­^ ¤å ºK ­n We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced naïve pheochrommocytoma cell (PC12) death. PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect. The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

The effect of heat shock was observed on the expression of hilA in Salmonella Typhimurium by creating a fluorescence-based reporter strain of Salmonella and by realtime reverse transcriptase polymerase chain reaction (RT-PCR). The hilA gene in Salmonella is known to play an important role in its pathogenesis. hilA is known to be activated when the bacteria encounter stress-inducing conditions. A number of factors have been identified that affect hilA expression, such as, pH, osmolarity, oxygen tension. When Salmonella enter their warm-blooded hosts, they encounter an increase in temperature. Therefore, heat is another stressor that is encountered by Salmonella during infection of their hosts. A fluorescence-based strain of Salmonella was created to study the effect of heat shock. The gene for green fluorescent protein (gfp) was placed under the control of the promoter of hilA on a plasmid. This plasmid was used to transform Salmonella cells to create a fluorescent strain. In this strain, when the hilA promoter is activated, gfp is transcribed, which encodes the green fluorescent protein. This protein can be measured by a fluorescence assay. The results of this study indicated that at 45ºC, hilA is activated. RT-PCR was used to look at hilA expression at different temperature. The results of this study indicated that, compared to 37ºC, higher temperatures like 45ºC and 55ºC significantly activate hilA.

YDL100cp is the ArsA homologous protein found in Saccharomyces cerevisiae. Previous studies show that deletion of YDL100c was not lethal but unable to grow at 40¢XC. To study the role of YDL100c in response to lethal heat shock, the wild type strain (WT) and YDL100c disrupted strain (KO) were exposed to 50¢XC for 30 min. The growth and survival rate of KO cells at 30¢XC after heat-shock was lower than that of WT cells, and the difference was complementated by introducing the plasmid carrying YDL100c. The oxidative stress has been shown to be involved in the heat-induced cell death in S. cerevisiae. Therefore, the intracellular molecular oxidation level, expression of antioxidant genes, trehalose accumulation, and glutathione (GSH) content were further examined. The intracellular molecular oxidation was increased in KO compared to WT when exposed to 50¢XC, suggesting heat-shock-induced cell death is related to oxidation of intracellular components. The results also demonstrated that both WT and KO had a decreased GSH content and trehalose accumulation after heat-shock, indicating that GSH and trehalose are not directly involved in the slow growth of KO after heat-shock. However, CTT1 expression is decreased in KO compared to WT when exposed to 50¢XC, suggesting that decreased CTT1 expression resulted in the increased intracellular oxidation and YDL100c is likely involved in the activation of CTT1 expression.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 174-188).

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The heat shock response was studied in the intestinal parasitic protozoan Entamoeba histolytica. Temperature shifts from 37$ sp circ$C to 44$ sp circ$C enhanced the synthesis of five major heat shock (or stress) proteins (HSP) of 100, 50, 42, 37, and 28 kDa. Similarly, exposure of amebae to lymphokine activated macrophages and hydrogen peroxide caused HSP expression. Heat shock caused the reversible inhibition of amebic adherence to Chinese hamster ovary cells and human colonic mucin binding to trophozoites by ${>80 %}$. This was due to a decrease in the surface expression of the Gal/GalNAc adherence lectin and a marked reduction in the lectin mRNA expression. However, the presence of target Chinese hamster ovary cells during recovery at 37$ sp circ$C augmented amebic adherence. These results suggest that E. histolytica trophozoites produce a variety of HSP in response to different stimuli and can modulate the expression of the surface adherence lectin which maybe important in pathogenesis.

Thesis (M.S.)--West Virginia University, 1998. / Title from document title page. "December, 1998." Document formatted into pages; contains v, 40 p. Vita. Includes abstract. Includes bibliographical references (p. 32-39).

Thesis (Ph. D.)--University of Washington, 1998. / Includes bibliographic references (leaves [97]-124).

Thesis (Ph.D.)--University of Wollongong, 2006. / Typescript. Includes bibliographical references: leaf 228-250.