Cadmium is a highly toxic chemical and has been classified by the International Agency for Research on Cancer as a human carcinogen. Cadmium is abundant in the environment, at specific work places, and in food and water. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. The present study examined the effects of cadmium on heat shock protein (HSP) accumulation in Xenopus laevis A6 kidney epithelial cells. HSPs are molecular chaperones involved in protein folding and translocation. In response to environmental stress these proteins bind to unfolded protein and inhibit their aggregation. Stress-inducible hsp gene transcription is mediated by the heat shock promoter element (HSE), which interacts with heat shock transcription factor (HSF). In the present study, hsp30 and hsp70 mRNA and protein were induced by heat shock, as determined by northern and western blot analysis. Exposure of A6 cells to cadmium chloride also induced the expression of hsp genes. For example, northern and western blot analysis revealed that exposure of A6 cells to cadmium chloride induced the accumulation of hsp30 and hsp70 mRNA and their respective proteins. Western blot analysis also revealed that A6 cells recovering from a cadmium chloride treatment retained relatively high levels of HSP30 and HSP70 protein accumulation over 24 h after the removal of the stress. Treatments combining a mild heat shock and cadmium chloride resulted in a synergistic increase in hsp30 and hsp70 gene expression at mRNA and protein levels. Further experiments in which two stressors were combined revealed that synergistic effects occurred with varying cadmium concentrations and different temperatures. Immunocytochemistry and confocal microscopy were used to confirm the results attained from western blot analysis. Further, this technique allowed the determination of intracellular localization of HSP30 in A6 cells and the examination of cellular morphology and cytoskeletal structure during cadmium chloride treatments. A 2 h heat shock at 33??C resulted in the accumulation of HSP30 in the cytoplasm, whereas a 2 h heat shock at 35??C resulted in some HSP30 accumulation in the peripheral region of the nucleus. This is in contrast to cells treated with cadmium chloride, where HSP30 accumulation was restricted to the cytoplasm. A 14 h 50 ??M cadmium chloride treatment resulted in the accumulation of HSP30 in approximately 10% of cells. The proportion of cells displaying HSP30 accumulation increased to 80% and 95% in cells treated with 100 ??M and 200 ??M, respectively. HSP30 accumulation frequently occurred in large granular structures. High concentrations of cadmium chloride resulted in cell membrane ruffling at areas of cell-cell contact, as well as actin disorganization. This study characterized the pattern of hsp gene expression, accumulation and localization under various cadmium chloride conditions. These results suggest that hsp30 and hsp70 gene expression can be used as potential biomolecular markers for cadmium exposure.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. The present study examined the pattern of accumulation of HSP30 and HSP70 in cells recovering from heat shock as well as the effect of proteasome inhibition on cytoplasmic/nuclear and endoplasmic reticulum (ER) molecular chaperone accumulation, large multimeric HSP30 complexes, stress granule and aggresome formation in Xenopus laevis A6 kidney epithelial cells. Initial immunoblot analysis revealed the presence of elevated levels of HSP30 after 72 h of recovery. However, the relative levels of HSP70 declined to near control levels after 24 h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24 h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. To verify if proteasome inhibition in A6 cells induced the formation of similar HSP30 granules, immunoblot and immunocytochemical analyses were performed. MG132, celastrol and withaferin A enhanced ubiquitinated proteins, inhibited chymotrypsin-like activity of the proteasome and induced the accumulation of cytoplasmic/nuclear HSPs, HSP30 and HSP70 as well as ER chaperones, BiP and GRP94 and heme oxygenase-1. Northern blot experiments determined that proteasome inhibitors induced an accumulation in hsp30, hsp70 and bip mRNA but not eIF1α. The final part of this study demonstrated that treatment of A6 cells with proteasome inhibitors or sodium arsenite or cadmium chloride induced HSP30 multimeric complex formation primarily in the cytoplasm. Moreover, these stressors also induced the formation of RNA stress granules, pre-stalled translational complexes, which were detected via TIA1 and polyA binding protein (PABP), which are known stress granule markers. These stress granules, however, did not co-localize with large HSP30 multimeric complexes. In comparison, proteasome inhibition or treatment with sodium arsenite or cadmium chloride also induced the formation of aggresome-like structures, which are proteinaceous inclusion bodies formed as a result of an abundance of aggregated protein. Aggresome formation was identified by monitoring the presence of vimentin and γ-tubulin, both of which are cytoskeletal proteins and serve as markers of aggresome detection. Aggresome formation, which was also verified using the ProteoStat assay, co-localized with large HSP30 multimeric complexes. Co-immunoprecipitation experiments revealed that HSP30 associated with γ-tubulin and β-actin in cells treated with proteasome inhibitors or sodium arsenite or cadmium chloride suggesting a possible role in aggresome formation. In conclusion, this study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation. Finally, proteasome inhibition, sodium arsenite and cadmium chloride treatment of A6 cells induced cytoplasmic/nuclear and ER chaperones as well as resulting in the formation stress granules and aggresome-like structures which associated with large HSP30 multimeric complexes.

