The independent effects of acute heat and hypoxic stress on human physiological function and performance are relatively well documented. Although in the field these environmental stressors rarely occur in isolation the effects of combined or sequential exposure to them has not been extensively studied in humans. Animal models have however shown that acclimation to one stressor can induce ‘cross acclimation’ a positive adaptive response upon exposure to a different stressor. The three studies within this thesis were conducted in humans to assess how exposure to acute and repeated exposures to heat affects the later physiological and cellular responses to acute exercise in normobaric hypoxia. A possible site for any cross-acclimatory affects and conferred cellular tolerance resides in the heat shock response (HSR) and the increased expression of heat shock proteins (HSPs). The 72 kilodalton HSP, HSP72 has been implicated in heat acclimation mediated cross acclimation in rodent models, and also shown to be important in the human adaptation to heat and hypoxic stressors. Study One determined the physiological and HSR to exercise in both heat (HEAT; 40°C) and hypoxia (HYP; FIO2 0.14) alone, and in combination (COM) as well as a normothermic normoxic control (NORM). 24 hours after the initial exposure a hypoxic stress test (HST; 15 minutes of seated rest and 60 minutes of cycling exercise at 50% normoxic peak) was conducted to determine what effect the prior stress exposure had on both whole body physiological responses and the cellular HSR. It was hypothesised that the stressor that elicited the greatest physiological strain and HSR on day one would have the biggest effect on reducing physiological strain in a subsequent HST. Twelve male participants completed 4 trials consisting of a 15 minute rest period in normoxic temperate conditions, followed by 30 minutes seated rest and 90 minutes cycling exercise at 50% Npeak within NORM, HEAT, HYP and COM. 24 hours after completing this exercise bout, participants undertook a HST. Exercise duration was reduced in HEAT (78 ± 12mins), HYP (81 ± 13mins) and the CON (73 ± 19mins) trial compared to the NORM (89 ± 3mins). HR and core body temperature (Tcore), and thus physiological strain, were greater in the HEAT and COM trial compared to HYP alone. This response was also observed with post exercise monocyte HSP72 (mHSP72). Basal HSP72 was elevated 24 hours after the HEAT and COM and attenuated post HST. Exercising HR, Tcore and PSI was reduced during the HST 24 hours after a heat stressor had been applied, but unaffected by a prior hypoxic exposure. Therefore the hypothesis was accepted. It was concluded that at the temperature and level of hypoxia studied, a prior exposure to exercise heat stress was beneficial when conducting subsequent acute hypoxic exercise. Study Two investigated the effect of short-term heat acclimation (STHA) on subsequent hypoxic tolerance in 16 male participants divided equally into 2 matched groups. This study also examined the response of extracellular HSP72 (eHSP72) to acute hypoxic exercise. It was hypothesized that STHA would increase basal HSP72 and that the post HST increase in HSP72 would be attenuated in this group, indicating conferred cellular tolerance. Eight males completed a HST one week before undertaking 3 consecutive days of STHA (60 min/day, 40°C, 50%peak) followed by a final HST 48-hours after the last acclimation day. The matched controls (CON) completed an identical protocol in normothermic, normoxic conditions. The initial HST induced a post exercise increase in HSP72 in both groups. HSP72 was increased after the first day of heat acclimation and unchanged in the control group. After acclimation day 2, basal HSP72 was increased from on day 1 basal values and the post exercise increase observed on day 1 was absent in the heat group. The increase in basal HSP72 persisted until the post acclimation HST for the STHA group and post exercise HSP72 was attenuated. eHSP72 increased immediately after the HST in both groups, however large inter-individual variation was evident. Mean exercising HR, Tcore and physiological strain was reduced during the HST in the STHA group, indicating that a short period of heat acclimation can improve both cellular and physiological tolerance to exercise in acute normobaric hypoxia. Study Three examined how a prior period of long term heat acclimation (LTHA) or time and absolute exercise intensity matched hypoxic acclimation (HA) affects both tolerance and performance to a HST and 16.1 km time trial (TT). Plasma hypoxia inducible 1 alpha (HIF-1α) was assessed before and after the acclimation periods as this transcription factor plays an important role in heat acclimation mediated cross tolerance. Twenty-one male participants completed ten 60-minute cycling bouts (50% Npeak) in thermoneutral, normoxic conditions (CON, 18°C, FIO2 0.209; n = 7), heated conditions (LTHA, 40°C, n = 7) or hypoxic conditions (HA, FIO2 0.14, n = 7). A HST immediately followed by a 16.1 km TT was completed one week before and 48 hours after the acclimation period. Both LTHA and HA induced increases in basal HSP72 by the end of the 10-day period. Increases in basal HSP72 occurred earlier in the acclimation period and to a greater magnitude with LTHA. Prior to the post acclimation HST both basal HSP72 and plasma HIF1-α were elevated in the LTHA and HA groups, with no changes observed in CON compared to the initial HST. Post HST mHSP72 and HIF1-α was attenuated in LTHA and HA. Mean exercising HR, Tcore and PSI were reduced in the LTHA group with no changes in these physiological variables observed in the HA or CON groups. During the TT, mean power output (MPO) was elevated at each kilometer in the HA group, leading to an improved performance after acclimation. The LTHA group produced greater power outputs between km 1 – 8 and 14-16 and consequently were faster overall compared to their pre acclimation TT. This indicates an altered pacing strategy following the LTHA period. The data suggests that, at the levels studied herein, LTHA induces a faster accumulation of basal mHSP72 over a 10-day period, occurring to a greater magnitude. This is the first study to examine the plasma HIF-1α response to both heat and hypoxic acclimation in humans. The data suggest that each environmental stressor induces an increase in resting levels of this transcription factor, however further study is required due to the large variation in response. It is not yet known whether the benefits conferred from heat to acute bouts of hypoxia would translate to more prolonged hypoxic exposures. Both the mechanisms of cross-acclimation and the effects of extended or prolonged hypoxic exposure following heat acclimation require further study. The immediate post exercise mHSP72 increase to exercise was consistently shown to be greater following a heat stress condition when compared to hypoxia. STHA induced greater increases in basal mHSP72 compared to the acute exposure, further attenuating post HST mHSP72 elevations and physiological strain. LTHA increased basal mHSP72 at a faster rate and magnitude than HA and 16.1km time trial performance improved to a similar magnitude following both heat and hypoxic acclimation It is speculated that heat acclimation mediated activation of HIF-1α may hold a key mechanistic role in the observed cross-acclimatory response. From a practical perspective, the use of heat-stress based acclimation/training programs may provide a cheaper and more effective means of preparing individuals for subsequent hypoxic exposure. Future studies should confirm these observations hold true in a hypobaric environment and establish how prior heat acclimation may impact on longer term exposures and adaptations to hypoxic environments.

