Thesis (M.M.A.)--University of Washington, 2009. / Includes bibliographical references (leaves 41-45). Also available on the World Wide Web.

Because of the eutrophication resulting from increasing anthropogenic activities, hypoxia (i.e. dissolved oxygen < 2.8 mg O2 L-1) is on the rise globally. The objective of this research was to understand more about the effects of hypoxia on the marine benthic communities. Particularly, it focused on the latent effects and indirect effects of hypoxia by investigating how early exposure to hypoxia affect the later life stage of a marine gastropod Crepidula onyx, and how hypoxia alter the bacterial composition of biofilms and the subsequent larval settlement of marine invertebrates. In the first study, the larvae of C. onyx were exposed to 2, 3, and 6 mg O2 l-1. Under low food concentration (Isochrysis galbana at 1 × 105 cells l-1), larvae in both hypoxic treatments (2 and 3 mg O2 l-1) required a longer time to become competent to metamorphose. But when they did, they had a similar size and total lipid content to the control larvae. Moreover, the latent effects of early hypoxic exposure on the juvenile growth were evident. After 2 weeks development in field, the growth rate, mean dry weight and filtration rate of juveniles were significantly reduced in the hypoxic treatments. However, there was no discernible effect on larvae or juveniles when the food concentration during the larval stage was doubled (I. galbana at 2 × 105 cells l-1), suggesting that the latent effects of hypoxia can be offset by larval access to high algal concentration. In the second study, the biofilms were exposed to hypoxia and normoxia in microcosms for up to 7 days, and their bacterial community composition was analysed by terminal-restriction fragment length polymorphism (T-RFLP). The results suggested that hypoxia altered the bacterial community structure within biofilms, and the difference between the hypoxia and normoxia treatments increased through the length of exposure period. The resulting changes in biofilms did not alter the larval settlement response of a model species (i.e. C. onyx) in laboratory assays. Nevertheless, when the biofilms were deployed in the field to allow natural larval settlement and recruitment, biofilms that had been exposed to hypoxia altered the overall larval settlement pattern of different marine invertebrates, potentially leading to a shift in the benthic invertebrate community. This research suggested that periodic hypoxic events and the resulting exposure of organisms to hypoxia during their early development might have effects that persist across the life history. Moreover, it highlighted the possibility that the effects of hypoxia on species composition and structure of benthic invertebrate communities might be mediated through changes in biofilms and subsequently larval settlement and recruitment. To conclude, this research demonstrated that hypoxia could affect the growth in the later life stages of marine invertebrates and the recruitment of the benthic communities. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Many experimental therapies have been used in the search for effective approaches to improve recovery after spinal cord injury (SCI). One of the most promising approaches is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH-brief exposures to reduced O2 levels alternating with normal O2 levels) elicits plasticity in respiratory and non-respiratory spinal systems in experimental animals. AIH treatment has also been shown to improve walking abilities in persons with chronic incomplete SCI. In this thesis, I first examined the effect of AIH treatment, alone or in combination with motor training, on functional recovery in a rat model of incomplete cervical SCI. Second, I examined the effect of AIH on the expression of plasticity- and hypoxia-related proteins in the spinal cords of SCI rats. In a randomized, blinded, normoxia-controlled study, rats were trained to cross a horizontal ladder and footslip errors were measured before surgery for SCI, 4 wks post-surgery, each day of daily AIH treatment, and 1, 2, 4 and 8 weeks after treatment. dAIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 wks post-SCI. AIH-treated rats made fewer footslips on the ladder task compared to normoxia-treated control rats after 4 days of treatment and this improvement was sustained for 8 wks post-treatment. Importantly, daily ladder training was required for AIH treatment to facilitate recovery. AIH treatment + motor training also increased the expression of Hypoxia-inducible factor-1α (HIF-1α), Vascular endothelial growth factor (VEGF), Brain-derived neurotrophic factor (BDNF), tyrosine kinase B receptors (trkB) and phospho-trkB in spinal motor neurons in SCI rats compared to normoxia-treated SCI rats. In particular these hypoxia- and plasticity-related proteins were differentially expressed both temporally and spatially in the spinal cord during AIH treatment. These findings demonstrate that AIH + motor training can augment neural plasticity and improve motor recovery in an animal model of SCI. Taken together with the promising findings from human SCI studies, the results of this thesis suggest that AIH has potential as an effective therapy to restore motor function after nervous system injury.

