Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

The study focuses on the effects of heat stress on <i>Serpula lacrymans</i> and <i>S. himantioides</i> (which have been previously shown to be relatively thermosensitive and thermotolerant respectively) in relation to the heat shock response. A part of this response is the production of heat-shock proteins (hsps). Comparative studies of chick embryo fibroblasts and <i>Saccharomyces cerevisiae</i> (wine strain, L-2226) were used to demonstrate possible differences between these three very distinct eukaryotic systems. The aims of the project were to determine whether <i>S. lacrymans</i> and <i>S. himantioides</i> undergo the heatshock response with respect primarily to induced thermotolerance and the production of hsps. The approach taken to establish the above was by systematic analysis of the different pathways involved in this response. Firstly, overall heat induced changes in protein and mRNA synthesis were analysed, in conjunction with the identification of specific proteins related to the heat-shock response, namely hsp60, hsp70 which have been shown in other eukaryotic systems to play key roles in this response. Secondly, immunological studies were also used to detect changes in the pattern of protein ubiquitination, which resulted from heat stress. Thirdly, changes in hsp70 mRNA were monitored by RT-PCR. The cDNA and genomic amplimer products generated by RT-PCR and PCR respectively were cloned and sequenced to identify a putative of hsp70 gene sequence. Fourthly, the AMP-activated protein kinase (AMPK) cascade has been demonstrated to be activated in response to stress and steps were made to detect and identify AMPK activity in cellular extracts of both <i>Serpula </i>species. Finally, the effect of heat upon trehalose accumulation and mobilisation during the heat-shock response was studied as there is growing evidence to link increased thermotolerance to transient increase in trehalose levels, which is now considered to be a stress metabolite.

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Thesis (Ph. D.)--Illinois State University, 1990. / Title from title page screen, viewed November 29, 2005. Dissertation Committee: Arlan Richardson (chair), Marjorie A. Jones, Lynne A. Lucher, Anthony J. Otsuka, Brian J. Wilkinson. Includes bibliographical references (leaves 168-187) and abstract. Also available in print.

The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Ecology and Biodiversity / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Medical Sciences