Hypoxia is a major stressor at high altitudes that limits tissue oxygen availability. High altitude environments are also extremely cold which increases thermogenic demand. Small mammals living at high altitude face the competing energetic challenge of maintaining thermogenesis in a hypoxic environment that can impair aerobic ATP supply. It has been suggested that hypoxia-induced impairments in ATP synthesis capacity and cold-induced increases in thermogenic demand could be counteracted by an increase in tissue oxidative capacity and/or fuel selection. As the organelle that consumes oxygen to produce ATP, changes in mitochondrial physiology can help offset physiological impairments at high altitudes. We explored this hypothesis in North American deer mice (Peromyscus maniculatus), from populations native to high and low altitude. We compared mitochondrial volume densities, intracellular distribution, respiratory capacities, enzyme activities of the mitochondrial complexes, capillarity, and fibre-type distribution in skeletal and cardiac muscles. To examine potential changes to mitochondrial physiology at high altitudes deer mice (P. maniculatus) were acclimated to: warm (25°C) normoxia; warm hypoxia (simulated altitude of 4300m); cold (5°C) normoxia; and cold + hypoxia. In skeletal muscle, highlanders had higher mitochondrial volume densities than lowlanders, entirely due to an increased abundance of mitochondria in a subsarcolemmal location next to capillaries. Mitochondria from highland mice also had higher mitochondrial respiratory capacities and cytochrome c oxidase activity in control conditions, but these values converged after hypoxia acclimation. Cold acclimation restored pyruvate and fatty acid respiratory capacity to control levels in highland mice, which also showed an increase in mitochondrial uncoupling. Cold increased respiratory capacities in lowland mice. Acclimation to cold+hypoxia did not change mitochondrial physiology beyond cold alone and appeared to counteract the effects of hypoxia on highland mice. In cardiac muscle highland mice had higher respiratory capacities, but after hypoxia acclimation lowland mice significantly increased respiratory capacities. In response to hypoxia, highland mice increased the relative capacity to oxidize carbohydrates compared to fatty acids. Our results suggest that both highland ancestry and plasticity affect mitochondrial physiology, and likely contributes to performance at high altitudes. / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24947号 / 生博第509号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 原田 浩, 教授 松本 智裕, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Understanding the respiratory physiology and energy metabolism is important for establishing the dissolved oxygen (DO) requirements of freshwater mussels, determining the metabolite(s) indicative of environmental stress, and interpreting environmental conditions based on physiological indicators of mussels. Three studies were undertaken to address these questions. The first study was conducted with seven mussel species collected from reservoir and riverine habitats. The two objectives were to determine the diurnal patterns of valve gaping of freshwater mussels from different habitats, and to monitor heart rate changes of a mussel species that exhibited the diurnal gaping. The results showed that night gaping is evident for the mussels collected from lentic areas, but not for those collected from lotic areas. The heart rate of Pyganodon grandis increased when they gaped. The second study was conducted with nine species of freshwater mussels from different habitats. The three objectives were to determine the patterns (i.e., regulator and conformer) of oxygen consumption (OC) rate under declining DO, evaluate the effects of temperature on ability to regulate OC under declining DO, and finally to use this information to predict DO criteria for maintaining freshwater mussels in captivity. The results showed that mussels living in the habitats subjected to low DO have a better ability to regulate the OC and were more tolerant to hypoxia. The third study assessed three mussel species from different habitats with different abilities to regulate OC under low DO. The two objectives in this study were to identify the energetic metabolite changes under different DO levels and air exposure for the three species, and to determine the appropriate tissue(s) and metabolite(s) to use for estimating the stress in mussels. The results showed that different biochemical strategies were used by Villosa iris, Elliptio complanata, and Pyganodon grandis. Villosa iris had the lowest anaerobic capacity. Elliptio complanata had a more efficient anaerobic metabolism, and P. grandis reduced energy metabolism under low DO and air exposure. posterior adductor muscle, gill and mantle were good tissues for evaluating hypoxic stress. The mantle tissue had the highest glycogen store and was the best tissue for non-lethal study of physiological condition. / Ph. D.

