As global change alters the chemical and physical dynamics of the ocean, it is increasingly necessary to determine ecological responses across environmental gradients. The benthic ecosystems of fjords often contain a multitude of environmental gradients conducive to multivariate field studies. In this thesis, I describe the benthic community structure of two British Columbian fjords in relation to markedly different environmental variables. In Chapter 2, I show a strong correlation between suspension-feeder abundance and flow structure on the steep fjord walls of Douglas Channel, BC. I also describe distinct assemblages with depth and with location along the fjord head-mouth axis. Using a suite of biological traits, I show that the deep portion (> 400 m depth) of the most seaward site is the most taxonomically and functionally diverse in the fjord. My results suggest fjord walls form an expansive ecosystem containing diverse and dense assemblages of suspension feeders relevant to the flow of energy through fjord basins and as biodiversity reservoirs. In Chapter 3, I extend a long-term hypoxia time-series (2006 -2016) to document the response of soft-bottom epibenthic megafauna of Saanich Inlet, BC to a prolonged hypoxic event in 2016 that caused abundance declines, community aggregation and shifts in species composition more extreme than those seen in the 2013 hypoxia cycle. I also assess community threshold responses along the oxygen gradient; I found community transitions consistent across years and with Northeast Pacific oxygen thresholds based in ecophysiological studies. Taken together, these studies show a strong coupling between oceanographic conditions and the community structure of fjord benthos. I suggest that climate-driven alterations in North Pacific oceanographic regimes may portend major changes in fjord ecosystems. / Graduate

Cancer develops in many organs and tissues in the body through genetic and environmental modifications to acquire the hallmarks of cancer. The hallmarks of cancer allow the cells to become malignant and progress to a tumorigenic state. It has previously been shown in various carcinomas that HIF-2a, a key component in hypoxia adaptation, has a role in autonomous growth, the first hallmark of cancer. Ovarian cancer is the most lethal of the gynecological malignancies and accounts for 3% of new cases in women annually but is the fifth most common cause of death due to cancer. Here, it is shown in two ovarian carcinoma cell lines that HIF-2a is involved in in vitro and in vivo growth. It is also shown that the effect of HIF-2a is due to its role in autonomous growth and not vascularization with the use of in vitro spheroids. From recent findings in the laboratory the oxygen-stimulated translation initiation complex was discovered and HIF-2a is one of its components. In the absence of HIF-2a there is a downregulation in translation in hypoxia in ovarian carcinoma. This is also seen in a HIF-2a translational target, IGF1R and its downstream signaling pathway, which may be involved in autonomous growth as well as other hallmarks of cancer. Taken together, the data in this thesis presents the importance of HIF-2a in autonomous growth and cancer progression in ovarian carcinoma, as well as verifying its role in translation.

Adipose tissue plays a central role in the regulation of lipid storage and mobilization. A tight control between adipose tissue lipid storage and mobilization functions must be exerted to prevent an overload of lipids at other organs such as the heart, liver and skeletal muscles, and favor the risk of developing metabolic disorders, such as Type 2 diabetes and cardiovascular diseases (CVD). There is strong evidence from animal studies that low oxygen levels (hypoxia) are noted in adipose tissue as the mass of the organ excessively expands and, in turn, exacerbates some adipose tissue functions. Whether hypoxia exposure, which could be derived from reduced environmental oxygen availability, disease or a combination of both, affects adipose tissue lipid storage and mobilization functions in humans is not well known. Using in vitro and in vivo approaches, this thesis aimed at characterizing the effects of hypoxia on human adipose tissue lipid storage and lipid mobilization functions. Study I investigated how hypoxia can modulate human adipose functions such as lipid storage and lipid mobilization in vitro. Study II examined whether acute intermittent hypoxia, which simulates obstructive sleep apnea, affects adipose tissue lipid storage/mobilization functions and triglyceride levels in healthy young men in postprandial state. Study III tested the effect of an acute 6-hour continuous exposure to hypoxia (fraction of inspired oxygen (FIO2) = 0.12)) on plasma triglyceride levels in healthy young men in the fasting state. Study I indicates that both acute (24h) and chronic (14d) hypoxia (3%, and 10% O2) modulate human adipose tissue lipid storage and mobilization functions in a different manner. Study II demonstrates that acute exposure to intermittent hypoxia (6h) is sufficient to increase plasma non-esterified fatty acids (NEFA) levels, as well as insulin levels, but does not alter circulating triglyceride or subcutaneous adipose tissue lipid storage and/or mobilization capacity ex vivo in healthy men. Study III shows that acute exposure to normobaric hypoxia increases circulating NEFA and glycerol concentrations but did not translate in altering circulating triglycerides in fasting healthy men. In conclusion, our observations suggest that an exposure to reduced oxygen levels impairs human adipose tissue storage and/or mobilization functions, a phenomenon known in the development of metabolic disorders, such as Type 2 diabetes and CVD.

