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Evidence suggesting that Hsp72 is actively participating in cellular signalling as well interacting with immune system dynamics has been increasing. This is true in healthy, stressed and diseased cells but to different degrees. Modulation of the plasma membrane association and secretion in the extracellular environment by different types of stressors is the key event that leads to different degrees of immune system activation. Hence a better understanding of the mechanisms of Hsp72 secretion and association with plasma membrane is crucial. This thesis investigated the tissue source and mechanism of Hsp72 surface presentation to plasma membrane structures and release in relation with different cellular and physiological stressors. In vivo models confirmed that different tissue types determine specific Hsp72 responses following the same stress and increase serum Hsp72 dependant on intensity and duration of the stress. Diseases models confirm that Hsp72 responses in specific cell populations is related to disease progression, while in vitro models clearly showed that there are multiple mechanisms of secretion and surface presentation, dependent on the nature of the stressor as well as the intensity and duration. This observations clearly change the view of extracellular Hsp72 as a danger signal and lead to a revision of the original danger model. It also suggests that manipulation of Hsp72 translocation through the different pathways involved may prove effective therapeutically.

Although a number of HSPs have been shown to be up-regulated in a wide range of human cancers, the full significance of this remains to be determined. The localisation of HSPs seems to be critical in determining their role in cancer cell survival; High intracellular levels (iHsp) appear to be advantageous to the tumour cell, inhibiting key steps in apoptosis, while in some circumstances, surface expression (sHsp) appears to be detrimental to the cell, aiding immune recognition by various effector cells. Consequently, clarifying the importance of HSP cellular location in the cancer setting may lead to the development of novel therapies based upon manipulation of HSP localisation. This thesis had two major aims; (1) to investigate the cellular localisation of HSPs in leukocytes from patients with both myelocytic and lymphocytic malignancies in order to establish relationships between apoptosis and stage of disease (2) to study the synergistic effect of four chemotherapeutic drugs with membrane fluidising agents, compounds which have the potential to modulate HSP localisation. Hsp90 and Hsp27 expression was shown to be restricted to the inside of peripheral blood leukocytes, while Hsp72 was localised both intracellularly and on the cell surface. In CLL, iHsp90 and iHsp27 levels were found to be significantly higher than in control subjects, while surface and intracellular Hsp72 was shown to be expressed either at very high levels or at very low levels. Furthermore, iHsp90 levels were found to be associated with stage of disease, while iHsp27 levels were shown to negatively correlate with levels of apoptosis. CLL patients with stable disease were found to express higher levels of iHsp72 than patients with progressive disease. However, in AML and MDS, levels of all HSPs in peripheral blood were found to be similar to those seen in control subjects, but disease patients showed a much wider range of expression. In AML, levels of sHsp72 positively correlated in all cell types, an observation not made in MDS patients or control subjects. HSP localisation was shown to be affected by membrane fluidising agents, with a movement of Hsp72 and Hsp60 to the cell surface. This effect was not due to proteotoxicity and supports data implicating the cell membrane in the regulation of HSP responses. This manipulation of HSP localisation and the increase in membrane fluidity resulted in increased sensitivity of CLL cells to three chemotherapeutic agents and points to the possibility that manipulation of membrane fluidity, may have significant value in the development of new treatment regimes.