Ovarian cancer is the fifth leading cause of all cancer deaths in women and the most lethal gynecologic cancer in the United States. During metastasis, cancer cells exfoliate from the primary tumor and aggregate to form spheroids, enhancing their survival within the peritoneal cavity during dissemination to a secondary outgrowth site. The inability of removal of these aggregates by traditional surgical interventions may contribute to the high recurrence and mortality rate of ovarian cancer diagnosed at late stages. Obesity, particularly abdominal obesity, has been shown to increase ovarian cancer risk and decrease survival. The recruitment of stromal vascular fraction (SVF) present in adipose tissue represents a growth and proliferation advantage to ovarian tumors, and endogenous sphingolipids like sphingosine-1-phosphate are increased in ovarian cancer patients. These conditions, combined with the physiological conditions within malignant ascites (hypoxia and low glucose), represent a physiological environment that can impact the metabolic responses of ovarian cancer spheroids. Here, we investigated the metabolic adaptations of serous ovarian cancer cells across the metastatic cycle and in conditions that mimic those of the peritoneal cavity and malignant ascites. We first investigated the different in metabolic responses between adherent monolayers and 3D spheroids. We confirmed that spheroids have a reduced metabolic rate and drug response that is affected by the incorporation of obese SVF into aggregates. To investigate these changes in the next stages of the metastatic cycle, we used time trials to observe how adherence of spheroids to a secondary site changes metabolic response and substrate utilization in physiological conditions. Adhesion of spheroids showed changes in energy metabolism and substrate utilization, switching from mainly glutamine oxidation to glucose oxidation that could support successful outgrowth. Spheroids also were resilient to culture conditions, even non-permissive conditions such as those found in the peritoneal cavity. Finally, we utilized human malignant ascites from ovarian cancer patients as a further investigation into conditions that imitate in vivo characteristics that could affect spheroid metabolism. Exposure to malignant ascites reduced spheroid viability as well as basal respiration and ATP synthesis. However, spare respiratory capacity was increased, and human spheroids changed their substrate utilization in response to ascites. Taken together, these studies provide an identification of metabolic switches across different stages of ovarian cancer metastasis that contribute to their survival, which represents an emerging target for prevention and treatment for individuals with ovarian cancer. / Doctor of Philosophy / Ovarian cancer is the deadliest reproductive cancer in women, and most women who are diagnosed will die from the disease because of its high recurrence rate and because it is typically detected at late stages. When ovarian cancer metastasizes, cells or cell clusters from the original tumor aggregate together to form balls of cells called spheroids, which move through the abdominal cavity to other sites to grow additional tumors. These spheroids are thought to contribute to recurrence of this cancer, since they cannot be removed by surgery. As these spheroids move through the abdominal cavity, they are exposed to an environment that has a low amount of oxygen and glucose. These spheroids may also be exposed to bioactive lipids and cells from the adipose tissue called stromal vascular fraction, both of which are related to obesity and may help cancer spheroids survive. The survival of these spheroids is in part related to how their metabolism functions, which may help them make energy and the building blocks needed to continue growing and form successful secondary tumors. Identifying how these spheroids change their metabolism at different points during the disease may help identify points that can be targeted to prevent changes in metabolism that could support their growth. This dissertation identified metabolic changes that occur in spheroids, in conditions that are similar to those spheroids would be exposed to in an abdominal cavity. First, we compared single layers of cells to spheroids and found that spheroids had a lower metabolic rate and lower drug response, which may help them survive in the abdominal cavity. Next, we allowed the spheroids to lay down and grow out, like they would when they found a new location during metastasis, to see how their metabolism changed and what substances they used to make energy in conditions that mimicked the abdominal cavity. As spheroids adhered, they changed their energy metabolism and switched the substances they used to make energy, all while continuing to survive and grow out even in conditions that were not supportive. These switches could help them grow out and successfully metastasize. Lastly, we used ascites fluid from human ovarian cancer patients and treated spheroids with this to see how their metabolism changed in response. While some aspects of metabolism and survival was reduced, their ability to increase their metabolism when stressed increased and human spheroids used nutrients to make energy differently. Overall, we show that across the stages of metastasis, ovarian cancer spheroids can change their metabolism in response to their environment. Identifying these metabolic switches helps us understand how successful metastasis happens, and can inform future targets to slow or prevent metastasis, prolonging the life of women who have been diagnosed with ovarian cancer.

Climate change is predicted to have many diverse effects on freshwater lakes and reservoirs by increasing both hypolimnetic hypoxia and runoff, which will increase nutrient concentrations and degrade water quality. Hypoxic conditions can trigger the release of metals and nutrients from the sediments, i.e., internal loading, while storms can increase external nutrient loading to a waterbody. One potential solution for combating hypoxia is to use side stream supersaturation (SSS), a novel form of hypolimnetic oxygenation. First, in Chapter 1, I tested the efficacy of SSS operation to improve water quality in Falling Creek Reservoir (FCR), a shallow, eutrophic, drinking water reservoir. I found that SSS operation successfully increased hypolimnetic oxygen concentrations in FCR and suppressed internal loading of iron, manganese, and phosphorus. In Chapter 2, I manipulated inflow volumes to FCR and used SSS as a tool to alter hypolimnetic oxygen conditions in whole-ecosystem manipulations of internal and external nutrient loading. I observed that internal nitrogen and phosphorus loading during hypoxic conditions largely controlled the hypolimnetic mass of nutrients in FCR, regardless of inflow volumes, presumably as a result of the accumulated nutrients in its sediment from historical agriculture. Additionally, FCR consistently functioned as net sink of N and P throughout almost all of the treatments and substantially reduced nutrient export to downstream ecosystems. In summary, my research demonstrates the sensitivity of reservoir water quality to global change. / Master of Science