Introduction: Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. Methods: In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. For this gas chromatography mass spectrometry (GC-MS) and ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) were used. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to elucidate novel metabolic pathways as potential therapeutic targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Results: Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers associated with hypoxia-induced chemoresistance were related to metabolic pathways including gluconeogenesis, DNA synthesis and fatty acid synthesis. Furthermore, network-based correlation analysis revealed specific metabolites in the fatty acid synthesis pathways were contributing to drug resistance, which included malonyl-CoA, 3-oxoeicosanoyl-CoA, stearoyl-CoA and octadecanoic acid. To facilitate the detection of metabolites in ToF SIMS datasets, a series of metabolites standard spectra were acquired. Hypoxic metabolite markers detected in ToF-SIMS data of cell lysates included glycine, lactic acid and succinic acid, which were also shown to be metabolite markers in GC-MS metabolic data. Furthermore, MTS sections were imaged using ToF-SIMS to profile the chemical response to chemotherapy treatment within the oxygen gradient. Loadings from image PCA were explored to determine the metabolic response in the highly oxygenated outer region and hypoxic inner region of the MTS. Conclusion: A multitude of analytical techniques were able to contribute to elucidating the metabolic mechanisms associated with hypoxia-induced chemoresistance. Metabolic profiling combined with a systems biology approach was further able to identify potential underlying metabolic regulation of resistance. Finally ToF-SIMS was developed as a tool for metabolite analysis in complex biological systems in situ.

The altitude at which oxygen supplementation should commence to be administered to aircrew in South Africa, flying in unpressurised aircraft is 12,000 feet. Above that altitude effects of reduced tissue oxygen content (hypoxia) become significant. Vision is particularly sensitive to hypoxia, and it was decided to test visual parameters at 12,000 feet to see if there were any subtle changes which might impair flight safety. It this were so, the level at which oxygen supplementation should commence would have to be lowered. The visual parameters to be assessed, all important in aviation, were colour vision, contrast sensitivity, and the presence of phorias (potential squints). 37 Healthy volunteers had these parameters assessed in an altitude chamber at ground level, 8,000 feet and 12,000 feet. Analysis of the results revealed no clinically significant degradation of vision at 12,000 feet, and it was concluded that the present altitude at which oxygen supplementation should begin (12,000 feet) is appropriate. It was advised that further testing should take place in subjects based at sea level. / Dissertation (MSc)--University of Pretoria, 2006. / School of Health Systems and Public Health (SHSPH) / Unrestricted

