No description available.

No description available.

No description available.

No description available.

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.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vi, 94 p. Includes abstract. Includes bibliographical references.

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.

Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration have yet to be investigated. Here, I show that Panx1 levels increase during development, becoming highly expressed in adult skeletal muscle. A switch in Panx3 expression pattern was observed as its ~70 kDa immunoreactive species was mainly expressed in embryonal and neonatal muscles while its ~40 kDa species was the main form expressed in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. Since Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, I next used mild and severe mouse models of this disease and found a significant down-regulation of both Panx1 and the lower MM form of Panx3 in dystrophic skeletal muscles, with an increase in the ~70 kDa immunoreactive species of Panx3. I also found that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Indeed, in vitro PANX1 levels may be increasing during human primary satellite cell differentiation and blocking PANX1 channel activity with the pharmacological compounds probenecid or carbenoxolone inhibited the differentiation and fusion of these satellite cells into myotubes. In addition, satellite cell proliferation was inhibited by probenecid and carbenoxolone. These findings are the first to demonstrate that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration and dystrophy. In addition to our laboratory’s previous reports, I now demonstrate that PANX1 levels may be modulated during satellite cell differentiation and that PANX1 channels regulate satellite cell differentiation and proliferation. Altogether, my studies suggest that Panx1/PANX1 and Panx3/PANX3 channels may play important and distinct roles in myoblasts and satellite cells in healthy and diseased skeletal muscles.