited from Harriet Shezi Children’s HIV Clinic at Chris Hani Baragwanath Hospital, Soweto, Gauteng Province, South Africa. The study population included a group of children receiving HAART (n=16) and a group of children not receiving HAART (n=16). A once off test of muscle strength was administered to each child using a hand-held dynamometer. A demographic questionnaire and the Household Economic and Social Status Index (HESSI) were administered to their primary caregiver. Results showed the sample population to be of low socio-economic status (average score=54%) and the children to be underweight and short for their age (p<0.001). The CD4 count of the group on HAART was significantly higher than the group not receiving HAART (p<0.05). The group not receiving HAART was significantly stronger than the HAART group (p<0.05). Length of time having received HAART and muscle strength showed no significant correlation (p=0.647). No significant correlation was shown between CD4 count and muscle strength in the group receiving HAART (p>0.1). A significant negative correlation was shown between CD4 count and muscle strength in the group not receiving HAART (p<0.05). As statistically significant normative muscle strength data for children not infected with HIV in this age group fails to exist, the study was unable to ascertain a quantitative measure of weakness in these children. Comparison of those values available, however, showed normative values to be double that of children who participated in the study. The implications of these findings are that as one observes this group of children’s CD4 count drop, so too does their muscle strength. HAART, once initiated, stems the decrease in muscle strength over a period of time but does not reverse it. Furthermore, children and caregivers who participated in this study were faced with the adversities of poor socioeconomic status, limited access to medication and ARV treatment and inadequate nutritional intake, most of which were largely beyond their immediate control. This pilot study has indicated the feasibility and importance of a full study to investigate the muscle strength of children infected with HIV. Further research is needed to establish the impact of earlier administration of HAART on muscle strength. The effect of exercise on the muscle strength of children who are infected with HIV has yet to be documented. The implication of these factors on gross motor development in children infected with HIV has yet to be investigated.

Thesis (Master of Science)--University of the Witwatersrand, Faculty of Health Sciences, 1963. / The rapid progress of the last decade has made possible the synthesis of the molar and molecular approaches to the mechanism of muscle contraction. It is now possible to offer a molecular explanation for many of the gross mechanical properties of muscle. As a result there is a necessity to re-examine the validity of the classical terminology used to describe these properties, and to define them more accurately. Since Pick (1882), various "types” of contraction have been ascribed to muscle, according to the changes in length and tension of the activated muscle. These are dependent upon the load opposing the muscle. If the load is less than the force developed in the muscle, shortening occurs at a constant tension just exceeding the load. This process is termed isotonic contraction. If, on the other hand, the load is equal to the tension developed in the muscle,there is no overall change in length, although tension in the system rises. This constitutes an isometric contraction. Recently, studies of the ultra-structure and mechanical properties of muscle have revealed inherent difficulties in the classical terminology. / WHSLYP2017

Duchenne Muscular Dystrophy (DMD) is a disorder caused by mutations in the dystrophin gene, preventing the production of the functional dystrophin protein which assures maintenance of the myofiber integrity throughout muscle contraction. A lack of dystrophin results in severe muscle degeneration and regeneration accompanied by a loss of muscle function. Many pre-clinical and clinical studies are focused on developing strategies to counteract the detrimental effects of DMD; however, there is no cure. One such approach consists of upregulating the endogenous protein utrophin A in dystrophic muscle, which, once highly expressed at the sarcolemma, could functionally compensate for the lack of dystrophin. Recent evidence demonstrates that utrophin A expression is regulated at its 3’ and 5’UTR through post-transcriptional and translational events. Therefore, in the work presented here, we hypothesized that repurposing FDA-approved drugs that target the signaling pathways involved in post-transcriptional and translational regulation of utrophin A will be an efficient approach in rapidly bringing new therapeutic interventions for DMD. In this work, we repurposed four promising FDA-approved drugs able to stimulate utrophin A expression levels in dystrophic muscles: the anti-coagulant drug Heparin, the anti-inflammatory drug Celecoxib, the β-adrenergic receptor blocking agent Betaxolol and the cholesterol-lowering drug Pravastatin. These drugs induce significant improvements in the dystrophic phenotype of mdx mice. This includes amelioration of muscle fiber integrity and muscle function as well as promoting morphological and fiber type changes in mdx mice muscles. Collectively, this thesis describes the potential of a repurposing approach to activate key post-transcriptional and translational pathways involved in utrophin A’s regulation in the hopes of developing new therapeutics for the treatment of DMD.

