The critical swimming speed (Ucrit) of rainbow trout parr (Oncorhynchus mykiss) and three life stages of Galaxias maculatus, larval (whitebait), postlarval inanga and adult inanga, were tested at temperatures from 5oC to 25oC. All fish were swum at their acclimation temperature under normoxic conditions to determine the optimal aerobic exercise temperature. To determine whether acclimation affected swimming ability, trout parr acclimated to either 10oC or 20oC were swum at 20oC and 10oC, respectively. The potential effect of mild hypoxia (75% saturation) on trout parr and whitebait was also examined at 10oC, 15oC and 20oC, and also tested separately and in combination were the effects of mild hypoxia and severe anaemia on the prolonged swimming ability of trout smolts at temperatures from 10oC to 20oC. For all trout experiments, blood samples were taken from non-exercised and exercised fish by acute caudal venepuncture to determine haematological responses to both acclimation and exercise. Under normoxic conditions, Ucrit max for trout parr (7.0 0.5 cm fork length) was calculated to be 5.8 body lengths per second (BL s-1) at 15.1oC, but declined at lower and higher temperatures. This result implies that swimming performance was limited by temperature below 15oC, whereas performance at higher temperatures was limited by oxygen availability. In support of this hypothesis, mild hypoxia (75% saturation) had no effect at 10oC or 15oC but caused a significant reduction in Ucrit at 20oC. However, fish acclimated at 20oC showed an adaptive elevation in oxygen carrying capacity due to an increase in mean erythrocyte volume and haemoglobin content. Furthermore, acclimation to 20oC improved warm water swimming performance. Trout parr acclimated to 10oC performed significantly worse than fish acclimated to 20oC when swum at 20oC. However, trout parr acclimated to 20oC performed as well as fish acclimated to 10oC when swum at 10oC. Following exercise, haematocrit was elevated under both normoxic and hypoxic conditions. However, the primary cause of this apparent increase in oxygen carrying capacity was splenic release of erythrocytes under normoxic conditions, whereas stress-induced erythrocytic swelling contributed to the observed increase in hypoxia. This contrasting response was most pronounced at 10oC. Larval whitebait (4.7 - 5.0 cm total length (TL)) also showed a temperature dependence of prolonged swimming ability with Ucrit max calculated to be 5.1 BL s-1 at 17.7oC. Hypoxia significantly reduced Ucrit at 15oC and 20oC, lowering the optimal aerobic temperature to 13.9oC and reducing Ucrit to 4.2 BL s-1. Mild hypoxia therefore had a more pronounced impact on inanga whitebait than trout. Postlarval inanga (3.9 - 4.0 cm TL) performed poorly at higher temperatures with Ucrit max of 5.6 BL s-1 at 9.4oC indicating an ontogenetic change in swimming ability, possibly resulting from a developmental shift in red muscle kinetics or a greater dependence on anaerobic muscle. Adult inanga (5.5 - 6.8 cm TL) prolonged swimming ability showed similar temperature dependence to that of inanga whitebait but lower relative swimming speeds due to their larger size. The dramatic decline in performance exhibited by juveniles at warmer temperatures was not apparent in adults. Ucrit max for adults was 4.0 BL s-1 at 18.3oC. The critical swimming speed of trout smolts, subjected to mild hypoxia (6.8 mg