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. / Science, Faculty of / Zoology, Department of / Graduate

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2alpha gene (EPAS1) in normal cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2alpha gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumours and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2alpha pathway in the hypoxic tumour microenvironment, which is necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumour growth in vivo. In addition, restoring HIF-2alpha expression in DNMT3a-reintroduced cancer cells restores full tumorigenic potential, including the capacity to traverse the hypoxic barrier. These data support a tumour-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2alpha oxygen-sensing pathway and the cellular response to hypoxia.

INTRODUCTION: Blood lipids, more precisely triglycerides (TG), are important fuel sources that are highly regulated since an exaggerate amount can lead to cardiovascular diseases. TG breakdown after a meal is mainly controlled by an enzyme expressed in adipose tissue called lipoprotein lipase (LPL). Recent evidence in animals report that adipose tissue LPL is inhibited after an exposure to an environment with reduced oxygen content, leading to a raised level of plasmatic TG. The objective of this thesis was to characterize the effects of an acute exposure to hypoxia on the plasmatic lipolytic activity level and on postprandial TG levels in humans. It was hypothesized that postprandial TG level and plasmatic lipolytic activity, a proxy of LPL activity, would be negatively affected by hypoxia. METHODS: Postprandial TG, non-esterified fatty acid (NEFA), glucose levels, and postheparin plasmatic lipolytic activity were measured on healthy young men (n=7) exposed for 6 h to either control (FiO2=0.2093) or hypoxia (FiO2=0.1200) in a randomized crossover fashion. RESULTS: Exposure to acute hypoxia led to a close to significant (p = .06) increase in postprandial plasmatic TG level and significant postprandial NEFA levels. Postprandial glucose levels were not affected by acute exposure to hypoxia. A significant increase in postheparin plasmatic lipolytic activity was observed after acute hypoxia exposure as compared to the control condition. CONCLUSION: Acute hypoxia in healthy men tend to negatively affects postprandial TG level while increasing plasmatic lipolytic activity. These results lend support to the increased blood lipid levels reported in individuals exposed to lower partial pressure of oxygen during sojourn at high altitude.

