No description available.

No description available.

O-mannosylation is a specific form of glycosylation, a post-translational protein modification with O-linked mannose attached to serine or threonine residues. O-mannosylation is implicated in crucial biological processes such as neuronal and muscle development, cell adhesion and cell migration. Two O-mannosyltransferase genes have been described in mammalian genomes so far, POMT1 and POMT2. Disruptions of O-mannosylation result in congenital muscular disorders in humans. The most severe, the Walker-Warburg Syndrome is associated with mutations in POMT1 and POMT2. Just like vertebrates, Drosophila has two O-mannosyltrasferase genes, DmPOMT1 (rt) and DmPOMT2 (tw), which share significant similarities with their mammalian counterparts. Mutations in both DmPOMT1 and DmPOMT2 cause the "rotated abdomen" phenotype, a clockwise rotation of abdominal segments in adult flies. In my dissertation, I analyzed the expression patterns of rt and tw during development. Both genes have similar essentially overlapping expression patterns. Immunostaining revealed that RT and TW proteins are co-localized in the ER compartment. The analysis of double mutants revealed a mutual epistatic relationship between rt and tw, which could be evidence for RT and TW functioning in the same molecular complex. Also, I studied temporal and spatial requirements of tw during development. I found a broad "developmental window competent to fully rescue the abdomen rotation in adult flies." The spatial studies of tw requirements demonstrated that tw expression is pattern-dependent and the function of tw is cell-autonomous or it has a very short-range effect. The analysis of rescue results with different drivers suggested that the tw requirement is not strictly limited to larval epidermis or muscles alone, but required a contribution from epidermal and muscle cells with a possible involvement of CNS. I have shown that Drosophila Dystroglycan is modified with mannose in the presence of RT-TW enzymatic complex in vivo and in vitro. The co-expression of RT and TW is required to generate high-molecular-mass bands of DG. The lectin staining revealed differences in glycan compositions of DG purified from different genetic backgrounds. Overall, this research work established Drosophila as a model system to study mannosylation, which may shed light on mechanisms of muscular dystrophies in humans.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder affecting 1 out of every 3500 male births. The underlying cause of DMD is mutations within the dystrophin gene resulting in loss of protein expression, which leads to myofiber instability and damage. The constant damage of skeletal muscle causes sustained immune infiltration, marked by increased levels of cytokines, such as TGF-beta. Interestingly, TGF-beta can decrease the myogenic potential of satellite cells, thus preventing muscle regeneration. Previously, our lab has shown that knockdown of the Ste20 Like Kinase, SLK, in normal mammary epithelial cells was sufficient to delay TGF-beta induced epithelial to mesenchymal transition. Therefore, we speculated that decreasing SLK levels would be sufficient to decrease the anti-myogenic effects of TGF-beta both in cultured myoblasts and in a mouse model of muscular dystrophy. In the first section of this study, we explored the effect of muscle specific deletion of SLK on muscle development and regeneration. Skeletal muscle specific deletion of SLK did not impair muscle development, but caused a myopathy in older mice. Additionally, muscle regeneration was delayed, but not inhibited by SLK deletion. These findings indicated that SLK has beneficial roles in skeletal muscle, but was not absolutely required for optimal muscle development and regeneration. In the second section, we investigated the potential for SLK knockdown to mitigate the anti-myogenic effects of TGF-beta in vitro. Decreasing levels of SLK restored myoblast differentiation in the presence of TGF-beta in a p38 dependent manner. In the final section, we determined that SLK levels are elevated in dystrophic muscle and that subsequent deletion of SLK in the mdx mouse enhances terminal differentiation of myoblasts without further exacerbating the pathology of the disease. Collectively, this work demonstrates that SLK inhibition can provide a protective effect against the anti-myogenic effects of TGF-beta via upregulation of p38 activity.

