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Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains x, 66 p.; also includes graphics. Includes bibliographical references (p. 57-61).

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 92-97.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 77-81).

(I) Recent research on Duchenne muscular dystrophy lends support to the notion that the genetic defect may affect the surface membranes of not only muscle cells but other cells particularly erythrocytes. Abnormalities in the content and compositions of fatty acids and phosphorylations of some erythrocyte membrane laroteins have been reported in support of this hypothesis. SDS gel electrophoresis patterns of erythrocyte membrane proteins from Duchenne muscular dystrophy were reported to be normal. In this report, SDS gel electrophoresis of erythrocyte membrane proteins prepared by hypotononic lysis of red blood cells at pH8, were not able to show any differences between samples from Duchenne muscular dystrophy patients and those from normal control subjects. The relative intensities of the protein bands varied from preparation to preparation but did not follow a pattern that could distinguish control samples from test samples. Two dimensional gel electrophoresis of completely denatured erythrocyte membrane proteins resolved over 150 peptides in 9% gels and over 200 peptides in gels above 12%, but no consistent differences were observed between Duchenne muscular dystrophy samples and those from control subjects. (II) Although cystic fibrosis is the most frequent lethal genetic syndrome among caucasian children, there is no known biochemical or structural defect to account for all its pathophysiological phenomena. Previous reports in other laboratories have shown that a cystic fibrosis protein (CFP) could be demonstrated in sera of over 90% of cystic fibrosis homozygotes by isoelectric focusing. The protein was characterised as having a molecular weight of between 3000-10 000 and pi near pH8.4. Our isoelectric focusing results demonstrated the protein, pI8.5, in 40% of the homozygotes, thus supporting the view that the procedure is not a useful diagnostic tool and that the "cystic fibrosis protein" is not a specific marker for the disorder. We extended this investigation by labellings, with 125I, all the serum Proteins that focused between pH8 and pH9 and then running the labelled proteins in some 15% SDS gels. The results we obtained could not distinguish cystic fibrosis samples from normal control ones. Some studies have suggested that a mutant form of alpha-2-macroglobulin could be the primary defect in cystic fibrosis and also in multiple sclerosis. To test for the presence of charge or size mutations, alpha-2-macroglobulin was subjected to extensive investigations by (i) SDS gel electropihoresis of its heat fragments, (ii) two dimensional gel electrophoresis following complete denaturation and (iii) immune- electrofocusing of its native form. Alpha-2-macroglobulin from cystic fibrosis patients could not be distinguished from that obtained from normal control subjects but that from multiple sclerosis patients showed some different patterns. The significance of the abnormality of ?2M from multiple sclerosis patients needs further investigations. Extensive two dimensional gel electrophoresis of whole serum following complete denaturation were performed but the results did not reveal any consistent differences between cystic fibrosis and multiple sclerosis patients and normal control subjects.

The words of Gowers are as true today as they were nearly a century ago. My interest in muscular dystrophy started in 1957 when, as Senior House Officer at Queen Mary's Hospital for Children, Carshalton, Surrey, I first observed a large number of children suffering from this tragic disease. The frustration of helplessly, watching its inevitable course stimulated the present study.After obtaining an initial impression or' the collection from my clinical observations and a review of the hospital records between 1920 and 1950, I started a more systematic enquiry. The present investigation compromises my personal observations on 65 cases ranging in age from 3 to 16 years. Of these, 57 were seen and followed up at Queen Mary's Hospital for Children, and 8 at the Southern Hospital, Dartford, Kent.

Duchenne Muscular Dystrophy (DMD) is a severe X-linked progressive muscle wasting disease resulting from the absence of the membrane-associated protein dystrophin and the secondary components of the dystrophin-glycoprotein complex. Although the genetic basis of the disease has been known for over 15 years, the onset mechanism of the disease is not yet known and no treatment is yet available to significantly increase the lifespan of DMD patients. Increased levels of intracellular calcium have been noted in dystrophic muscle (Turner et al., 1991) and increased intracellular levels of calcium in skeletal muscle lead to increased levels of calcium-dependent proteolysis (Zeman et al., 1985). Increased levels of calpain, a calcium-dependent protease have been reported as early as age 4 weeks in mdx (dystrophin-deficient) mice (Spencer et al., 1995). Increased calpain activity has been demonstrated in mdx myotubes (Alderton et al., 2000a). There is also evidence of a role for calpain in DMD, but the contribution of calpain activity to the onset of DMD has not yet been determined. The purpose of this study was to test the hypothesis that increased calpain activity contributes to the onset of DMD in maturing (birth to weaning) dystrophic skeletal muscles and to determine if increased calpain activity was due to the relative distribution of calpain and calpastatin, calpain's endogenous inhibitor. Calpain activity was assessed in quadriceps and diaphragm muscle homogenate supernatant and pellet fractions from C57BL/6 control and mdx mice at ages 7, 14, and 21 days. Total calpain and calpastatin content were determined by Western analysis. In both the quadriceps and diaphragm samples, calpain activity in the supernatant increased with age. There was a significant increase (47.7%; p<0.05) in calciumdependent calpain activity in mdx quadriceps pellet compared to control at age 7 days. In the quadriceps at age 7 days, calpain activity in the pellet in the presence of calcium was significantly greater than at age 14 (61.2%) and 21 days (52.6%; p<0.05). In the diaphragm, there were no significant differences in pellet activity in either the presence or absence of calcium at any age between control and mdx samples. In both control and mdx diaphragms, pellet calpain activity in the absence compared with the presence of calcium was significantly greater at both age 7 (control, 46.4%; mdx, 45.4%) and 14 days (control, 42.4%; mdx, 43.6%; p<0.05). At age 21 days, both control and mdx calpain activities in the diaphragm supernatants in the presence of calcium were significantly greater than those at ages 7 (control, 66.7%; mdx, 72.1%) and 14 days (control, 39.9%; mdx 49.5%; p<0.05). In general, there were no differences in total calpain and calpastatin content that would account for the differences in calpain activity. There were similar patterns of calpain activity and total calpain and calpastatin content in both control and mdx samples, suggesting a similar pattern of development in control and mdx muscle from ages 7-21 days. The increase in calcium-dependent calpain activity in mdx quadriceps pellet compared to control at age 7 days may be due to differences in regulation and/or distribution of the calpain system early in mdx maturation compared to control. From the present study, the role of calpain in the onset of DMD appears to be minor if global calcium-dependent activity is evaluated. / Master of Science

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