Water-soluble Pt(II) phosphors exist predominantly for photophysical studies. However, fewer are known to be candidates for cisplatin derivatives. If such a molecule could exist, it would be efficient at not only destroying the cancerous cells which harm the body, but the destruction would also be traceable within the human body as it occurred. Herein, research accomplished in chemistry describes the photophysical properties of a water-soluble phosphor. Spectroscopically, this phosphor is unique in that it possesses a strong green emission at room temperature in aqueous media. Its emission is also sensitive to the gaseous environment. These properties have been expanded to both analytical and biological applications. Studies showing the potential use of the phosphor as a heavy metal remover from aqueous solutions have been accomplished. The removal of toxic heavy metals was indicated by the loss of emission as well as the appearance of a precipitate. The gaseous sensitivity was elicited to be used as a potential cancerous cell biomarker. In vivo studies were accomplished in a wide variety of species, including bacteria (E. coli), worms (C. elegans), small crustaceans (Artemia), and fish (D. rerio and S. ocellatus). The phosphor in question is detectable in all of the above. This fundamental research lays the foundation for further expansion into bioinorganic chemistry, and many other possible applications.

In this project we study the role of RAGE in the melanoma and pancreatic cancer progression. Based on published studies, we hypothesized that RAGE localization in melanoma varies with different cellular architectures. To test this hypothesis, we utilized an in vitro spheroid model and a lung colonization mice model to compare the RAGE localization in 3D architecture vs 2D monolayer culture. RAGE was found at the cell surface in WM115 and B16F10 spheroids, whereas RAGE is mostly distributed intracellularly in WM266. We also observed that RAGE is present at the surface of B16F10 melanoma cells within tumor nodules in the lungs of mice colonized with B16F10 cells. Previously, our group has demonstrated that RAGE promotes pancreatic tumor cell survival under normoxic conditions, upon gemcitabine administration. Hypoxia is also associated with increased tumor aggressiveness. Based on published reports, we hypothesized that RAGE upregulation under hypoxic conditions contributes to autophagy and migration in pancreatic cancer cells. We observed that autophagy decreases after RAGE inhibition by FPSZM1. Moreover, we observed decreased cell migration after RAGE blockage, indicating that RAGE also mediates migration under hypoxia. We also investigated Advanced Glycation Endproducts (AGEs) on proliferation and migration of pancreatic cancer cells. Based on published reports, we hypothesized that RAGE activation by AGEs contributes to the proliferation and migration in pancreatic cancer cells. We employed ribose modified BSA to activate RAGE in the murine KPC 5517 pancreatic cancer cell line. We observed that AGE-treated samples showed significant increase in migration but no change in proliferation. As RAGE is involved in the progression of melanoma and pancreatic cancer, our results will help researchers to better understand the biology of RAGE. Our research can help to design RAGE-specific antibodies and inhibitors that could target RAGE more effectively. Moreover, our findings on AGE-RAGE interactions, and on the role of RAGE in pancreatic cancer progression under hypoxia, may contribute to reduce the progression of pancreatic cancer. Our results showing that a RAGE inhibitor can reduce autophagy and migration of pancreatic tumor cells, suggest that FPS-ZM1 could be utilized as a potential therapeutic aid for the treatment of pancreatic cancer.

