The present study investigated the hypothesis that maximal voluntary contractions (MVC) peak torque, VJ, muscle tenderness, and plasma creatine activity would be significantly less for the condition that subjects were exposed to hypoxic (H) condition for 4 hours after eccentric exercise compared with the normoxic (N) condition.

Inheritance of one mutant von Hippel-Lindau (VHL) allele gives rise to the development of the autosomal dominant VHL disease, which affects approximately 1 in 36 000 individuals. The VHL tumour suppressor protein plays a critical role in the E3 ubiquitin ligase-mediated destruction of hypoxia-inducible factor (HIF) and the promotion of fibronectin extracellular matrix assembly. A failure in either process is associated with oncogenic progression. Work included in this thesis provides evidence that these tumour suppressor functions are mutually exclusive. Additionally, post-translational modification of VHL by NEDD8 is shown to act as a ‘molecular switch’, altering VHL protein associations and providing a mechanism of pathway segregation. As a result of HIF stabilization, the expression of a homophilic adhesion molecule E-cadherin is significantly down-regulated in primary renal clear-cell carcinoma (RCC) upon VHL loss. E-cadherin down-regulation is shown to increase the invasive potential and is of prognostic value in RCC. Finally, VHL and SOCS1 are shown to dimerize and negatively regulate the JAK2-STAT5 signalling cascade. Defects in this dimerization are shown to underlie Chuvash polycythemia and provide a molecular understanding of the phenotypic observations associated with VHL-related polycythemias.

Inheritance of one mutant von Hippel-Lindau (VHL) allele gives rise to the development of the autosomal dominant VHL disease, which affects approximately 1 in 36 000 individuals. The VHL tumour suppressor protein plays a critical role in the E3 ubiquitin ligase-mediated destruction of hypoxia-inducible factor (HIF) and the promotion of fibronectin extracellular matrix assembly. A failure in either process is associated with oncogenic progression. Work included in this thesis provides evidence that these tumour suppressor functions are mutually exclusive. Additionally, post-translational modification of VHL by NEDD8 is shown to act as a ‘molecular switch’, altering VHL protein associations and providing a mechanism of pathway segregation. As a result of HIF stabilization, the expression of a homophilic adhesion molecule E-cadherin is significantly down-regulated in primary renal clear-cell carcinoma (RCC) upon VHL loss. E-cadherin down-regulation is shown to increase the invasive potential and is of prognostic value in RCC. Finally, VHL and SOCS1 are shown to dimerize and negatively regulate the JAK2-STAT5 signalling cascade. Defects in this dimerization are shown to underlie Chuvash polycythemia and provide a molecular understanding of the phenotypic observations associated with VHL-related polycythemias.

Cancer is one of the leading causes of death. There have been many investigations into therapeutic ways to prevent and reverse cancerous growth. We report a new approach in this thesis, which is to investigate the functions of Vitamin B1 (thiamine) in cancerous cells and their regulation. A number of thiamine analogs were synthesized to carry out the structure-activity relationship (SAR) studies with two transporters THTR1 and THTR2. Initial results show that the modifications of thiazole reduced the uptake of thiamine.

Heparan sulfate proteoglycans modulate many of the growth factor pathways that drive prostate cancer progression. Prior to being secreted into the extracellular matrix, the covalently attached HS chains are modified by sulfation which has been shown to increase the affinity of binding growth factors. The specific HSPG that I focus on in this dissertation is Perlecan (Pln). Previously, our group along with collaborators found that 54 percent of prostate cancer tumors had upregulated levels of Pln protein that correlated with increasing Gleason score [93]. The LNCaP-DU145-LN4 cell line series is introduced as a model for this subset of tumors because Pln levels increase 50-fold as the cells become more metastatic. It was found that three stress-induced transcription factors, HIF1α, NFkB, and ATF2, all stimulate Pln expression. ChIP analysis reveals that HIF1α and NFkB directly bind the Pln promoter while ATF2 does not. The ROS-generating NADPH Oxidase and the ROS-inducible p38 MAPK were also found to induce Pln expression. To address the subset of prostate cancer tumors that reach metastasis without upregulation of Pln, I focused on the 2-o-sulfotransferase enzyme and its effect on proliferation and invasion in the LNCaP-C4-2B cell model which does not show upregulation of Perlecan expression. 2OST RNAi resulted in a significant decrease in proliferation in each line of the series. 2OST RNAi in highly metastatic C4-2B cells caused a significant decrease in cell invasion. Cells with decreased levels of 2OST had increased accumulation of actin and E-cadherin suggesting the possible formation of adherens junctions. I also found that expression of 2OST increases four-fold as cells become more metastatic. I found HIF1α and ATF2 act in a direct manner while NFkB acts indirectly to stimulate 2OST expression. In summary, I have analyzed the effect of cellular stress on the expression of the Pln and 2OST genes and investigated the phenotype of 2OST knockdown in metastatic prostate cancer cells. These studies lead me to propose that the tumor stress response is necessary for prostate cancer progression due to the role of stress in the upregulation of extracellular HS that is required for growth factor signaling and metastatic behaviors.