Heat shock proteins (hsp) and heat shock transcription factors (HSF) have important roles in the development of the eye lens. Our lab previously demonstrated that knockdown of hsp70 gene expression using morpholino antisense technology (MO) resulted in a small lens phenotype in zebrafish (Danio rerio) embryos. A less severe phenotype was seen with knockdown of hsf1, suggesting other factors that regulate hsp70 are involved during lens formation. Both HSF1 and HSF4 are known to play a role in mammalian lens development. An expressed sequence tag encoding zebrafish HSF4, named hsf4a, has been identified and a second splice variant, hsf4b, has been predicted in the Ensembl database. The objectives of this thesis were to characterize the zebrafish HSF4 and compare its expression to other HSFs as well as investigate its role in lens development. Analysis of zebrafish HSF4 sequence was performed using standard in silico analytical software. The deduced amino acid sequence of HSF4a shares structural similarities with mammalian HSF4 including the lack of an HR-C domain. This domain is absent due to a C-terminal truncation within zebrafish HSF4a relative to the mammalian protein. HSF4b is identical to the HSF4a sequence with the exception of an additional 155 amino acids at the carboxyl end of the protein which contains an HR-C domain, unlike mammalian HSF4. Surprisingly, electrophoretic mobility shift assays (EMSA) demonstrated that the binding affinity of zebrafish HSF4 to discontinuous HSEs is more similar to HSF1 than to other HSF4 proteins. The amino acid sequence of zebrafish HSF4 DNA binding domain was also more similar to HSF1 than other HSF4 proteins. These results, along with a phylogenetic analysis of HSF proteins from eleven species, suggest that HSF1 was an evolutionary precursor of HSF4 and that functions of this protein may differ between zebrafish and mammals. The expression level for each of the three zebrafish HSFs was determined in adult tissues and in developing embryos by quantitative reverse transcription polymerase chain reaction (qPCR) analysis. Expression of both hsf4 transcripts was observed predominantly in the eye but only observed in developing embryonic tissue at 60 hours post fertilization or later. This, together with the lack of an observable phenotype following MO knockdown of hsf4, suggests that HSF4 likely has a role in later stages of lens development. Additionally, hsf1 and hsf2 expression were detected in all tissues and in all stages of development as well as being present as maternal transcripts in zebrafish eggs. The results presented in this thesis demonstrate that while zebrafish HSFs share some similarity with HSF proteins from other species, they also have structural characteristics and expression patterns unique to the zebrafish.