Adaptation to the shortage of oxygen at altitude (hypoxia) promotes physiological changes which could enhance endurance performance. Consequently, altitude training has become a popular practice among competitive endurance athletes. Since its inception, the live-high train-low paradigm (LHTL) has been widely regarded as the most effective approach to altitude training. Over the past decade, brief intermittent simulation of LHTL via the use of hypoxic inhalers and re-breathing devices has gained increased popularity, but the evidence supporting their use is limited and conflicting. The experimental studies in this thesis investigated the response of sea level exercise performance and related physiological measures following adaptation to the usual and a novel protocol of brief intermittent hypoxia. I intended to perform all experimental studies on flat-water kayakers. Therefore, an initial requirement of this thesis was to establish the smallest worthwhile effect in performance for this sport. The final study utilising a meta-analytic approach was conducted to compare the effectiveness of brief intermittent hypoxia to other natural and simulated protocols, and to investigate the topical issue of what physiological responses mediate performance changes following hypoxic exposure. In Study 1, the typical variation in competitive performance of elite flat-water canoeists was investigated using a repeated-measures analysis of race times. For individual flat-water canoeing events, the smallest worthwhile change in performance time was ~0.5%. In two separate experimental studies, adaptation to 60 min per day of brief intermittent hypoxia consisting of alternating 5 min intervals of hypoxia and normoxia for 3 weeks (5 days per week) using a nitrogen filtration device resulted in clear enhancement of endurance performance (~5%) for kayakers (Study 2) and cyclists (Study 3). Clear enhancements in repeat sprint performance were observed for kayaking only. The physiological mechanisms underlying performance changes were unclear. Modification of the hypoxic and normoxic intervals (Study 3) did not result in any clear alterations in performance or physiological mechanisms. The meta-analysis (Study 4) revealed clear enhancements in endurance power output of 1-3% in sub-elites following adaptation to hypoxia with the natural altitude protocols, and with two of the artificial-altitude protocols (LHTL-long and LHTL-brief-intermittent). In elite athletes the enhancements tended to be smaller and were clear only for the natural protocols. These enhancements could be mediated by VO2max, although other mechanisms may be possible.

The bluefin tuna have a variety of distinctive anatomical and physiological adaptations that enhance performance. However, our understanding of bluefin tuna physiology is limited by the logistical difficulties of studying these large pelagic fish. This thesis examines some aspects of the metabolic physiology of the southern bluefin tuna. It provides insight into the high-performance, high-energy demand physiology of bluefin. It also examines the metabolic physiology of the mulloway, another important aquaculture species for which physiological information is currently limited. 1. Routine metabolic rate (RMR) of southern bluefin tuna (SBT) (Thunnus maccoyii), the largest tuna specimens studied so far (body mass = 19.6 kg (± 1.9 SE)) was measured in a large (250,000 l) flexible polypropylene respirometer “mesocosm respirometer”. Mean mass-specific RMR was 460 mg kg⁻¹ h⁻¹ (± 34.9) at a mean water temperature of 19°C. When total RMR is added to published values of other tuna species at equivalent swimming speeds, there is a strong allometeric relationship with body mass (654 • Mb ⁰·⁹ ⁵, R ² = 0.97). This demonstrates that interspecific RMR of tuna scale with respect to body mass similar to that of other teleosts, but is approximately 5-fold higher than the standard metabolic rate (SMR) of other active teleost species. 2. This study reports on the first measurements of the metabolic cost of food digestion and assimilation (specific dynamic action, SDA) of a tuna species. Oxygen consumption (MO₂) and swimming velocity of southern bluefin tuna (SBT) (Thunnus maccoyii) were elevated for periods between 20-45 h (longest for the largest rations) post-ingestion of sardines (Sardinops sagax). It is suggested that the purpose of increased swimming velocity was to increase ventilation volume as a response to the enhanced metabolic demand associated with SDA. The magnitude of SDA as a proportion of gross energy ingested (SDA coefficient) averaged 35 ± 2.2 %. This demonstrates that the absolute energetic cost of SDA in SBT is approximately double that recorded in other teleost species. 3. This study examines the effects of sardines (Sardinops sagax) with high- (12.9%) or low- (1.8-4.0%) lipid level on specific dynamic action (SDA) and swimming velocity of southern bluefin tuna (SBT) (Thunnus maccoyii). Fish swam faster during the SDA period with the increase in velocity being greatest for the fish that ingested the high-lipid sardine. Magnitude of SDA was also greater for fish that ingested the high-lipid sardines. However, the energetic cost of SDA as a proportion of ingested energy was not significantly different between fish that ingested the high- (34.3 ± 2.4%) and low-lipid sardines (31.5 ± 2.9%). These results confirm that the high energetic cost of SDA is ecologically relevant. 4. In this study the metabolic and behavioural responses of both fasted and postprandial southern bluefin tuna (Thunnus maccoyii, SBT) to low dissolved oxygen (DO) was examined. In moderate hypoxia (4.44 and 3.23 mg l⁻¹), swimming velocity (U) and routine metabolic rate (RMR) of fasted fish was mildly enhanced. At 2.49 mg l⁻¹, U increase to over double in the normoxic speed, possibly as an escape response. At 1.57 mg l⁻¹, both U and RMR were suppressed and SBT failed to survive the entire 20 h exposure period. This reveals that SBT are remarkably well adapted to low DO. Feeding did not greatly influence their hypoxia tolerance. In a subsequent experiment there were no significant differences in U, RMR and gastric evacuation rates of postprandial SBT in hypoxia (2.84 mg l⁻¹) compared to those in normoxia (7.55 mg l-¹). 5. In this study, 768 h of simultaneous recordings of metabolic rate (MR, = heat production) and visceral temperature were made in both fasted and postprandial southern bluefin tuna (SBT, Thunnus maccoyii) of two sizes (~10 and 20 kg) and at two water temperatures (~19 and 16°C). Duration and magnitude of specific dynamic action (SDA) were strongly related to duration and magnitude of postprandial visceral warming providing the first empirical evidence of a link between SDA and postprandial visceral warming. Visceral temperature of fasted SBT was also directly related to MR. In this case, source of heat is thought to be metabolic work performed within the red muscles which warmed the viscera through thermal conductance. Visceral excess temperatures were over 1°C warmer in larger than smaller SBT. Better heat retention ability of the larger SBT is likely attributed to improved retia mirabilia development and greater thermal inertia. SBT at 16°C maintained visceral excess temperatures significantly warmer than similarly sized fish at 19°C. This demonstrates some ability of SBT to physiologically regulate visceral warming. 6. In this study, the effect of progressively severe hypoxia levels on the swimming performance and metabolic scope of juvenile mulloway (Argyrosomus japonicus) were investigated. In normoxic conditions (6.85 mg l⁻¹), standard metabolic rate (SMR) and cost of transport were typical for subcarangiform fish species. Mulloway had a moderate scope for aerobic metabolism (5 times the SMR). The critical dissolved oxygen level was 1.80 mg l⁻¹ revealing that mulloway are well adapted to hypoxia. In all levels of hypoxia (5.23, 3.64, and 1.86 mg l⁻¹) the active metabolic rate was reduced however, the critical swimming velocity was reduced only at 3.64, and 1.86 mg l⁻¹. Mulloway metabolic scope was significantly reduced at all hypoxia levels, suggesting that even mild hypoxia may reduce growth productivity. / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2007