"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T19:19:46Z : No. of bitstreams: 1 aguiar_af_dr_botib.pdf: 679778 bytes, checksum: c5aff2254c12cb4b62de64431ebf700b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Embora fortes evidências demonstrem que os fatores de regulação miogênica (MRFs) e o fator de crescimento semelhante à insulina (IGF-I) apresentem um importante papel na resposta hipertrófica após treinamento resistido (TR) agudo, permanece desconhecido se a resposta dos MRFs e IGF-I também ocorre durante a adaptação ao TR a longo-prazo. Portanto, o objetivo deste estudo foi testar a hipótese de que a resposta hipertrófica e modulação das fibras do músculo esquelético após TR a longo-prazo poderia estar associada ao aumento na expressão gênica dos MRFs e IGF-I. Ratos Wistar (80 dias de idade, 250-300 g) foram divididos em quatro grupos: Controle 8 semana (C8, n = 8), Treinado 8 semanas (T8, n = 8), Controle 12 semanas (C12, n = 8) e Treinado 12 semanas (T12, n = 8). Os grupos T8 e T12 foram submetidos a um programa de TR progressivo (3 dias/semana) durante 8 e 12 semanas, respectivamente. O protocolo de treinamento consistiu de quatro séries de 10-12 repetições, com um período de descanso de 40 segundos entre cada série, realizado a 65-75% de uma repetição máxima (1RM). Ao término do experimento, os animais foram sacrificados e o músculo plantar coletado para as análises morfológica e molecular. O TR durante 8 e 12 semanas não promoveu qualquer alteração (p > 0,05) significante no ganho de peso corporal e consumo alimentar dos grupos T8 e T12 em relação aos grupos C8 e C12, respectivamente. Após 8 e 12 semanas de TR, a força absoluta (T8: 69,7% and T12: 126,0%, p < 0,05) e relativa (T8: 36,1% and T12: 57,7%, p < 0,05) foi significantemente elevada nos grupos T8 e T12, em comparação aos seus respectivos controles. No entanto, houve um similar aumento da área de secção transversal (AST) das fibras musculares (T8: 29% vs. T12: 35%, p > 0,05) entre os grupos T8 e T12, comparados aos grupos C8 e C12, respectivamente... / Although strong evidence show that the myogenic regulatory factors (MRFs) and insulin-like growth factor (IGF-I) have important roles in the hypertrophy response after acute resistance training, it is still unclear if response of MRFs and IGF-I also occurs during the adaptation to prolonged periods of resistance training (RT). Therefore, the purpose of this study was to test the hypothesis that fiber-types transition and hypertrophy during long-term RT could be associated with increased MRFs and IGF-I mRNA expression in the skeletal muscle. Male Wistar rats (80 days old, 250-300 g) were divided into four groups: 8 weeks control (C8, n = 8), 8-weeks trained (T8, n = 8), 12-weeks control (C12, n = 8), 12-weeks trained (T12, n = 8). T8 and T12 groups were submitted to a progressive RT program (3 day/week) for 8 and 12 weeks, respectively. The training protocol consisted of four sets of 10–12 repetitions, with a 40 s rest period between each set, performed at 65–75% of one repetition maximum (1RM). At the end of the experiment, animals were sacrificed and the plantaris muscle collected for morphological and molecular analysis. The RT did not change (p > 0.05) in body weight gain and food intake in the T8 and T12 compared to the C8 and C12 groups, respectively. After 8 and 12 weeks of RT, the absolute (T8: 69.7% vs. T12: 126.0%; p < 0.05) and relative (T8: 36.1% vs. T12: 57.7%; p < 0.05) strength (relative 1RM) was significantly elevated in the T8 e T12 groups, compared to respective control groups. RT for 8 and 12 weeks induced similar increase in myogenin (T8: 44.8% vs. T12: 37.7%; p > 0.05), MyoD (T8: 22.9% vs. T12: 22.3%; p > 0.05) and muscle fiber crosssectional area (CSA) (T8: 29% vs. T12: 35%; p > 0.05) in the T8 and T12, compared to C8 and C12 groups, respectively. After 8 weeks of RT, IGF-I increased in 30.1% in the T8 compared to C8 group, but returned to baseline after 12 weeks... (Complete abstract click electronic access below)