At the cellular level, the stress response involves the synthesis of a highly conserved family of heat shock proteins (Hsps). These proteins are essential for maintenance of cellular homeostasis, both in times of stress and in normal cell functioning. Some of the most abundant forms of Hsps in the cell are members of the 70 kDa family. Intracellular heat shock protein 70 (Hsp70) expression in response to proteotoxicity is a highly conserved cellular stress response, but little is known about the role of extracellular Hsp70 (eHsp70) in fish. In order to begin characterizing eHsp70 in fish, the hypothesis that an acute stressor will elevate plasma Hsp70 levels in rainbow trout (Oncorhynchus mykiss) was tested. Subsequent in vitro studies examined whether eHsp70 level was modulated by cortisol and if this involved the action of the glucocorticoid receptor (GR), a ligand-activated transcription factor. The effect of cortisol on the eHsp70 response is important to consider because this steroid is elevated as a result of stressor exposure to allow for short-term allocation of energy stores to cope with stress. Cortisol is the primary corticosteroid in fish and exerts its main effects by binding to either GR or mineralocorticoid receptors (MR). Furthermore, eHsp70 has been previously implicated as having important immunoregulatory roles in mammalian models, but nothing has yet been reported in fish. To this end, a hypothesis tested here was that eHsp70 levels will increase after exposure to the bacterial endotoxin lipopolysaccharide (LPS), and that this response is modulated by cortisol. Finally, research on the effects of exogenous Hsp70 has not been reported in lower vertebrates; however, the relevance of this protein in intercellular signaling, especially in regards to immune regulation, is gaining increasing importance in mammalian models. Therefore, an experiment to determine whether Hsp70 would elicit upregulation of key immunoregulatory cytokines was also conducted. To accurately measure the low levels of Hsp70 in the plasma, a competitive antibody-capture enzyme-linked immunosorbent assay (ELISA) was developed. In the in vivo study, fish exposed to an acute heat shock (1h at 10°C above ambient temperature) exhibited a significant elevation in red blood cell Hsp70 levels over a 24 h period. There was also a significant increase in plasma Hsp70 levels at 4 h, but not at 24 h post-heat shock. To more specifically determine how cortisol affected the release of Hsp70, in vitro studies using primary cultures of hepatocytes demonstrated that cortisol significantly decreased eHsp70 levels in the medium at 24 h when compared with untreated controls, and this response was abolished in the presence of a GR antagonist, mifepristone (RU486). This result for the first time established a link between cortisol signaling and eHsp70 release in any animal model. When hepatocytes were exposed to LPS in vitro, eHsp70 levels were significantly lower in the LPS (30 µg/ml) group; however, heat shock abolished this effect at 24 h. Though eHsp70 levels in the heat shocked hepatocytes treated with low-dose LPS (10 µg/ml) was similar to untreated control levels, high-dose LPS treated hepatocytes showed significant elevation of eHsp70 levels above the low dose group. The ability of LPS to modulate eHsp70 release was not observed to be further regulated by cortisol. While this work suggests the modulation of eHsp70 by LPS, the physiological role remains to be elucidated. Finally when hepatocytes were exposed to exogenous Hsp70, there was no effect on key immunoregulatory genes (IL-1β and IL-8) transcript levels; however, the effect of this protein remains to be tested using other cell systems, including immune cells in fish. Overall, eHsp70 concentration was measured in trout plasma using a competitive ELISA and demonstrates for the first time that stressor exposure affects plasma eHsp70 levels in fish. Furthermore, cortisol, the primary corticosteroid in teleosts, modulates eHsp70 release in trout hepatocytes and this is action is mediated by GR signaling. Also, while trout hepatocytes secrete eHsp70 in response to endotoxin shock, a role for eHsp70 in eliciting an immune response is not clear in lower vertebrates. Taken together the results from this study suggest a role for eHsp70 in acute stress adaptation in fish, but the target tissues involved and the physiological responses remain to be elucidated. Further work on the effects of eHsp70 on target tissues effects, and the mechanisms involved, may have important implications in our understanding of the role of this stress protein in cell signaling and stress adaptation in fish.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 104-124). / Microfiche. / xii, 124 leaves, bound photos. 29 cm

Acute quadriplegic myopathy (AQM), or critical illness myopathy, is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients characterized by generalized muscle wasting and weakness of limb and trunk muscles. A preferential loss of the thick filament protein myosin is considered pathognomonic of this disorder, but the myosin loss is observed relatively late during the disease progression. In attempt to explore the potential role of factors considered triggering AQM in sedated mechanically ventilated (MV) ICU patients, we have studied the early effects, prior to the myosin loss, of neuromuscular blockade (NMB), corticosteroids (CS) and sepsis separate or in combination in a porcine experimental ICU model. Specific interest has been focused on skeletal muscle gene/protein expression and regulation of muscle contraction at the muscle fiber level. This project aims at improving our understanding of the molecular mechanisms underlying muscle specific differences in response to the ICU intervention and the role played by the different triggering factors. The sparing of masticatory muscle fiber function was coupled to an up-regulation of heat shock protein genes and down-regulation of myostatin are suggested to be key factors in the relative sparing of masticatory muscles. Up-regulation of chemokine activity genes and down-regulation of heat shock protein genes play a significant role in the limb muscle dysfunction associated with sepsis. The effects of corticosteroids in the development of limb muscle weakness reveals up-regulation of kinase activity and transcriptional regulation genes and the down-regulation of heat shock protein, sarcomeric, cytoskeletal and oxidative stress responsive genes. In contrast to limb and craniofacial muscles, the respiratory diaphragm muscle responded differently to the different triggering factors. MV itself appears to play a major role for the diaphragm muscle dysfunction. By targeting these genes, future experiments can give an insight into the development of innovative treatments expected at protecting muscle mass and function in critically ill ICU patients.

