Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

The heat shock response is a highly conserved genetic mechanism which is induced by a wide range of environmental stimuli. Although intensively studied in both prokaryotes and eukaryotes, no regulatory mechanism has been identified by which the environmental stimuli affect expression of the heat shock genes. In addition, although many inducers of the heat shock response are known to cause DNA damage, the role of heat shock in repair of DNA damage remains unclear. Mutants of Escherichia coli defective in the recA, uvrA, and xthA genes are more sensitive to heat than wild type. However, these mutants are able to develop thermotolerance, suggesting that thermotolerance is an inducible response capable of repairing heat-induced DNA damage independent of recA, uvrA, and xthA. Thermotolerance itself is shown to depend on the dnaK gene, directly linking the E. coli heat shock response to thermotolerance. In addition, the dnaK mutant is sensitive to heat and H₂O₂, but not to UV suggesting that the DnaK protein may function to protect cells from the specific DNA damage caused by heat and H₂O₂. An E. coli grpE mutant was found to be substantially more resistant to 50°C heat treatment than wild type. However, grpE⁻ cells have the same H₂O₂ and UV sensitivity as wild type. This implies that the conditions, for which a grpE mutation is beneficial, are unique to heat exposure and are not caused by H₂O₂ or UV exposure. Furthermore, heat shock protein synthesis occurs sooner in the grpE mutant than in wild type, indicating that the grpE gene product of E. coli may act as a negative regulator of the heat shock response. An adenyl cyclase deletion mutant of E. coli (cya) failed to exhibit a heat shock response even after 30 min. at 42°C. Furthermore, a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. Together, these results suggest that the cya gene may regulate the heat shock response, through cyclic AMP, by directly affecting the level of expression of the heat shock sigma factor.

M.Sc. / Heat shock proteins (HSP) are a group of highly conserved proteins induced in pro- and eukaryotes by a wide variety of environmental stresses such as heat shock (HS) and oxidative injury. HSP are classified into families according to their apparent molecular mass and respective inducers. Induction of HSP is primarily regulated on transcriptional level through multiple copies of a conserved cis-acting heat shock element (HSE) in the promoter region of all hsp genes to which the heat shock transcription factor (HSF) binds. Members of the HSP family function collectively as molecular chaperone systems, and fulfil essential roles under normal conditions and provide protection and adaptation during and following stress. The induction of HSP following stress and the subsequent protection confer HSP the potential application in stress therapy and in biomarking of stress. During a previous study in which the effect of Mycobacterium tuberculosis (M.tb) on the stress response of peripheral blood moncytes (PBM) from different donors was investigated, it was observed that different individuals from different South African populations showed differential a HSP synthesis in response to M.tb. This compelled us to investigate the following: Variation in HSP synthesis in peripheral blood monocytes (PBM) from different individuals in response to the classical HSP inducer, HS. The most appropriate technique to study HSP expression on protein level. HSP synthesis was studied in PBM from 36 individuals (European (E): n=22; non-Europeans (nE): n=14) using biometabolic labelling. Three techniques were compared in the determination of HSP expression in six donors in terms of HSP synthesis, which is measured by biometabolic labelling, and accumulation of hsp70 that were measured by Western blot analysis and flow cytometry. Results obtained are : European (E) and non-European (nE) populations differed significantly (p < 0.05) from each other in spite of a prominent variation in HSP synthesis within donors ; Flow cytometry is the technique of choice for the analysis of HSP levels, since it allows fast and safe measurement of HSP levels in single cel populations within a mixed population. Data from flow cytometry correlate with Western blot analysis, but not with biometabolic labelling. The means and ranges for different HSP synthesis in different populations reported in this study, set a standard for the use of HSP as biomarker of pa environmental stress for populations inhabiting southern Africa. Efficient measurement of HSP expression as biomarker of stress can therefore be implemented in routine analysis of environmental stress, as well as investigations concerning the implications of HSP in pathology.