Thesis (M.S.)--Western Washington University, 2010. / Includes bibliographical references (leaves 34-41).

A comparative phylogenetically independent contrast (PIC) analysis was employed to investigate the adaptive role of traits involved in hypoxia tolerance in sculpins, a group of closely related fish species that live in the nearshore marine environment. I demonstrated that there was a tight correlation between critical oxygen (O₂) tension (P-crit) and the distribution of species across an environmental gradient. Species of sculpins with the lowest P-crit inhabit the 0₂ variable intertidal zone, while species with higher P-crit inhabit the O₂ stable subtidal zone. Low P-crit values in sculpins were associated with enhanced O₂ extraction capacity, with three principal traits accounting for 83% of the variation in P-crit: low routine O₂ consumption rate (MO2 ), high mass specific gill surface area and high whole cell hemoglobin-oxygen (Hb-0₂) binding affinity. Variation in whole cell Hb-O₂ binding affinity was strongly correlated with the intrinsic affinity of Hb for O₂ and not to differences in the concentration of the allosteric Hb modulators ATP and GTP. When environmental O₂ dropped below a species' P-crit, some species of sculpins behaviorally responded to the severe hypoxia by performing aquatic surface respiration (ASR) and aerial emergence. Although intertidal sculpins consistently performed these behaviors, the clustering of these species into a single phylogenetic Glade did not allow us to draw conclusions regarding the relationship between ASR, aerial emergence and P-crit using PIC analysis. Three species of sculpins, which were chosen because of their low, medium and high P-crit values, exhibited dramatically varied mortality rates when exposed to severe hypoxia equivalent to 40% of their respective P-crit. Although ATP turnover rates were similar between the three species in the initial two hours of hypoxia exposure, the differences in the ability of the three species to survive severe hypoxia appeared to be associated with the concentration of on-board liver glycogen and the degree of liver glycogen depletion. However, when liver glycogen was assessed in twelve species of sculpins at normoxia and compared with P-crit, there was nosignificant PIC correlation between P-crit and liver glycogen. Overall, I have shown that there is a clear relationship between P-crit and the distribution of sculpins along the nearshore environment and that this is primarily related to differences in O₂ extraction capacity. When O₂ tensions are well below their P-crit, there are dramatic differences in behavioral, physiological and biochemical responses among these species of sculpins.