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancy due to the insufficient accurate screening programs for the early detection of EOC. To improve the accuracy of the early detection, there is a need to deeply understand the mechanism of EOC progression and the interaction between cancer cells with their unique microenvironment. Therefore, this work investigated the metabolic shift in the mouse model for progressive ovarian cancer, and evaluated the effects of hypoxic environment, spheroid formation as well as stromal vascular fractions (SVF) on the metabolic shift, proliferation rate, drug resistance and protein markers in functional categories. The results demonstrated an increasingly glycolytic nature of MOSE cells as they progress from a tumorigenic (MOSE-L) to a highly aggressive phenotype (MOSE-FFL), and also showed changes in metabolism during ovarian cancer spheroid formation with SVF under different oxygen levels. More specifically, the hypoxic environment enhanced glycolytic shift by upregulating the glucose uptake and lactate secretion, and the spheroid formation affected the cellular metabolism by increasing the lactate secretion to acidify local environments, modulating the expression of cell adhesion molecules to enhance cell motility and spheroids disaggregation, and up-regulating invasiveness markers and stemness makers to promote ovarian cancer aggressive potential. Hypoxia and spheroid formation decreased ovarian cancer cells growth but increased the chemoresistance, which leads to the promotion of aggressiveness and metastasis potential of ovarian cancer. SVF co-cultured spheroids further increased the glycolytic shift of the heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer heterogeneous of ovarian cancer spheroids, induced the aggressive phenotype by elevating the corresponding protein markers. Decreasing the glycolytic shift and suppression of the proteins/pathways may be used to inhibit aggressiveness or metastatic potential of ovarian cancer. / Master of Science

No / BACKGROUND AND PURPOSE: Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti-cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs). EXPERIMENTAL APPROACH: The effect of hypoxia (0.1% oxygen) on the activity of conventional cytotoxic drugs (5-fluorouracil, doxorubicin and vinblastine), the hypoxia-activated prodrug tirapazamine and 9 TKIs was determined in a panel of cell lines. Where hypoxia had a marked effect on chemosensitivity, Western blot analysis was conducted to determine the effect of hypoxia on target expression and the effect of TKIs on cell signalling response under aerobic and hypoxic conditions. KEY RESULTS: Three patterns of chemosensitivity were observed: resistance under hypoxia, equitoxic activity against hypoxic and aerobic cells, and preferential cytotoxicity to hypoxic cells. Significant hypoxia selectivity (independent of HIF1) was observed in the case of dasatinib and this correlated with the ability of dasatinib to inhibit phosphorylation of Src at tyrosine 530. Sorafenib was significantly less effective under hypoxic conditions but resistance did not correlate with hypoxia-induced changes in Raf/MEK/ERK signalling. CONCLUSIONS AND IMPLICATIONS: Hypoxia influences the activity of TKIs but in contrast to conventional cytotoxic drugs, preferential activity against hypoxic cells can occur. The search for hypoxia-targeted therapies has been long and fruitless and this study suggests that some clinically approved TKIs could preferentially target the hypoxic fraction of some tumour types.

No / Hypoxia is a common feature observed in solid tumours. It is a target of interest in oncology as it has been found to be closely associated with tumour progression, metastasis and aggressiveness and confers resistance to a variety of chemotherapeutic agents as well as radiotherapy. AQ4N, also known as banoxatrone or 1,4-bis-[2-(dimethylamino-Noxide) ethyl]amino-5,8-dihydroxyanthracene-9,10-dione is a very promising bioreductive prodrug. This paper, describes an application of MALDI-MSI combined with ion mobility separation and an "on-tissue" bottom up proteomic strategy to obtain proteomic data from AQ4N dosed tumour xenograft tissue sections. These data are then correlated with the drug distribution determined also using MALDI-ion mobility separation-mass spectrometry imaging (MALDI-IMS-MSI). PCA-DA and OPLS-DA have been used to compare treated and untreated xenografts and of note is the marked increase in expression of Histone H3.

Yes / Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease. / European Research Council-INTERRUPTB starting grant nr.311725 (to BdJ, BO, FG, MA, CM).

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.