by Lam Wai Yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 184-195). / Contents --- p.I - IV / Abstract --- p.V / Abbreviations --- p.VI / List of Tables and Figures --- p.VII - XV / Chapter Chapter One: --- Introduction / Part I / Chapter 1.1 --- Human genome project --- p.1 / Chapter 1.2 --- Progress of human genome project --- p.2 / Chapter 1.3 --- Human heart cDNA sequencing --- p.3 / Chapter 1.4 --- Significance of the human heart cDNA library project --- p.5 / Chapter 1.5 --- Homology search tools for cDNA sequences alignment --- p.5 / Part II / Chapter 1.6 --- Investigation of a human heart cDNA clone A076 --- p.7 / Chapter 1.7 --- General introduction of Heat Shock Proteins (HSPs) --- p.7 / Chapter 1.7.1 --- Definition of HSP --- p.8 / Chapter 1.7.2 --- Discovery of HSP --- p.10 / Chapter 1.7.3 --- Transcriptional regulation of heat shock genes --- p.11 / Chapter 1.7.4 --- Nomenclature of HSPs --- p.13 / Chapter 1.7.5 --- HSP110 --- p.13 / Chapter 1.7.6 --- HSP90 --- p.14 / Chapter 1.7.7 --- HSP70 --- p.15 / Chapter 1.7.8 --- HSP60 --- p.17 / Chapter 1.7.9 --- Ubiquitin - HSP8 --- p.19 / Chapter 1.7.10 --- HSP27 --- p.20 / Chapter 1.8 --- The theme of this thesis --- p.28 / Chapter Chapter Two: --- Method and Materials / Chapter 2.1 --- The human heart cDNA library --- p.29 / Chapter 2.2 --- Plating out the cDNA library --- p.29 / Chapter 2.3 --- DNA amplification --- p.31 / Chapter 2.4 --- DNA sequencing reaction - Cycle sequencing reaction --- p.32 / Chapter 2.5 --- Operation of the A.L.F. DNA sequencer --- p.33 / Chapter 2.5.1 --- Preparation of the gel cassette --- p.33 / Chapter 2.5.2 --- Preparation of the acrylamide gel --- p.34 / Chapter 2.5.3 --- Fitting the gel cassette into the electrophoresis unit --- p.35 / Chapter 2.5.4 --- Settings of electrophoresis --- p.36 / Chapter 2.6 --- Comparison of DNA sequences to databases --- p.37 / Chapter 2.7 --- Programming for sending cDNA sequences to NCBI --- p.38 / Chapter 2.8 --- Storage of sequence data --- p.39 / Chapter 2.9 --- Synthesis and purification of primers --- p.40 / Chapter 2.10 --- Connection of cDNA clones using Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.11 --- Purification of DNA fragment from agarose gels by GENECLEAN´ёØ --- p.42 / Chapter 2.12 --- "Preparation of competent Escherichia coli for transformation (Hanahan, 1986)" --- p.43 / Chapter 2.13 --- Transformation of Plasmid into Competent Escherichia coli --- p.44 / Chapter 2.14 --- "Small scale preparation of plasmid DNA (Sambrook et al.,1989" --- p.45 / Chapter 2.15 --- Large scale plasmid preparation by QIAGEN´ёØ --- p.46 / Chapter 2.16 --- DNA sequencing reaction - Unicycle sequencing reaction --- p.48 / Chapter 2.17 --- Synthesis of Radiolabeled DNA probe --- p.49 / Chapter 2.18 --- "Isolation of genomic DNA from human blood cells (Thomas A. Ciulla, 1988)" --- p.51 / Chapter 2.19 --- Southern blotting --- p.52 / Chapter 2.20 --- Prehybridization and hybridization procedure for Southern blot analysis --- p.54 / Chapter 2.21 --- "AGPC-RNA extraction method (Chomczynski and Sacchi 1987, modifed)" --- p.56 / Chapter 2.22 --- Electrophoresis of RNA through gels containing formaldehyde --- p.58 / Chapter 2.23 --- First-Strand cDNA synthesis --- p.59 / Chapter 2.24 --- Use of T7 RNA polymerase to direct expression of the cloned hsp27b gene (A076&B490) --- p.60 / Chapter 2.25 --- "Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (Laemmli, 1970)" --- p.61 / Chapter 2.26 --- Staining of the Gel by the Commassie Blue Method --- p.63 / Chapter Chapter Three: --- Results / Part I / Chapter 3.1 --- Sample results of Sequencing a few clones --- p.64 / Chapter 3.2 --- A Catalogue of 497 cDNA clones obtained from human heart cDNA sequencing --- p.71 / Chapter 3.3 --- Submission of novel sequences to genbank --- p.81 / Chapter 3.4 --- A Catalogues of genes that are expressed in the adult human heart --- p.83 / Chapter 3.5 --- The use of the programmes to assist the sending and receiving of sequence data E-mail message --- p.90 / Chapter 3.5.1 --- The use of the SENDMAIL.EXE programme --- p.91 / Chapter 3.5.2 --- "The use of the EDITBLN.EXE, ALLFILE.EXE and DATABASE.EXE" --- p.95 / Part II / Chapter 3.6 --- DNA sequence profiles of cDNA clones A076 and B490 --- p.105 / Chapter 3.7 --- Ligation of cDNA clones using Polymerase Chain Reaction (PCR) --- p.112 / Chapter 3.8 --- Cloning of the PCR product A076&B490 into the pAED4 expression vector --- p.117 / Chapter 3.9 --- Unicycle sequencing of the subcloned insert A076&B490 --- p.121 / Chapter 3.10 --- Southern hybridization of hsp27b (A076&B490) --- p.125 / Chapter 3.11 --- Results of RT-PCR and PCR --- p.127 / Chapter 3.12 --- Expression pAED4-A076&B490 in E.coli --- p.133 / Chapter Chapter Four: --- Discussion / Part I / Chapter 4.1 --- EST characterization --- p.138 / Chapter 4.2 --- Further investigation --- p.140 / Chapter 4.3 --- Disadvantage of randomly picked cDNA sequencing --- p.141 / Chapter 4.4 --- Problem of GenBank database searching --- p.141 / Part II / Chapter 4.5 --- The DNA sequence of A076 and B490 --- p.143 / Chapter 4.6 --- Ligation of HSP27B by using PCR --- p.144 / Chapter 4.7 --- Analysis of the DNA and protein sequence ofhsp27b (A076&B490) --- p.145 / Chapter 4.8 --- Southern hybridization of human hsp27b --- p.153 / Chapter 4.9 --- "RT-PCR and PCR of first strand cDNA with primers A076-ATG, A076-mid and oligo dT" --- p.153 / Chapter 4.10 --- Expression of human hsp27b --- p.154 / Chapter 4.11 --- The possible roles of human hsp27b --- p.156 / Chapter 4.12 --- Further analysis --- p.160 / Appendix I --- p.161-182 / Appendix II --- p.183 / References --- p.184-195