Orientador: Antonio Carlos de Moraes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Educação Física / Made available in DSpace on 2018-08-18T17:14:16Z (GMT). No. of bitstreams: 1 Fontes_EduardoBodnariuc_D.pdf: 9503115 bytes, checksum: ace5f37f3ad02cc8337e62c4be60c8b2 (MD5) Previous issue date: 2011 / Resumo: O presente trabalho buscou um maior entendimento da formação da percepção subjetiva de esforço (PSE) durante esforços exaustivos. Dessa forma, o primeiro estudo verificou as associações da atividade muscular (EMG) com a PSE, bem como a determinação do limiar de esforço percebido (LEP) e de fadiga neuromuscular (LFN). Esse estudo analisou 11 adultos jovens durante testes de carga constante até a exaustão voluntária máxima com monitoramento constante de PSE e EMG. A taxa de aumento dessas variáveis (EMGslope e PSEslope) foram significativamente correlacionados e inversamente associados ao tempo de exaustão. LEP e LFN e não se diferiram significativamente. Assim, indicamos a estreita relação do recrutamento adicional de fibras com o aumento da PSE. O segundo estudo foi realizado durante estágio no exterior (sanduíche) na África do Sul no ano de 2009. Nesse trabalho, foi analisado os efeitos da diminuição de oferta de oxigênio (hipóxia) sobre variáveis centrais e periféricas e suas associações com PSE. Seis ciclistas realizaram testes exaustivos de carga constante em normoxia e hipóxia com contínua aquisição de respostas de PSE, EMG e oxigenação muscular (MOX) e cerebral (COX). Foi demonstrado que na condição hipóxia ocorre um significativo aumento sobre PSE em seus diferentes modos (local, respiração e geral), EMG e COX, mas não em MOX. Os slopes de PSE e valores finais de COX foram relacionados ao desempenho em normóxia, no entanto ainda maiores foram apresentados em hipóxia. Além disso, COX foi ainda significativamente relacionada RPE local em normóxia e novamente, hipóxia exerceu efeitos maiores nessas associações, mas dessa vez para todos os modos de PSE. No terceiro estudo, foram utilizado os mesmos dados do estudo anterior para verificamos os possíveis efeitos de hipóxia ao estimarmos LEP de maneira diferenciada (local, respiração e geral) e LFN pelo mesmo protocolo. Todos os modos de LEP diminuíram significativamente sob hipóxia, com maiores efeitos sobre LEP local. Já LFN não respondeu aos efeitos da condição experimental. Dessa forma, expandiu-se a utilização de LEP para altitudes moderadas e foi apresentado uma nova forma de predizer capacidade aeróbia referente aos membros envolvidos e respiração, além de PET para o corpo como um todo. Associando os achados dos estudos, podemos inferir a estreita relação de respostas periféricas e centrais sobre a formação de PSE, senda essas fortalecidas em condições de diminuídas ofertas de oxigênio. Mais adiante, essas associações justificam a ampliação de utilização prática de PSE, podendo ser para o exercício de alta intensidade ou monitoramento localizado da capacidade aeróbia / Abstract: The present study aimed to bring better understanding of ratings of perceived exertion (RPE) during exhaustive exercise. Thus, the first study verified the associations of the neuromuscular responses (EMG) with RPE, as well as the determination of the perceived exertion threshold (PET) and neuromuscular fatigue threshold (NFT). Eleven adults performed exhaustive constant-load tests with RPE and EMG recordings. The rate of increase of these variables (EMGslope e RPEslope) were significantly related and associated to performance. Além disso, PET and NFT did not differed. Therefore, it was shown the close relationship of the additional muscle recruitment and RPE. The second study was completed during the international internship in South Africa in 2009. At this investigation, were demonstrated the effects of decreased fraction of inspired oxygen (hypoxia) on central and peripheral responses, as well their relationship with RPE. Six trained cyclists completed exhaustive constant-load tests under normoxia and hypoxia having continuously monitoring of RPE, EMG and cerebral (COX) and muscle (MOX) oxygenation. It was shown that under hypoxia there is a significant increase for all RPE modes (legs, breathing and overall), EMG and COX, but not MOX. The RPE slopes and end values for COX were related to performance under normoxia, however higher associations were found under hypoxia. In addition, COX was significantly related to RPE for legs under normoxia, but again, hypoxia exert higher effects on this association, but this time to all RPE modes. During the third study, the data from last investigation was used to verify the possible effects of hypoxia when estimating differentiated PET (legs, breathing and overall) and NFT during same protocol. All PET modes decrease significantly under hypoxia, with higher effects of PET legs, however, NFT estimation was not affects by this experimental condition. Thus, PET's used was expanded to moderated altitudes and presented a new method to predict aerobic capacity associated to active limbs and breathing, in addition to whole body PET. Associating the studies' findings it is possible to conclude that there is a strict relationship of peripheral and central responses to RPE construct, being this sthrengthed by decreased oxygen availability. Furthermore, these relationship justifies the practical use RPE, as for prescription of high intensity exercise or localized monitoring of aerobic capacity / Doutorado / Ciencia do Desporto / Doutor em Educação Física