Introduction: Exposure to hypoxia may alter substrate utilization through diverse mechanisms. Acute hypoxia is known to increase circulating nonesterified fatty acid (NEFA) levels and reduce systemic sensitivity to insulin. The hepatic fate of NEFA is dictated by major pathways such as esterification to triglycerides and complete/partial oxidation, the latter leading to ketogenesis. To our knowledge, the effect of hypoxia on ketogenesis, more specifically ß-hydroxybutyrate (ßOHB), remains unknown in humans. Moreover, adipose tissue is a significant site of NEFA liberation into circulation, and insulin inhibits this process. Under acute hypoxia, systemic insulin resistance develops, and the suppression of lipolysis is impeded. Therefore, the objective of this study was to determine the effect of acute hypoxia on plasma circulating ßOHB levels. Furthermore, to better understand how hypoxic and prandial conditions may modulate plasma NEFA and ketonemia, we calculated the βOHB:NEFA ratio and the adipose tissue insulin resistance index (Adipo-IR), which respectively gives indications of the partial hepatic oxidation of NEFA and the adipose tissue insulin sensitivity. Methods: Plasma samples from 3 different randomized crossover studies were retrospectively assessed for ßOHB concentrations. In the first study, 14 healthy men (23 ± 3.5 years) were exposed to 6 hours of normoxia or intermittent hypoxia (IH) (15 hypoxic events per hour) following an isocaloric meal (IH-Fed). In the second study, 10 healthy men (26 ± 5.6 years) were exposed to 6 hours of continuous normobaric hypoxia (CH) (FiO2= 0.12) or normoxic conditions in the fasting state (CH-Fasted). In the third study, 9 healthy men (24 ± 4.5 years) were exposed to 6 hours of CH in a constant prandial state. ßOHB, NEFA and insulin levels were measured during all sessions (CH-Fed). The adipose tissue insulin resistance index (Adipo-IR) was also calculated from NEFA and insulin levels. Results: In study 1 (IH-Fed), ßOHB and NEFA levels tended to be greater over 6 hours of IH (condition x time interaction, p = 0.108 and p = 0.062, respectively) compared to normoxia. In study 2 (CH-Fasted), ßOHB and NEFA levels increased over time in both experimental conditions, and this effect tended to be greater under CH (condition x time interaction, p = 0.070 and p = 0.046, respectively). In study 3 (CH-Fed), ßOHB levels slightly increased up to 180 min before falling back to initial concentrations by the end of the protocol in both normoxia and CH (p = 0.062), while NEFA slightly increased under CH (p = 0.006). Adipo-IR tended to increase after 6 hours of hypoxia compared to normoxia in the first two studies (main effect of condition, p = 0.024; p = 0.097, respectively), and significantly increased over time under hypoxia in CH-Fed (condition x time interaction, p = 0.004). Conclusion: Acute normobaric hypoxia exposure significantly increases plasma ßOHB concentrations over time in healthy men. The stimulating effect of hypoxia on plasma ßOHB levels is however attenuated during postprandial and postprandial states. Contribution to advancement of knowledge: To our knowledge, this research provides some of the first evidence that an acute exposure to hypoxia increases plasma ßOHB levels in humans. It also reveals potential underlying mechanism that modulate ketogenesis upon hypoxia exposure. Overall, this thesis provides further insights into the homeostatic response of healthy men to oxygen deprivation.

Hypoxia occurs when the dissolved oxygen concentrations in water fall below 2 mg/L, and negative impacts to flora and fauna can result. In this study, sediment core and water column measurements were collected and processed through several laboratory and field methods to better understand the biogeochemistry of Weeks Bay (WB), AL. Results show that salinity, temperature, and dissolved oxygen fluctuate on a diurnal cycle. Results also show a significant difference in water column and sediments between different years and sites. The western side of WB was found to more likely to go hypoxic than other portions. Conclusions of this research include that major differences in the water column are shown during a diurnal cycle, but sediments appear to be less dynamic. The implications of this work include WB hypoxia tendency is heterogeneous over space and time. However the sediments, although important, indicate that the pelagic and benthic components are decoupled.

Animals who live in the high-altitude environment are faced with the harsh conditions of low oxygen availability. This can make it difficult to perform daily tasks that require aerobic metabolism. However, deer mice (Peromyscus maniculatus) have evolved physiological adaptions that allow populations to survive in the high-altitude environment. One activity important for survival is aerobic locomotion and the rapid recovery of muscle metabolism after a bout of exercise. Hypoxia acclimated high-altitude mice have a greater reliance on carbohydrates to power exercise than low altitude mice and show a significant depletion of muscle glycogen. However, it is unclear how quickly after exercise is finished that these mice can replenish muscle glycogen stores. The gastrocnemius muscle of high-altitude deer mice has a more aerobic phenotype and a greater capacity to oxidize lipids than in low altitude mice. This suggests that high altitude mice may recover more quickly from exercise than their lowland counterparts due to a greater capacity to power glycogen replenishment through aerobic metabolism using the lipids stored in muscle as intramuscular triglycerides (IMTG). Using low- and high-altitude native deer mice born and raised in common lab conditions and acclimated to chronic hypoxia, I determined changes in oxygen consumption (VO2) following aerobic exercise and sampled skeletal muscle at various time points during recovery to examine changes in key metabolites, including glycogen and IMTG. I found there was depletion in glycogen stores during exercise in lowlanders and glycogen did return to resting levels following 90 minutes of recovery. In contrast, IMTG’s did not significantly change with exercise or during the recovery period in either population. These data suggest that muscle recovery from aerobic exercise may be influenced by altitude ancestry in deer mice. / Thesis / Master of Science (MSc)

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