by Wing-Hoi Tam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 95-99). / Abstracts in English and Chinese. / Title --- p.1 / Acknowledgement --- p.2 / Contents --- p.3 / Abbreviations --- p.5 / List of figures --- p.7 / List of tables --- p.9 / Primers and their sequences used in the projcet --- p.10 / Abstract --- p.12 / Introduction / Small heat shock proteins --- p.16 / Human heart sequencing EST project --- p.17 / Small heat shock protein B3 (HSPB3) --- p.18 / The alpha crystallins --- p.18 / Point mutation of a conserved arginine in the alpha-crystallin domain perturbs the overall alpha crystallins structure and function --- p.19 / HSP27 Confers thermotolerance in cells --- p.19 / Chaperone properties of sHSP --- p.20 / In Viro-chaperone function of sHSPs --- p.21 / Does the alpha-crystallin domain necessary for chaperone activity? --- p.21 / Expression HSPB3 in heart diseases patients and in porcine model --- p.22 / Small heat shock proteins in development and differentiation --- p.23 / HSPB3 role in mammalian embryogenesis --- p.24 / Structure of the small heat shock proteins --- p.25 / Crystal structure of a small heat shock protein --- p.25 / Subunit contacts --- p.26 / Muscle is the only tissue that simultaneously expresses all five sHSPs --- p.26 / "Two independent sHSP systems in muscle cells, to serve and to protect" --- p.27 / Methods and Materials / Small scale preparation of plasmid DNA --- p.29 / Large scale preparation of plasmid DNA --- p.29 / Amplification of mouse and rat HSPB3 coding region --- p.30 / Northern Blotting and Hybridisation --- p.31 / Purification of DNA fragments by GENECLEAN --- p.31 / Preparation of competent Escherichia coli for transformation --- p.32 / Transformation of DNA plasmid into competent Escherichia coli --- p.32 / 5´ة Rapid Amplification of cDNA Ends (5´ةRACE) --- p.33 / 3´ة Rapid Amplification of cDNA Ends (3´ةRACE) --- p.35 / Upstream and Downstream Genomic sequence --- p.36 / Isolation of genomic DNA from human tissue --- p.37 / Multiple tissue cDNA panel (MTC) --- p.37 / The yeast two hybrid screenings and confirmation --- p.43 / Isolation of total RNA from human tissues --- p.43 / Automated Sequencing --- p.44 / Alignment of DNA and amino acid sequence --- p.44 / Radiation hybrid mapping --- p.45 / Results / Determination of the foll length of human HSPB3 cDNA by 5' and 3' RACE --- p.46 / Identification of human HSPB3 gene in mouse and rat heart tissues --- p.49 / "Sequence homology between human, mouse and rat HSPB3" --- p.51 / Chromosomal mapping of the HSPB3 gene --- p.60 / Expression of human HSPB3 is not confined to the heart and skeletal muscle --- p.62 / Mouse HSPB3 expression is confined to the heart tissue --- p.65 / HSPB3 genomic PCR of downstream sequence --- p.67 / HSPB3 upstream genomic sequence --- p.67 / No intronic sequences between the open reading frame --- p.68 / The yeast two hybrid screening --- p.71 / HSPB3 can not form dimers --- p.82 / Expression of Prepro-CDD-ANF in human adult and fetal --- p.83 / Discussions / Previously reported sequence HSPL27 is a chimera --- p.87 / HSPB3 monomers do not dimerises --- p.88 / No intronic sequence in the HSPB3 gene --- p.89 / HSPB3 and CDD-ANF may play an important role during stress to the heart --- p.91 / Change of expression pattern of Prepro-CDD-ANF during development --- p.93 / Reference --- p.95

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

The gastrointestinal epithelium normally sealed by tight junctions, which act as a structural barrier and paracellular channels. Inflammation can increase the permeability of microvasculature that result in plasma leakage. Mammalian intestinal epithelium has many goblet cells which discharge mucus in the inflammatory response. The discharging mucus functioning is as a defensive barrier and lubricant. The mucus layer is the anatomical site at which the host first encounters gut bacteria, physical damage, and chemical stimulant. The heat shock response is one of the most primitive cellular defense mechanism. A variety of stressful situations including environmental (ultraviolet radiation or heavy metals), pathological (infections or malignancies), or physiological (growth factors) stimuli induce heat shock proteins. This study investigated the effect of heat shock on endotoxin-induced plasma leakage and goblet cell mucus discharge in the small intestine of rats of Sprague-Dawley (SD) and Long-Evans (LE) strains. India ink was used as the tracer to detect leaky microvessels. The mucus secretion of the goblet cells of intestinal villi was observed with scanning electron microscopy and calculated with digital morphomertric software SimplePCI. Our results showed that endotoxin-induced plasma leakage and goblet cell discharging in the two strains increased significantly as compared to rat groups receiving saline. Numerous openings on the epithelial surface of villi resulted from compound exocytosis of mucus granules in goblet cells. Either 30 min or 1h after LPS injection, heat shock pretreatment in LE rats LPS-induced plasma leakage in the duodenum and ileum was reduced by 58-80% (P<0.01). 1 h after LPS injection in LE rats pretreated with heat shock, the number of discharging goblet cells in the ileum was reduced (P<0.05). In SD rats, heat shock inhibited LPS-induced plasma leakage in the duodenum and ileum at 1h after LPS injection by 56-68% (P<0.01), and the number of discharging goblet cells was reduced in the duodenum and ileum (P<0.05). In conclusion, heat shock could protect rat intestine from endotoxin-induced inflammation and mucus secretion.