Hypoxia is one of the hallmarks of cancer. It was first postulated to occur in solid tumours by Thomlinson and Gray in 1955.1 The presence of hypoxia has been demonstrated in different types of solid tumours.2 Intratumoral hypoxia is caused by the lack of functional blood vessels in proliferating tumour tissue, resulting in low intratumoral oxygen concentrations. If hypoxia is severe or prolonged, cell death occurs.3 Malignant cells can undergo genetic and adaptive changes that allow them to escape from dying of oxygen deprivation. These changes are associated with a more aggressive malignant phenotype 4,5 conferring resistance to radiation 6,7 and chemotherapeutic agents.3,8,9 Hence hypoxia is known to be a key factor responsible for tumour resistance in humans. / Invasive polarographic oxygen sensor measurements have demonstrated hypoxia in solid tumours and it is generally defined to occur at an oxygen tension less than ten mmHg.10 Perhaps of more importance is that hypoxia has been demonstrated to be a prognostic indicator for local control after treatment with radiotherapy in glioma, head and neck and cervical cancers.11-13 It has also been able to predict for survival and the presence of distant metastases in soft tissue sarcomas.14 Finally, the significance of hypoxia in the activation and induction of functional molecules such as hypoxia inducible factors (HIFs) and VEGF, the modulation of gene expression (e.g. carbonic anhydrase IX), increased proto-oncogene levels, activation of nuclear factors and accumulation of other proteins (e.g. TP53) although progressing, is yet to be defined.15,16 / Thus, it is of clinical interest to understand the levels of hypoxia and numbers of hypoxic cell populations in tumours, particularly those resistant to radiation and chemotherapy. In doing so clinicians and researchers may formulate more accurate prognostic information and develop treatments targeting hypoxic cells. Renal cell carcinoma (RCC) is a tumour resistant to radiation and chemotherapy that is yet to have its oxygen status investigated. / Although the “gold standard” of oxygen tension measurement is the Polarographic Oxygen Sensor (POS or Eppendorf pO2 histograph), non-invasive means of measuring oxygen status via imaging, immunohistochemistry or serum tumour markers are more practical. As highlighted by Menon and Fraker, it is imperative that reliable, globally usable, and technically simplistic methods be developed to yield a consistent, comprehensive, and reliable profile of tumour oxygenation. Until newer more reliable techniques are developed, existing independent techniques or appropriate combinations of techniques should be optimized and validated using known endpoints in tumour oxygenation status and/or treatment outcomes.17 / Hanahan and Weinberg 18 surmised that the field of cancer research has largely been guided by a reductionist focus on cancer cells and the genes within them- a focus that has produced an extraordinary body of knowledge. Looking forward in time, they believe that progress in cancer research would come from regarding tumours as complex tissues in which mutant cancer cells have conscripted and subverted normal cell types (endothelial cells, immune cells, fibroblasts) to serve as active collaborators in their neoplastic agenda. The interactions between the genetically altered malignant cells and these supporting coconspirators will prove critical to understanding cancer pathogenesis and to the development of novel, effective therapies.18 / Essentially, the background outlined here not only highlights the core aim of this thesis: to better understand the oxygen status of renal cell carcinoma and the relationship of this to angiogenesis so that better targeted therapies may be pursued in the future; but it also places this research in the context of the future proposed by Hanahan and Weinberg,18 by clearly focusing on collaborators in the neoplastic agenda, rather than just tumour cells themselves, to better understand RCC.