The history of human muscular dystrophy with reference to clinical, histological and biochemical studies is reviewed. The value of the recent discovery of an experimental mouse with hereditary muscular dystrophy with clinical, histological, and biochemical similarities to human muscular dystrophy is discussed. The histology of normal muscle, with special reference to ultrastructure, is also reviewed. For this investigation mouse muscle samples for the electron microscope were fixed in Palades osmic acid solution, embedded in methacrylate or epon and stained with either lead hydroxide or phosphotungstic acid. A method for obtaining one day old muscle samples, while keeping the animals alive until the dystrophic symptoms are noted clinically, is outlined. Light microscope sections were obtained from electron microscope blocks and examined after staining with toluidine blue. Experimental results by light and electron microscope observations showed there to be no histological differences between one day old dystrophic and normal muscle. However one day old muscle showed histological differences compared to older muscle. The chief differences were the smaller size of fibers, random distribution of mitochondria and enlarged nuclei, and the presence of abundant interfibrillar sarcoplasm with a conspicuous granular components. The regular repeating pattern of the endoplasmic reticulum of adult muscle fibers was hot seen. Some "atypical" fibers showed similarities to altered fibers seen in older dystrophic mice. The mitochondria were swollen and vacuolated with few cristae and pale matrix. Endoplasmic reticular components were vacuolated and adjacent myofibrils were disorganized. Atrophy of fibers was first noted at 14 days of age and were conspicuous at 63 days. By 63 days alterations were also noted in mitochondria and endoplasmic reticulum and these changes became more prominent with the progress of the disease. Atrophy of the myofibrils was evident and the Z-bands were often irregular and out of register. Connective tissue also increased greatly. Several miscellaneous structures are also discussed. The histological findings are compared to those found by other workers. Several suggestions put forward by others as to the possible cause of the disease are summarized. On the basis of morphological findings it is suggested that ribonucleo-protein synthesis in the nuclei of dystrophic muscle fibers is increased but that there may be some intermediate stage at fault which prevents the conversion of amino acids to myofilaments. / Medicine, Faculty of / Graduate

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Robea Balle, Department of Human Genetics, MRC Unit for Skeletal Disorders, UCT Medical School, Observatory, Cape Town, South Africa. Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder, characterised by progressive muscle wasting and weakness. DMD has its onset in early childhood, leading to physical handicap by the mid-teens and usually death by the age of twenty years. Becker muscular dystrophy (BMD) is the allelic form of DMD and is differentiated by its age of onset and milder phenotype. DMD and BMD are incurable and the most effective way of managing affected families is by preventing the recurrence of the di9order. The use of intragenic and closely linked flanking markers facilitates the identification of the defective X chromosome in female carriers and their affected male foetuses. DMD is thought to be the most common of the heritable muscle disorders, having an incidence of l in 3 300. When extrapolated to the large South African population, it presents a significant socioeconomic problem. For this reason, it was decided to develop a molecular genetic service for carrier identification and diagnostic predictions. The first step in the South African study involved the collection of biological material from affected individuals. In so doing, minimum prevalence's in the four major ethnic groups of Black, Caucasian, Indian and Mixed ancestry, were established. Although ascertainment was incomplete for a number of reasons, a markedly increased DMD frequency in the Indian population and a low frequency in the Black population was apparent. In the Caucasian group, an unexpectedly high BMD frequency, compared to DMD, was observed. 110 males affected with DMD and 18 with BMD were screened for deletions using genomic and cDNA probes and multiplex polymerase chain reaction (PCR) technology. Deletions were detected in the dystrophin gene of 47 DMD and 6 BMD patients, occurring predominantly in the 3' region of the gene (65%) and to a lesser extent in the 5' region of the gene (287.). The deletion frequency within individual ethnic groups,

Duchenne muscular dystrophy (DMD) is the most common type of muscular dystrophy caused by the loss of functional dystrophin. DMD is characterized by scoliosis, muscle wasting, loss of ambulation and a reduced life span. The first line of treatment for DMD is glucocorticoids (GCs). GCs are prescribed primarily for their anti-inflammatory and immunosuppressive effects; however, GC treatment is known to cause significant muscle atrophy. In DMD, GC treatment has been shown to improve muscle strength for the first 6 months and stabilization of the disease for up to 3 years. However, long term treatment reduces muscle function and accelerates disease progression. It is paradoxical that we use a medication that causes muscle wasting to treat a muscle wasting disease. The regeneration and function of muscle is dependent on the proper regulation and functioning of muscle satellite cells (MuSCs) to restore and repair muscle tissue. The impact GCs have on MuSCs from activation to proliferation and differentiation into muscle fibers is not well understood. GCs have many mechanisms of action by acting as a ligand to the glucocorticoid receptor (GR) to cause downstream effects by direct DNA binding or indirectly by regulating proteins. To study the role of GCs, we examined the effects of GC treatment on myoblast morphology, the cytoskeletal network, post-translational modifications (PTMs) of tubulin subunits, and the organization of microtubule organizing centers (MTOCs) in proliferating and differentiating myoblasts. This study shows that the GR is an essential regulator of myotube morphology and proper myonuclei placement. Furthermore, dexamethasone (DEX) treatment causes branching of the MT network, as well as an increase in the expression of the stabilizing MT markers, acetylated and detyrosinated tubulin during early differentiation. DEX treatment was also found to misposition the Golgi complex, a primary MTOC for the cytoskeletal network, from the periphery of the nucleus to the center of the nucleus during early differentiation. Finally, we found very few differentially expressed genes between WT and GRMuSC-/- myoblasts between early and late differentiation, indicating that these morphological defects we see are not due to GCs regulating gene expression. Thus, GCs act through the GR to modify the MT network during early differentiation, causing morphological changes in myoblasts that persist throughout differentiation.

No description available.

No description available.