Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 106-118).

Fibronectin (FN) is a critical component of the extracellular matrix (ECM) of fibroblasts and muscle cells and contributes to the maintenance of cell cytoskeleton, shape, migration, growth and differentiation. Extensive accumulation of both collagen and FN occurs in skeletal muscle of patients with Duchenne's muscular dystrophy (DMD). Several researchers have recently claimed that FN can interfere with proper muscle differentiation. Our hypothesis is that DMD fibroblasts or muscle cells fail to regulate properly the type, synthesis, or secretion of FN and that the increased accumulation FN in the ECM of fibroblasts in muscle may interfere with myogenesis, thus presenting the typical picture of regenerating but largely non-functional muscle in DMD. This was tested by labeling cultures of fibroblasts from DMD patients and controls with ³⁵S-methionine and quantitating basal levels of FN synthesis and degradation by immunoprecipitation. Collagen, FN and other components were measured in DMD sera and the metabolic effects of culturing DMD and control fibroblasts in the presence of DMD sera were studied. Muscle biopsies from DMD patients were examined for factors which might contribute to the accumulation of connective tissue. Increased levels of FN were measured in sera and cultured fibroblasts from DMD patients. Concomitant increases in other ECM components (collagen, glycosaminoglycans) were measured. The FN accumulation is at the expense of other cellular proteins as RNA and protein synthesis are reduced in DMD fibroblasts. Protein degradation studies indicate that reduced catabolism of FN may account for significant elevations in cells and sera. DMD sera cultured on control fibroblasts caused metabolic alterations reminiscent of dystrophic cells (increased FN and collagen accumulation, decreased RNA synthesis). Many of the observed metabolic changes are age-related in DMD, increasing in older patients. These results suggest that overproduction by fibroblasts and increased serum levels of components of the ECM in DMD may influence the accumulation of connective tissue in DMD muscle. DMD may not be of myopathic origin but an increased compartment of fibroblasts and macrophages as observed in DMD muscle may result from chemotactic mobilization caused by elevated FN and/or collagen in the tissue.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

No description available.

abstract / Pathology / Doctoral / Doctor of Philosophy

Normal human skin fibroblast cells were used to study the effect of zinc supplementation of the media on cell growth and the competitive binding activity of low density lipoprotein (LDL). Cells were grown in the media containing Dulbecco's Modified-Eagle Medium (DMEM), 5% (v/v) fetal calf serum (FCS), and various levels of zinc. Cell counts and protein determination revealed that there was no stimulatory effect of zinc on the growth of cells, showing a flat growth curve with up to 6 μg/ml zinc supplementation. However, zinc supplementation of greater than 6 μg/ml to the medium appeared to be toxic to the cell and thereby prevented growth. When zinc was removed from the medium using Epoxy-activated Sepharose 6B coupled with iminodiacetate, zinc concentration in the medium was markedly reduced to 0.045 μg/ml from 0.210 μg/ml. The cell growth study using this zinc depleted medium exhibited a growth curve similar to that obtained from the earlier study, suggesting that 0.045 μg/ml of zinc in the control medium was still sufficient to support normal cell growth. For the LDL binding study, cells were grown in the media with various levels of zinc supplementation for 7 days and the competitive binding activity of LDL was determined. When cells were grown in the zinc removed medium with 1.5 μg/ml zinc supplementation, the maximum amounts of ¹²⁵I-LDL bound and internalized in the cells were observed; however, higher levels of zinc supplementation to the growth medium caused decreased ¹²⁵I-LDL binding to the cell receptors. These results suggest that zinc may be involved in the binding of LDL to the receptors. / Graduation date: 1983

In response to a variety of hyperthermic treatments, rat embryonic fibroblasts synthesize heat shock proteins (hsps), including those with molecular weights of 68,000 (hsp 68), 70,000 (hsp 70) and 89,000 (hsp 89). Hyperthermic stresses, which produce the hsps, also cause expression of thermotolerance. The dependence of thermotolerance expression on hsp synthesis was investigated in this mammalian cell line under different heating conditions. Temperature shift experiments showed that hsp synthesis and thermotolerance expression were dependent not only on the absolute hyperthermic temperature, but also on the difference between the initial incubation temperature and the hyperthermic temperature. Small temperature differences which produced no cell killing did not cause detectable synthesis of hsp 68. Increasing the difference of the initial and hyperthermic temperatures reduced cell survival and increased the synthesis of hsp 68. Thermotolerance could be expressed by surviving cells following an initial heat stress even when both heat shock and general protein synthesis were inhibited. Cells exposed to cycloheximide were heated, incubated at their initial temperature for six hours and reheated in the presence of the drug. The inhibitor was then removed and the cells plated for colony formation. The hsps were expressed during this latter incubation period. The regulation of hsp 70 in rat fibroblasts was investigated next. Hsp 70 synthesis rates correlated with the amount of hsp 70 encoding mRNA. The time course of heat shock synthesis and general protein synthesis recovery were each dependent on the duration of the heat stress. Inhibiting protein synthesis with cycloheximide resulted initially in the accumulation of the RNA encoding hsp 70 but did not effect the normal turnover of this RNA species. The conclusions based on these findings are that thermal survival adaptation can be expressed in the absence of hsp 68 synthesis. Hsp 68 is expressed by cells that will ultimately die (see Chapter 2). The hsps do not appear to protect cells against subsequent heat stress. They may function in a repair capacity (see Chapter 3). Hsp 70 expression is primarily regulated by transcription in Rat-1 cells. Hsp 70 does not act to regulate its own turnover (see Chapter 4).

No description available.

by Wai-kee Cheung. / Bibliography: leaves 307-332 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984