Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 151-162).

In mammals, the neurogenic inflammatory response can be induced by stimulation or activation on the peripheral sensory C-fibers to release neuropeptides from the peripheral terminals, at the same time their afferent functions are enhanced. There are several neuropeptides stored and released from peripheral terminals of the afferent fibers, such as substance P (SP), neurokinin A, and calcitonin gene related peptide (CGRP). SP is one of the major inflammatory mediators of neurogenic inflammation that can act on neurokinin-1 receptors on smooth muscles and endothelial cells of blood vessels, causing vasodilatation, endothelial gap formation, and local plasma leakage. There are many studies and reports indicate that animals pretreated with a short period non-lethal hyperthermia can induce heat shock response and activate the expression of a group of inducible proteins called heat shock proteins (HSPs), and this stress response reduces the injury by same or other following stresses. In this study, the hyperthermia treatment (HT) was implemented by 42¢J hot water bath and the core body temperature of anesthetized rat was elevated and maintained around 42.0¡Ó0.5¢J for 15 min, and the normothermia control treatment (NT) was implemented by 37¢J warm water bath with the same period. 24 hours after NT or HT, the neurogenic plasma leakage was induced by intravascular injection with capsaicin (90 £gg/kg), SP (3 £gg/kg), or electrical stimulation on the right thoracic vagus nerve. The blood pressures of each animal were continually recorded during the neurogenic inflammation induction or sham operation. The amount of neurogenic inflammation of airway was evaluated by the area density leaky blood vessels. The leaking vessels were labeled with India ink and quantitative analysis by morphometric method. Plasma leakage was also measured by interstitial Evans blue concentration. The results indicated that HT could reduce plasma leakage and hypotension of the neurogenic inflammation that induced by capsaicin, SP or electrical stimulation on vagus nerve. Animals pretreated with aminoguanidine (a selective inhibitor of iNOS) had no significant effect on the neurogenic inflammation by following systemic SP infusion, but that could eliminate the anti-neurogenic inflammatory effect of HT. Animal applied with diphenhydramine (an antagonist of histamine H1 receptor) could attenuate the neurogenic inflammation by following systemic SP infusion, and HT could attenuate the neurogenic inflammation that with or without H1 receptor antagonist. This result indicates that NO synthesis and the activity of iNOS have few effects on neurogenic inflammation of airway, but it plays a critical factor on the initiation of heat shock response. The neurogenic inflammation induced by SP not only direct act on blood vessels but have other indirect effect by the histamine H1 receptor to enhance inflammation. Neonatal rats received high dose capsaicin treatment would induce irreversible sensory C-fiber denervation. The adult rats that were neonatally treated with capsaicin showed a more serious inflammatory response to systemic SP infusion as compared with animals neonatally treated with vehicle. HT still had the anti-inflammatory effects on the neurogenic inflammation that induced by SP. The results indicated that animals with sensory C-fiber denervation might conserve their neurogenic inflammatory responses and were hypersensitive to SP. In conclusion, the HT could attenuate the neurogenic inflammation that induced by different drugs or methods, and the anti-inflammatory effects were correlated with the increase in HSP72 expression. In the neurogenic inflammation induced by SP, the activation of histamine H1 receptors may enhance inflammation, but the activity of endogenous iNOS was less effective.

Heat shock response (HSR) is an ancient and highly conserved signaling pathway in cells that regulates the expression of heat shock proteins (HSPs) in the presence of thermal and other environmental stresses. HSPs function to prevent the formation of non-specific protein aggregates and to assist proteins in acquiring their native structures. Although HSR has been extensively studied, key aspects of this pathway remain a mystery. In particular, how HSR is activated and regulated by the master transcription factor HSF1 is not well understood. The broad goal of this thesis is to develop a quantitative framework aimed at elucidating the HSF1-mediated activation of HSR in yeast cells. Understanding this process has important implications for development, physiology and disease. Indeed, HSF1 is conserved from yeast to human, has been shown to play an important role in stress resistance, health and disease, and is a therapeutic target for neurodegenerative diseases. Broadly, there are two putative (not mutually exclusive) models for activation in response to heat shock: (1) HSF1 dissociation from chaperone proteins and (2) hyper-phosphorylation and the subsequent activation of HSF1. However the relative contribution of each of these events in the activation process is not characterized. Thus far, there is no direct evidence linking either of these two events to activation, and the relative contribution of each mechanism to the activation process has not been quantitatively characterized. To address these issues, we develop a quantitative model of HSR in yeast cells. We use the model to make a series of quantitative predictions and, in a collaborative effort, experimentally test these predictions in a yeast model of HSR. Critically, we provide the first direct evidence for chaperone dissociation of HSF1 in response to heat shock. Moreover, we find that HSF1 phosphorylation is dispensable for activation of HSR, but is able to modulate its activity. Taken together, our work leads to a model for two “orthogonal” mechanisms regulating HSR in yeast, in which chaperone dissociation acts as an ON/OFF switch, whereas phosphorylation functions to tune the gain of the response. Finally, to complement and further test this quantitative model, we develop a novel microfluidic system to explore in more depth the behavior of individual cells in the presence of heat shock inputs. This includes (1) a microfluidic device with microscale on-chip heaters enabling programmable thermal perturbations and (2) a custom image analysis platform to follow single cells through heat shock time courses. In preliminary single-cell studies, we find a relationship between HSF1 phosphorylation state and cell-to-cell variability in HSR activation level (as measured by a transcriptional reporter). These preliminary results suggest that HSF1 phosphorylation may be generating and tuning noise in the HSR in order to promote phenotypic plasticity and increased survivability of a cell population in the face of stress.