Glicose e oxigênio desempenham um importante papel na regulação do metabolismo celular. Dada a importância de ambos no metabolismo - o primeiro como fonte de carbono preferencial da maior parte das células, e o segundo como aceptor final de elétrons na cadeia respiratória, em diversos organismos desenvolveram-se métodos adequados para detectar sua presença de modo a ajustar o metabolismo em função de sua disponibilidade. Neste trabalho foi realizado o estudo da expressão, no nível transcricional, dos genes envolvidos nas vias metabólicas primárias e genes envolvidos em morte celular, em células humanas, com o intuito de determinar as alterações no metabolismo energético em resposta a condições de hipóxia e anóxia, por meio da técnica de microarrays de cDNA. Utilizamos, inicialmente, células normais de fibroblasto humano ASl98 e células de fibroblasto humano MRC-5 imortalizadas por transfecção por SV40, e por fim células provenientes de ilhotas pancreáticas humanas, para a elaboração de um protocolo de cultura celular em que as mesmas crescessem aderidas a microcarregadores Cytodex I. Numa segunda etapa, células de ilhotas pancreáticas humanas foram cultivadas em suspensão, aderidas aos microcarregadores, num biorreator, sendo então realizada a análise do perfil transcricional dos genes escolhidos, frente às condições de baixa tensão de oxigênio. É apresentada a análise da expressão gênica de aproximadamente 160 genes na qual foram verificados um comportamento de indução daqueles envolvidos no metabolismo de lipídios e alguns na morte celular e um comportamento inicial de indução, e posterior inibição, do metabolismo primário como um todo. Em vista dos dados obtidos é de interesse ressaltar que essas células deveriam ser mantidas em saturações de oxigênio acima de 5% para evitar o efeito deletério observado na baixa concentração de oxigênio sobre a viabilidade celular, em termos da indução de alguns genes envolvidos na morte celular e da repressão geral dos relacionados ao metabolismo energético. Também foi verificado que, em saturações de oxigênio de até 10%, as células adotaram um padrão transcricional que indicou uma resposta ao estresse por falta de oxigênio, este por sua vez reflete-se na viabilidade celular, característica crucial para o sucesso do transplante clínico de ilhotas. / Glucose and oxygen have important roles on the regulation of cellular metabolism. Due to their importance in metabolism, the former as the preferential carbon source and the later as the final electron acceptor of the respiratory chain, many organisms have developed suitable processes to detect their presence in order to adjust the cellular metabolism to their availability. In this work, we have studied, in human cells and at the transcriptional level, the expression of genes involved in primary metabolism pathways and some of those related to cell death, aiming to resolve alterations in the energetic metabolism as a response to hypoxic and anoxic conditions, by means of cDNA microarrays. We initially used AS198 human fibroblastic normal cells and MRC-5 human fibroblastic cells immortalized by SV40, and later on cells from human pancreatic islets, to develop a cell culture protocol in which they would grow on the surface of Cytodex 1 microcarriers. As a second step, cells from human pancreatic islets were cultured on microcarriers in suspension inside a bioreactor. This culture was then used to carry out the transcriptional profile analysis of selected genes in response to low levels of oxygen. This work presents the analysis of gene expression of approximately 160 genes that can be divided into two distinct groups. The first group, the expression of which is induced, comprises genes involved in lipid metabolism and some of those related to cell death. The expression of the second group, consisting of diverse genes of the primary metabolism, suffers an initial induction followed by repression. Given the data acquired it is interesting to note that the human pancreatic islets should be maintained under at least 5% dissolved oxygen to avoid the deleterious effects on cell viability observed at lower oxygen concentrations, resulting in the induction of some genes involved in cell death and the repression of those related to energetic metabolism. It was also verified that, under oxygen saturation of at least 10%, these cells adopted a transcriptional profile that indicated a response to the stress created by the lack of oxygen, which would in turn reflect on cell viability, a crucial characteristic for success in clinical islet transplantation.

Background: Obstructive sleep apnea (OSA) consists of repeated, involuntary breathing suspension during sleep. These events induce rapid depletion/repletion of blood/tissue oxygen content, a phenomenon known as intermittent hypoxia. Aside from causing daytime sleepiness, the most important health consequence of OSA is a 2-fold increase in cardiovascular (CVD) risk. Animal studies provide evidence that intermittent hypoxia, a simulating model of OSA, causes important rise in plasma TG, especially in the postprandial state. However, the underpinning mechanisms linking intermittent hypoxia to altered postprandial TG levels remain unknown. As such, the objective of this study was to characterize the effects of acute intermittent hypoxia on postprandial TG levels in 2 distinct lipoprotein subtypes in humans: chylomicrons which are secreted by the intestine and carry dietary lipids, and denser TG carriers (mainly VLDL) which are secreted by the liver and carry endogenous lipids. Methods: The research consisted of a randomized crossover design. In collaboration with the Sleep laboratory at Montfort Hospital, 7 individuals diagnosed with moderate sleep apnea were recruited through phone calls as well as 8 healthy individuals without OSA from the University of Ottawa. While lying on a bed, participants were given a meal after which they were exposed for 6 hours to normoxia or intermittent hypoxia corresponding to moderate OSA, e.g. 15 hypoxic events per hour. Blood lipid levels were measured hourly.  Results: Plasma TG levels increased over time in both experimental conditions and tended to be greater under 6-h exposure to intermittent hypoxia (p=0.093, effect size ηp2= 0.383.). This trend toward higher total plasma TG under intermittent hypoxia was attributable to increased levels in denser TG carrying lipoproteins such as VLDL and CM remnants (p= 0.009, ηp2 = 0.173).  Conclusion: Acute intermittent hypoxia, a simulating model of obstructive sleep apnea, tends to negatively affect postprandial TG levels, which is attributable to an increase in denser TG carrying lipoprotein levels such as VLDL and CM remnants. These results lend support to the increase in blood lipid levels in animal studies observing the effect of acute hypoxia in mice.  Contribution to advancement of knowledge: This proposed research will allow a better understanding of the mechanisms by which obstructive sleep apnea may alter blood lipid profile. This information will be beneficial to the treatment of obstructive sleep apnea related dyslipidemia and contribute to reduce CVD risk in the large proportion of obstructive sleep apnea patients who are reluctant to current treatment avenues.