Myogenesis, or skeletal muscle formation, begins during embryogenesis and involves the proliferation of myoblasts followed by their exit from the cell-cycle to differentiate and form myotubes. This formation of skeletal muscle is a complex process involving many genes and various signalling pathways. Mighty is a novel myogenic gene discovered at AgResearch by the Functional Muscle Genomics (FMG) group in a genetic screen performed on the muscle of myostatin null and wild-type mice. It was found that heavily muscled mice, lacking myostatin, had increased expression of the mighty gene. This gene was found to be conserved, with cognates found in mammals, amphibians, teleosts, and arthropods. Mighty was found to be expressed in a variety of tissues, but only skeletal muscle showed increased mighty mRNA expression in myostatin null mice, indicating the specific regulation of mighty by myostatin in skeletal muscle (Marshall, 2005). The aim of this study was to characterise the mighty protein and examine its role in myogenesis to elucidate mighty's function. To undertake this study, antibodies specific for the full-length mighty protein and antibodies specific for a peptide region of mighty were characterised. Results using these antibodies, showed endogenous mighty, from myoblasts, to be a low-abundant, nuclear protein which shows a mobility of ~52 kDa in SDS gels, different to that of recombinant mighty protein. The mobility difference of endogenous mighty compared to recombinant mighty appears to be due to phosphorylation and may involve other post-translational modifications. In agreement, the determined isoelectric point (~5.7) of endogenous mighty also appears to be the result of phosphorylation. Interestingly, 52 kDa mighty was not detected in muscle extracts, but a ~30 kDa protein was specifically detected, indicating multiple forms, and subsequent roles, for mighty protein. Mass spectrometry (MS) was also performed for further characterisation of the mighty protein and possible post-translational modifications. Although hits were achieved with both recombinant mighty proteins, endogenous mighty MS analysis was not accomplished due to its low-abundance. The function of the mighty protein in myoblasts was investigated during proliferation and differentiation. The results indicate that proliferating myoblasts have low levels of mighty in G0 and increased levels in G1/S during the cell cycle. This differential expression of mighty may involve cell cycle exit at the G1/S phase. Differentiation results showed mighty to be upregulated before MyoD during differentiation, placing mighty very early in the differentiation hierarchy. This agrees with previous results by Marshall (2005) which showed mighty to upregulate MyoD through IGF-II expression. Enhanced differentiation was also seen in double muscle bovine myoblasts concomitantly with increased mighty expression. In conclusion, mighty appears to be a post-translationally modified protein that plays an early role in myogenic differentiation. This role in differentiation appears to be upstream of MyoD through the upregulation of IGF-II and may be linked to cell cycle exit in the G1 phase of the cell cycle.

Copies of author's previously published articles inserted. Bibliography: leaves 172-204. H-relexes are used to determine the reflex connections of muscle spindle afferents, the exitability of the motorneuron pool and the integrity of the reflex pathways. However, H-relexes are small and can be difficult to elicit in the masseter, limiting their use in the investigation of the masticatory system. This study investigated the recruitment of masseter motorneurons into the H-reflex, compared to the recruitment occuring during voluntary isometric biting, to determine the distribution of the effective muscle spindle input.