En aquesta tesi s'han dut a terme dos tipus d'estudis diferents. L'objectiu del primer era la preservació del semen de porcí a 15ºC i el del segon eren els co-cultius homòlegs de cèl·lules epitelials de l'oviducte i espermatozoides de porcí. Pel que fa al primer estudi, s'ha observat que l'addició de la prostaglandina F2α i àcid hialurònic a les dosis seminals no malmena la qualitat espermàtica i que la tolerància dels espermatozoides als canvis d'osmolalitat del medi es pot correlacionar proves de fertilitat i prolificitat..Respecte el segon, s'ha determinat que les cèl·lules oviductals afecten els paràmetres espermàtics i que la presència d'espermatozoides sobreexpressa els gens que codifiquen per les proteïnes de xoc tèrmic. Així, se suggereix que aquestes proteïnes tenen algun paper en els processos reproductius que tenen lloc a l'oviducte, malgrat que s'hagi observat, mitjançant la tècnica de la interferència de l'RNA, que la HSP90AA1 no està implicada en el perllongament de la viabilitat espermàtica. / In this thesis, two different studies have been conducted. The aim of the first experimental chapter was boar sperm preservation at 15ºC, the second dealing with in vitro homologous co-culture of oviductal epithelial cells (OEC) and spermatozoa. Regarding the first, it has been observed that the addition of prostaglandin F2α and hyaluronic acid do not cause any harm on sperm quality, and the osmotic tolerance of spermatozoa can be correlated with fertility and prolificacy rates of a given ejaculate.As far as the second study is concerned, OEC specifically affect sperm functional parameters and the presence of spermatozoa upregulates the expression of some genes encoding for heat shock proteins. Some role in the reproductive processes taking place in the oviduct is therefore suggested for this protein family, even though it has been observed, by means of RNA interference, that HSP90AA1 is not the protein involved in prolonging sperm survival.

The urokinase receptor (uPAR) plays a central role in metastatic process. It???s evident uPAR is overexpressed across a variety of tumour cells and leads to the increased aggressiveness and poor prognosis of cancer. Inhibition of uPAR expression can block metastatic potential in many tumours. In addition, besides uPA, there are several other proteins which have been confirmed to interact with uPAR, such as vitronectin and integrins. These interactions also contribute to signal transduction and the functions of uPAR complex. Therefore, downregulation of uPAR expression by targeting uPAR mRNA or protein, or by regulating the uPAR partners would be potential therapeutic strategies for prevention of cancer metastasis. There are two main aspects contained in this thesis. Firstly, three specific DNAzymes targeting uPAR mRNA were designed to downregulate uPAR expression in vitro and their effects to decrease cancer cell invasion studied in a human osteosarcoma cell line Saos-2. The results showed that two of them (Dz483 and Dz720) cleaved uPAR transcript in vitro with high efficacy and specificity and the Dz720 inhibited uPAR protein levels by 55% in Saos-2 cells. Besides, the Dz720 significantly suppressed Saos-2 cell invasion using an in vitro matrigel assay. Secondly, two potential uPAR partners from yeast two-hybrid screening, a heat shock protein MRJ and an anti-apoptosis protein HAX-1, were characterised and their functions binding with uPAR investigated. The interactions were confirmed by co-immunoprecipitation, GST-pull down assay and confocal microscopy in cancer cells. In addition, there was a 50% increase in cell adhesion after transfection with MRJ. This increase in adhesion is dependent on the uPAR/full length MRJ interaction as cells transfected with the mutant construct containing only N-terminal region or C-terminal region of MRJ had no increase in cell adhesion. The observed increase in adhesion to vitronectin by MRJ was also blocked by an anti-uPAR domain I antibody suggesting that the induced adhesion is at least in part contributed by uPAR on the cell surface. Together, the identification of both MRJ and HAX-1 as uPAR interactors provides further insight into the intricate relationship between uPAR and other proteins which may develop potential approaches for cancer therapy.

Bibliography: leaves 172-185. / xii, 185 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the induction of heat shock protein (HSP) by some of the anti-inflammatory agents and antirheumatic agents used in the management of rheumatoid arthritis. Presents HSP induction in peripheral white blood cells cultured in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 179-196). Also available for download via the World Wide Web; free to University of Oregon users.