L’hypoxie est une caractéristique majeure des glioblastomes (GB). Elle est la cause principale de la résistance aux traitements observée dans ces tumeurs. Les conséquences de la baisse en oxygène au niveau tumoral, sont médiées par les facteurs de transcription Hypoxia Inducible Factors (HIF). Ces facteurs sont des protéines hétérodimériques HIF-α/HIF-1β responsables de la transcription de nombreux gènes cibles. Certaines de ces cibles participent à la progression tumorale et à la mise en place d’un phénotype agressif. L’une des cibles de l’isoforme HIF-2α, est la βIII-tubuline (βIII-t). Cette protéine, qui compose les microtubules, est décrite comme surexprimée dans les gliomes de haut grade, comme les GB. Ces travaux de thèse s’intéressent au rôle de la βIII-t dans la progression tumorale ainsi qu’au développement de stratégies permettant d’inhiber l’expression de HIF-2α. Les résultats obtenus montrent que la βIII-t a une importance centrale dans le développement tumoral. En effet, les tumeurs issues de l’implantation de cellules humaines de GB invalidées pour la βIII-t, se développent significativement moins vite comparées aux tumeurs contrôles. In vitro, nous avons montré que cette protéine est impliquée dans la prolifération, la migration et l’invasion cellulaires. En revanche, nous n’avons pas pu confirmer que la βIII-t est impliquée dans la résistance aux traitements (chimio- ou radiothérapeutiques). Les deux composés testés comme inhibiteurs de HIF-2α (SR2933 et PT2385) ont montré des résultats prometteurs sur la βIII-t, gène cible spécifique de HIF-2α. Cependant, malgré les stratégies développées, nous n’avons pas pu évaluer l’efficacité directe de ces deux composés sur l’hétérodimérisation de HIF-2α avec HIF-1β. / Hypoxia is a major feature of glioblastoma (GB). It is the main cause of the resistance to treatments observed in these tumors. The consequences of the decrease in oxygen at the tumor level, is mediated by the Hypoxia Inducible Factors (HIF). These transcription factors are heterodimeric proteins HIF-α/HIF-1β responsible for the transcription of many target genes. Some of these targets are responsible for setting up an aggressive phenotype and in tumor progression. One of the targets of the HIF-2α isoform is βIII-tubulin (βIII-t). This protein, which is a constituent of microtubules, is described as overexpressed in high grade gliomas, such as GB. In the present thesis, we examined the role of βIII-t in tumor progression and we developed strategies to inhibit the expression of HIF-2α. Our results show that βIII-t is of central importance in tumor development. Indeed, tumors resulting from the implantation of GB human cells invalidated for βIII-t, developed significantly less rapidly compared to control tumors. In vitro, we have shown that this protein is involved in cell proliferation, migration and invasion. However, we could not confirm that βIII-t is involved in resistance to treatments (chemotherapeutic or radiotherapeutic).The two compounds tested as inhibitors of HIF-2α (SR2933 and PT2385) showed promising results on βIII-t, a specific target gene of HIF-2α. However, despite the different tested strategies, we could not evaluate the direct inhibitory action of these two compounds on the heterodimerization of HIF-2α with HIF-1β.

Early exposure to hypoxia is related to a variety of physiological and metabolic changes that have lasting effects on organisms’ physiology and life history. We measured the effects of maternal and embryonic mild, intermittent hypoxia on the life history of four clones of microcrustacean Daphnia magna, an emerging model organism for the studies of senescence and longevity. Daphnia individuals were produced parthenogenically, maintained in individual vials, and fed standard algal concentration daily. The cohort consisted of 189 individuals. We measured body size at first reproduction, fecundity (including late-life fecundity peak), offspring sex ratio, and longevity. We found no effect of maternal and embryonic hypoxia on body size and longevity; however, there was a slight but statistically significant increase in age-specific mortality in the early hypoxia treatment cohort. Daphnia from the hypoxia group showed higher early fecundity which disappeared by the age of 100 days. A late-life spike in fecundity was observed at the age of 100 days when hypoxia group individuals showed significantly lower fecundity. There was little evidence of a trade-off between early- and late-life fecundity. Finally, early hypoxia affected mid-life male production in one of the four clones, and we discuss possible physiological changes triggered by maternal and embryonic exposure to hypoxia.