Cancer cells have the ability to survive when oxygen is limiting, and upregulate the pathway of fatty acid synthesis, owing in part to alterations in their metabolism. I compared the metabolic phenotypes of the prostate cancer cell lines LNCaP, DU145, and PC3 assessing energy metabolism, and metabolic gene expression. I also explored the plasticity of the metabolic phenotype following passage, selection and in vivo growth. Finally, I explored the sensitivity of the fatty acid synthesis pathway to low oxygen. LNCaP cells had a more oxidative phenotype based on oxygen consumption, lactate production, enzyme assays, and mRNA levels. While DU145 and PC3 cells were more glycolytic, they were unresponsive to dichloroacetate (DCA), and dinitrophenol (DNP), stimulators of oxygen consumption. Mitochondrial dysfunction in the PC3 and DU145 cells may explain this phenomenon, for they possessed normal cardiolipin levels but lower mitochondrial enzyme activities (cytochrome oxidase (COX), citrate synthase (CS)). When LNCaP cells were subjected to high passage, with and without clonal selection, the derived lines acquired a dysfunctional oxidative phenotype, becoming more glycolytic. Clonal selection appeared to have the most dramatic effect on cellular metabolism. This finding is supported by decreased oxygen consumption, increased lactate production, and a reduction in the activity of the oxidative enzymes CS and COX in the clonally selected LNCaP-luc cell line. Similar to the DU145 and PC3 cells, NAO fluorescence indicates that the oxidative impairment in these LNCaP-derived lines may be due to a reduction in mitochondrial activity. The pattern of metabolic gene expression iii seen in vitro was unaffected when LNCaP cells were grown as subcaspular and muscle xenografts in immunodeficient mice, though xenografts did exhibit indications of an hypoxic response (elevated VEGF mRNA). Oxygen deprivation in vitro increased mRNA for HIF and responsive genes but not SREBP responsive genes. Similarly, oxygen deprivation had no influence on triglyceride levels in any of the lines suggesting that the SREBP axis may not be directly modulated by oxygen levels. Collectively these studies demonstrate differences in the metabolism of these prostate cancer models, with important ramifications of therapeutic strategies involving metabolic targets. / Thesis (Master, Biology) -- Queen's University, 2008-09-25 17:28:13.418

Monocarboxylate transporters (MCTs) have been shown to be important in regulating metabolism during hypoxia in mammals. However, the role of MCTs in hypoxic survival in lower vertebrates is currently unclear. The goal of this study was to investigate the coordination of MCTs along with other metabolic proteins during hypoxia. Therefore, we subjected zebrafish (Danio rerio) to 1.5 mg L-1 O2 over 48 and 96-hr and measured tissue-specific transcriptional changes of MCTs (1, 2 and 4), lactate dehydrogenase A (LDHa), citrate synthase (CS), and other metabolic proteins using real-time RT-PCR. There were no changes in mRNA in muscle at 48 and 96-hr. When data from both time points were pooled in brain, a significant increase was found in MCT4 (+102%) and LDHa (+28%) mRNA indicating a preference towards glycolysis. In gills, there were increases in LDHa at 48-hr (+101%) and MCT1 (+24%) mRNA from pooled data suggesting that both anaerobic and aerobic metabolism is being utilized. Heart had the greatest changes in transcriptional levels compared to other tissues. At 48-hr, increases were found in MCT1 (+117%), MCT4 (+86%), LDHa (+197%), and pooled data showed an increase in CS (+18%) mRNA. These results indicate that the influx and efflux of lactate are both employed as strategies in cardiac tissue during hypoxia. This study has shown that fish utilize tissue-specific regulation of MCTs along with other metabolic genes during hypoxia. / Thesis (Master, Biology) -- Queen's University, 2009-08-24 13:44:06.114

Hypoxic condition is a naturally occurring environmental stressor in aquatic ecosystems. However, due to modern anthropocentric activities, hypoxia has been increasing in prevalence and severity. Rainbow trout, a keystone species in many North American lakes, is hypoxia intolerant. As a result, this species is of particular concern when studying the effects of hypoxia on an organism’s physiological functioning. Chronic starvation was used as a tool to induce metabolic depression to determine the effect that depressed metabolic rate had on hypoxia tolerance. Juvenile rainbow trout were deprived of food for five weeks at 15oC. Each week, routine metabolic rate (RMR) and critical oxygen tension (Pcrit) were measured. Concomitantly, resting and post-hypoxia fish (8 h at ~50% air saturation) were sampled to measure metabolites in blood, liver and muscle, as well as enzyme activities in select tissues. Food deprivation resulted in a decrease in routine metabolic rate (RMR) and shift towards an increased reliance on aerobic metabolism. Pcrit decreased significantly following four weeks of food deprivation respectively, indicating that metabolic depression induced by food deprivation may confer an increased tolerance to low environmental oxygen concentration ([O2]). However, marginal metabolic scope (MMS), another indicator of hypoxia tolerance, did not change in response to metabolic depression. Furthermore, subjecting trout to O2 limitation resulted in mobilization of carbohydrates from the liver subsequently leading to hyperglycemia. This was likely a survival technique ensuring that if severe hypoxia ensues, anaerobic substrates are ready for transport to the necessary tissues. / Thesis (Master, Biology) -- Queen's University, 2011-10-12 23:21:04.517