<p>Environmental stress during embryonic development could lead to growth restriction of the embryo, and act as a risk factor for the development of cardiovascular disease in adult life. A common environmental stressor that causes growth restriction is prenatal hypoxia, which has been shown to adversely affect adult health in mammalian models. Prenatal hypoxia causes an increase in catecholamines which results in over stimulation of the cardiac β-adrenergic receptors. Previous work on chickens has shown that prenatal hypoxia causes an increase in the sensitivity of β-adrenergic receptors to epinephrine in the embryonic heart. The sensitivity of these receptors was found to be decreased in prenatal hypoxic juvenile. Prenatal hypoxia has no significant effect on the density of these receptors in neither the embryo nor the juvenile. The lack of change in receptor density implies that the effects of hypoxia are further down stream in the signalling cascade. The β2 adrenergic receptor can couple to both the stimulatory Gα subunit (Gsα) and the inhibitory Gα subunit (Giα). We hypothesized that prenatal hypoxia would cause an increase in the Gsα in the sensitized embryos, while increasing Giα in the desensitized juveniles. This study evaluated the relative levels of Gsα and Giα in the hypoxic chicken embryo, and in the prenatally hypoxic juvenile, Using western blotting. Hypoxia considerably increased Giα in the chicken embryo while having no effect on Gsα. In the prenatally hypoxic juvenile Gsα was significantly increased while no changes were found in Giα. This dissociation between the levels of Gα subunit and receptor sensitivity implies that that hypoxia affects the signaling cascade downstream of the Gα subunit.</p>