No description available.

Physical activity of moderate intensity and duration leads to healthy biological adaptations in humans. However, very intense and prolonged exercise may induce disruption in redox balance, potentially increasing oxidative stress. In addition, exposure to environmental heat stress and associated hyperthermia further increases oxidative stress and may induce the expression of heat shock proteins. However, antioxidant supplementation is believed to minimise the effect of oxidative stress and may therefore help reduce or limit the heat shock response to exercise heat stress. The first study (Chapter 4) examined whether exertional heat illness (EHI) casualties among military recruits may exhibit greater disturbances in redox balance following exercise compared to non-EHI controls. Nine (n=9) recruits were identified as having suspected EHI during the Loaded March (LM) on day 1, with a peak mean (SD) body core temperature of 40.1 (0.5) °C. Fifteen (n=15) recruits were identified as having suspected EHI during the Log Race (LR) on day 2, with a peak mean (SD) body core temperature of 39.7 (0.5) °C. A further twenty-one (n=21) recruits, which successfully finished both LM and LR events, were treated as controls (CON). Interestingly, the plasma antioxidant concentration was significantly elevated from pre to post-exercise (p < 0.001) for EHI and CON groups, during both LM and LR events, with no changes on lipid peroxide protein carbonyl concentrations. These data suggest there is no increase in lipid peroxide or protein carbonyl level damage in response to intense hyperthermic military exercise, regardless of acute heat illness. It is possible that military training augments the body's defence capabilities, thus reducing oxidative stress and damage induced by free radical production. To date there is a scarcity of data examining the effects of acute intake of antioxidant supplements on oxidative stress and heat shock response during continuous exercise in a hot environment. Hence, the aims of the second study (Chapter 5) were to examine the effects of acute ingestion of Quercetin (Q), Quercetin + vitamin C (QC) or placebo (P) 14 hours before, 2 hours before and every 20 minutes during trials on oxidative stress and heat shock response. In this randomised, crossover study 10 recreationally active males (age 21±2 y, V̇ O2max 54.9±8.4 ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (33.0±0.3°C; 28.5±1.8% relative humidity). Exercise heat stress significantly elevated plasma quercetin (p=0.02), antioxidant power (FRAP) (p < 0.001),plasma heat shock protein 70 (HSP70) (p=0.009) and plasma heat shock protein 90α(HSP90α) (p < 0.001) over time, but no differences were detected between trials. Also, no changes were observed in protein carbonyl concentration. Acute intake of quercetin significantly increased the level of plasma quercetin however, this did not affect the plasma antioxidant capacity or heat shock response to exercise heat stress. The increases in plasma HSP70 and HSP90α concentrations might act as supplementary antioxidants, reducing the oxidative damage reflected in the absence of changes in protein carbonyl. Exercise heat stress is effective in inducing both intracellular HSP70 (muscle and peripheral blood mononuclear cell (PBMC)) and extracellular HSP70 (plasma) concentrations. Thus, the third study (Chapter 6) tested the hypothesis that this acute quercetin supplementation would induce similar trends in plasma HSP70 and intracellular HSP70 concentrations 2 days following exercise heat stress. In this randomised, crossover study, 9 recreationally active males (age 22±2y, V̇ O2max 50.3±3.3ml.kg.min-1) completed three running trials at 70% V̇ O2max for 60 minutes in the heat (32.9±0.3°C; 28.3±1.2% relative humidity). This study demonstrated that there is no positive relationship between both intracellular of HSP70 (muscle and PBMC) and plasma HSP70 (eHSP70) 2 days following exercise heat stress. These data suggest that the release of eHSP70 could originate from others tissue or cells. Additionally, the absence of differences between trials in the expression of muscle HSP70, PBMC HSP70 and plasma HSP70 might indicate it is implausible that quercetin might inhibit the expression of HSP70 in plasma, muscle and PBMC 2 days following the exercise heat stress stimulus. Overall, the results from this thesis emphasise that the hyperthermia experienced in response to exercise and environmental heat stress could potentially influence the human redox response and heat shock response. Besides, there is reasonable evidence that acute quercetin co-ingestion with vitamin C has the potential to improve the bioavailability and bioactive effects of quercetin, however, the effects of quercetin supplementation in reducing oxidative stress in response to exercise heat stress remains to be elucidated. In addition, the anti-oxidative ability of acute ingestion of quercetin to suppress the intracellular and extracellular heat shock response remains uncertain and worthy for further investigation.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.