Healthy cells continuously produce proteins to accomplish various functions, including immune responses, reaction catalyses, transmitting signals, structural supports and molecular transport. Protein needs to fold correctly into three-dimensional shape in order to function well, using the information stored in the amino acid sequence. Proteins may fold spontaneously in solution, but the situation in living cells can be complicated. Cells are filled with nucleic acids and proteins thus they are usually in a stressful environment. Under such circumstances, proteins can be unfolded or misfolded, leading to non-function or even toxicity. Cells employ molecular chaperones to solve protein folding problems. Among the many types of chaperones, heat shock proteins of approximately 70KDa (Hsp70s) act as a hub, because its functions feed into other members of the chaperone network. Hsp70s help to stabilize nascent polypeptides, facilitate cross-membrane translocation, refold the misfolded proteins, and guide non-recoverable denatured proteins to degradation. Hsp70s have explicit role in cancer cells, because elevated metabolism requires increased Hsp70s’ activity to avoid apoptosis and ensure survival. Hsp70s also help to prevent neurodegenerative diseases, and decreased level of Hsp70s is found in age-related symptoms and diseases. In general, it is well understood what Hsp70s can do, but little is known how Hsp70s do the job. Hsp70s are present and highly conserved in all living species, comprised of two structural domains. The nucleotide binding domain (NBD) binds and hydrolyzes ATP, while the substrate binding domain (SBD) binds and releases hydrophobic peptides. Although Hsp70s are known to act as an allosteric molecular machine, the details are elusive about how the domains are regulated. Besides, how nucleotide binding affects the Hsp70s’ function, and how ATP hydrolysis is performed are also unknown. In this thesis, I first introduce salient background on the Hsp70 subject, then explore previously unclear aspects of Hsp70 allosteric regulation and catalytic activity in two chapters describing my dissertation research, and finally conclude with my perspectives on future directions.

The production of Heat Shock Protein 72, the inducible for~ of the highly conserved 70 kilodalton heat shock protein family, was investigated in MRL++ mouse intestine during the two weeks of a Trichinella spiralis infection. Within hours of an oral infection using the encysted Trichinella spiralis found in the diaphragm of an infected mouse, the larvae are released from the cyst in the stomach. They travel to the intestine and burrow into the epithelial layer of the intestine. The jejunum is the primary site of the intestinal phase of trichinosis (Despommier 1983). This stage of infection in the jejunum was the focus of this study. Heat shock protein (HSP) synthesis is precipitated by stressful stimuli: in vitro by chemicals such as sodium arsenite and in vivo by cytoskeletal disturbance and/or toxic 02 radicals (Linquist 1986). The latter in vivo studies lend support to the inflammatory response induction of HSPs. Heat shock protein 72 (HSP72) is rarely expressed constitutively especially in non-primates and is a good indicator of various stresses. This study hypothesized that HSP72 would be induced by cells in the jejunum of the MRL++ mouse during a Trichinella spiralis infection due to the stress of the parasitic infection. Different techniques were employed to investigate this hypothesis. Immunohistochemistry and immunoblots facilitated this study. Although immunoblots did not demonstrate HSP72 induction, immunohistochemical analysis suggested the presence of HSP72 in various cells in the lamina propria of the jejunal villi.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Graduation date: 1995