Extrinsic repair techniques for the treatment of acute chondral injuries continue to yield suboptimal repair. The inability of these techniques to produce hyaline cartilage underscores the limitations in our understanding of basic chondrocyte biology. Conversely, intrinsic repair tissue has not been extensively studied despite the fact that it can yield hyaline-like cartilage and is commonly observed in osteoarthritis. Attempts at extrinsic repair could therefore benefit from a better understanding of the successes and failures inherent in the intrinsic repair process. Chondrocyte culture has typically been conducted under non-physiologic conditions whereby chondrocytes readily dedifferentiate. Consequently, much of the knowledge gained about chondrocytes has been misleading thus hindering advancements in chondrocyte biology and attempts at extrinsic articular cartilage (AC) repair. Hypoxic culture conditions, which are beneficial towards the preservation of the chondrocyte phenotype, remain insufficient due to elevated collagen I gene expression. As such, an appropriate model system does not yet exist in which to study physiologically-relevant chondrocyte biology. The presence and prevalence of collagen I in both degenerate and de novo osteoartritic tissue was examined immunohistochemically. Collagen I deposition during osteoarthritic progression was compared against IHC staining for collagen II and aggrecan. A novel model system was also evaluated for chondrocytic phenotype retention. To this end, hypoxic, high-density-monolayer-chondrocyte (HDMC) cultures were compared to freshly isolated chondrocytes for their ability to maintain a chondrocytic extracellular matrix (ECM) gene expression profile. HDMC culture conditions prevented the severe loss of the phenotype typically associated with conventional monolayer culture. Moreover, prolonged HDMC culture resulted in the formation of a complex ECM and a marked suppression of collagen I expression. This study also demonstrated that collagen I deposition occurs in osteoarthritic AC at the onset of structural damage and increases in response to increasing structural damage. Collagen I deposition was also found in different types of de novo cartilage associated with osteoarthritic joints and suggests that it plays an important role in intrinsic cartilage repair. Taken together, this work demonstrates that collagen I is a common feature in the ECM of structurally immature and structurally damaged AC and hence may play a role in tissue stabilization. / Experimental Surgery

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: George W. Luther, III., College of Marine and Earth Studies. Includes bibliographical references.

The hypoxic area in the Gulf of Mexico is the second largest in the world, which has received extensive scientific study and management interest. Previous modeling studies have concluded that the increased hypoxic area in the Gulf of Mexico was caused by the increased anthropogenic nitrogen loading of the Mississippi River; however, the nitrogen-area relationship is complicated by many other factors, such as wind, river discharge, and the ratio of Mississippi to Atchafalaya River flow. These factors are related to large-scale climate variability, and thus will not be affected by regional nitrogen reduction efforts. In the research presented here, both statistical (regression) and numerical models are used to study the influence of anthropogenic and climate factors on the hypoxic area variability in the Gulf of Mexico. The numerical model is a three-dimensional, coupled hydrological-biogeochemical model (ROMS-Fennel). Results include: (1) the west wind duration during the summer explain 55% of the hypoxic area variability since 1993. Combined wind duration and nitrogen loading explain over 70% of the variability, and combined wind duration and river discharge explain over 85% of the variability. (2) The numerical model captures the temporal variability, but overestimates the bottom oxygen concentrations. The model shows that the simulated hypoxic area is in agreement with the observations from the year 1991, as long as hypoxia is defined as oxygen concentrations below 3 mg/L rather than below 2 mg/L. (3) The first three modes from an Empirical Orthogonal Function (EOF) analysis of the numerical model output results explain 62%, 8.1% and 4.9% of the variability of the hypoxic area. The Principle Component time series is cross-correlated with wind, dissolved inorganic nitrogen concentration and river discharge. (4) Scenario experiments with the same nitrogen loading, but different duration of upwelling favorable wind, indicate that the upwelling favorable wind is important for hypoxic area development. However, a long duration of upwelling wind decreases the area. (5) Scenario experiments with the same nitrogen loading, but different discharges, indicate that increasing river discharge by 50% increases the area by 42%. Additionally, scenario experiments with the same river discharge, but different nitrogen concentrations, indicate that reducing the nitrogen concentration by 50% decreases the area by 75%. (6) Scenario experiments with the same nitrogen loading, but different flow diver- sions, indicate that if the Atchafalaya River discharges increased to 66.7%, the total hypoxic area increases the hypoxic area by 30%, and most of the hypoxic area moved from east to west Louisiana shelf. Additionally, if the Atchafalaya River discharge decreased to zero, the total hypoxic area increases by 13%. (7) Scenario experiments with the same nitrogen loading, but different nitrogen forms, indicate that if all the nitrogen was in the inorganic forms, the hypoxic area increases by 15%. These results have multiple implications for understanding the mechanisms that control the oxygen dynamics, reevaluating management strategies, and improving the observational methods.

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.

Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.