One unique feature of cancer cells that can be exploited for anticancer drug discovery is their dependence on their own cellular stress responses to survive the stressful acidotic, hypoxic and nutrient-deprived conditions within the tumor. Reasoning that desert organisms surviving under stressful conditions may have evolved to produce small molecule metabolites capable of modulating heat shock protein 90 (Hsp90) function, and/or other cell stress responses, we employed the cellular heat shock response in a moderate-throughput phenotypic assay. This strategy has resulted in the isolation and characterization of a number of small molecule natural products with heat shock induction activity from these organisms. Three such natural products are the subject of this study.In a limited structure-activity relationship (SAR) study, a previously known Hsp90 inhibitor radicicol (RAD), and several structurally related molecules including the fungal metabolite monocillin 1 (MON) were found to interact with Hsp90. In addition, RAD and MON were shown to lead to the degradation of Hsp90 client proteins involved in the cancer cell survival the estrogen receptor (ER) and the insulin-like growth factor receptor 1 (IGF-1R).We further characterized MON and showed that by targeting the molecular chaperone Hsp90, this compound induces components of the heat shock response at the transcriptional and translational levels, and leads to the acquisition of a thermotolerant phenotype in seedlings of the plant Arabidopsis thaliana. These findings support our hypothesis that there is ecological significance to the elaboration of small molecules that target stress responses.A number of extracts active in our phenotypic assay contained small molecules with no apparent Hsp90 activity. One such extract afforded terrecyclic acid A (TCA) with significant anti-tumor activity against a panel of human cancer cell lines. To characterize the biological activities of TCA we examined three key stress responsesthe heat shock, oxidative, and inflammatory responsesand show that TCA destabilizes these pathways associated with cancer cell survival through induction of oxidative stress (ROS), and inhibition of NF-kappaB transactivation.The isolation of RAD, MON and TCA from Sonoran desert organisms provides proof of principle that we have developed an effective strategy for the discovery of small molecule modulators of cellular stress responses that can serve as leads for the development of new anticancer drugs with novel mechanisms of action.

There is a thermal range for the operation of neural circuits beyond which nervous system function is compromised. Poikilotherms are particularly vulnerable to thermal stress, since their body temperature can fluctuate with ambient temperature. Animals that experience frequent hyperthermia have various coping mechanisms such as the thermoprotective effect of a prior exposure to sublethal temperatures (heat shock response). The molecular mechanisms of this thermoprotection have yet to be understood. This project studies the changes in protein expression in the nervous system of gregarious Locusta migratoria subjected to heat shock. For this purpose, proteins were extracted from metathoracic ganglia (MTG) by different methods and a proteomic map was subsequently obtained by 2-D gel electrophoresis which was compared between control (CON) and heat-shocked (HS) animals. Additionally, the localization pattern of Hsp70 was studied in the MTG of CON and HS gregarious locusts. Although 2-D gels showed changes in the amount of different isoforms of ATP-synthase β, the overall amount of this protein subunit was found to be unchanged. My experiments also revealed no significant change in the distribution of Hsp70 in the MTG of locusts caused by HS. However, new findings show that this protein is constitutively expressed at higher levels in perineurium, glia and tracheal cells than in neurons. In separate experiments, isolated locusts were also examined in order to measure any stress-associated increase of Hsp70 in the tissues of animals not previously exposed to crowding pressure. Quantitative western blots did not show a consistent change of the Hsp70 level in the MTG of isolated locusts following heat shock. Results of my research suggest that the change in the protein profile of the metathoracic ganglion following heat shock, if it exists, is subtle or occurs in very low-abundance proteins whose monitoring requires the application of special techniques. Alternatively, the thermoprotective effect of heat shock on the nervous system might be promoted through other pathways which can change the protein activity at the post-translational level and may work independently from protein synthesis. / Thesis (Master, Biology) -- Queen's University, 2009-03-20 12:28:32.962