Bone remodelling, the process by which the skeleton adapts to environmental changes, is dependent on the actions of osteoclasts that resorb bone and osteoblasts which make new bone matrix. Aberrant remodelling underpins bone loss in several debilitating skeletal diseases such as osteoporosis, metastatic breast cancer and multiple myeloma. Changes in remodelling activity can also arise as a consequence of therapeutic intervention for instance intravenous bisphosphonate treatment is associated with osteochemonecrosis of the jaw and localised osteoradionecrosis is a common side effect of radiotherapy. Hyperbaric oxygen is often used as an adjunctive therapy in the treatment of these disorders. HBO involves the administration of 100% oxygen at atmospheric pressures greater than one in sealed chambers. The following studies aimed to evaluate the effect of HBO, hyperoxia, and pressure on RANKL-induced osteoclast differentiation and bone resorption from RAW264.7 and human peripheral blood mononuclear cells (PBMC), and osteoblast differentiation in vitro. The study also aimed to further examine the effect of HBO on ex vivo osteoclast formation from peripheral blood monocytes obtained from patients undergoing HBO. Daily exposure to HBO for ninety minutes significantly suppressed osteoclast differentiation and bone resorption in mouse and human monocytes in normoxic and hypoxic conditions in vitro. The suppressive action of HBO on osteoclast formation was associated with a significant reduction in HIF-1α and RANK mRNA expression and HBO also caused a significant reduction in NFATc1 and DC-STAMP expression. This study has for the first time shown that HBO is able to reduce the ability of precursors to form bone resorbing osteoclast. HBO also suppressed the ability of peripheral blood monocytes to develop into RANKL-induced resorptive osteoclasts. In an ex vivo culture system the suppressive effect of HBO was meditated by an action prior to activation of osteoclast differentiation by RANKL and must therefore be an inhibitory effect on the ability of precursors to differentiate along the osteoclastic lineage. HBO also accelerates the rate of osteoblast differentiation and augments early stages of mineralization and has a more pronounced effect than hyperoxia or pressure alone. HBO enhanced bone nodule formation and ALP activity in human osteoblasts. Furthermore HBO promoted the expression of type I collagen and Runx-2 in both normoxic and hypoxic conditions. HBO had a greater effect on these key markers of osteoblast differentiation than hyperoxia or pressure alone. This study suggests that HBO suppresses osteoclast activity and promotes osteoblastic bone formation, which may at least in part mediate its beneficial effects on necrotic bone. This provides evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation in a range of skeletal disorders associated with low oxygen partial pressure. The study also provides further support for the use of HBO in the treatment of skeletal disorders associated with excessive resorption such as osteomyelitis, and also provides a potential mechanism through which short term HBO may help fracture healing.

The cellular microenvironment consists of soluble and insoluble factors that provide signals that dictate cell behavior and cell fate. Limited characterization has hindered our ability to mimic the physiological or pathophysiological environment. While stem cells have vast promise in the areas of regenerative medicine and disease therapy, harnessing this potential remains elusive due to our limited understanding of differentiation mechanisms. Similarly, many in vitro cardiac disease models lack the critical structure- function relationships of healthy and diseased cardiac tissue. The goal of this work is to induce cardiomyogenesis and pathogenesis in vitro by recapitulating features of the native microenvironment during development and disease. / Engineering and Applied Sciences

Corpus Christi Bay in Texas is a wind driven system, and under most conditions winds over the bay mix the water column vertically. However, seasonal, episodic, bottom-water hypoxia has been observed in the bay in conjunction with vertical salinity stratification. This stratification may be caused by dense gravity currents entering the bay. Understanding and modeling the mechanisms that result in stratification in Corpus Christi Bay may help predict hypoxia, and for this reason that is the focus of this dissertation. An evaluation of existing gravity current modeling techniques shows that most currently available models are designed to capture either phenomena local to a gravity current, such as gravity current entrainment and spreading, or larger scale phenomena such as wind mixing and large-scale circulation, but not both. Because gravity current mixing in Corpus Christi Bay is enhanced by wind-induced turbulence, both local gravity current physics and wind mixing effects are critical elements governing gravity current propagation in Corpus Christi Bay. As existing models do not represent gravity current entrainment and wind mixing together, this dissertation develops a coupled model system that accounts explicitly for turbulent wind mixing of a bottom-boundary layer, in addition to representing other local features of dense gravity current propagation such as entrainment and spreading. The coupled model system consists of a 2D depth-averaged hydrodynamic model that calculates gravity current mixing and spreading, coupled with a 3D hydrodynamic model whose domain includes a lighter ambient fluid surrounding the gravity current. The coupled models have flexible boundary conditions that allow fluid exchange to represent mixing from both gravity current entrainment and wind mixing. The coupled model system’s development, verification and application in Corpus Christi Bay advances understanding of gravity current mechanisms, and contributes to our scientific understanding of hypoxia in Corpus Christi Bay. This modeling technique has the flexibility to be applied to other density-stratified systems that are shallow and potentially wind-driven, such as shallow desalination brine disposal sites. / text

Oxygen is a necessary part of our everyday lives and is important for normal eye function. Blood flow through the retinal vasculature supplies oxygen to the inner retina. The resistance of the retinal vessels can change, increasing and decreasing blood flow by dilation and constriction of the vessel. The response of retinal hemodynamics to vasoactive stimuli is termed vascular reactivity. To investigate vascular reactivity characteristics, a system that prospectively targets a certain level of oxygen is employed. We characterize how the retinal vessels respond over time to hypoxia as well as define vascular reactivity to different oxygen concentrations in healthy participants. We demonstrate that the vessels increase diameter fully after 6 minutes and flow after 10 minutes. The relationship between retinal hemodynamics and arterial partial pressure of oxygen (PaO2) is demonstrated in healthy humans. Future studies should investigate these changes in diseased models to better understand when the retinal vasculature response may be insufficient.

Oxygen is a necessary part of our everyday lives and is important for normal eye function. Blood flow through the retinal vasculature supplies oxygen to the inner retina. The resistance of the retinal vessels can change, increasing and decreasing blood flow by dilation and constriction of the vessel. The response of retinal hemodynamics to vasoactive stimuli is termed vascular reactivity. To investigate vascular reactivity characteristics, a system that prospectively targets a certain level of oxygen is employed. We characterize how the retinal vessels respond over time to hypoxia as well as define vascular reactivity to different oxygen concentrations in healthy participants. We demonstrate that the vessels increase diameter fully after 6 minutes and flow after 10 minutes. The relationship between retinal hemodynamics and arterial partial pressure of oxygen (PaO2) is demonstrated in healthy humans. Future studies should investigate these changes in diseased models to better understand when the retinal vasculature response may be insufficient.

Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation.

Exposure to chronic hypoxia (CH) leads to ventilatory acclimatisation to hypoxia (VAH) which is a time-dependent increase in breathing. This study examined the role of the GABAA receptor in establishing VAH. Rats were exposed to CH or control (normoxic) conditions for 10 days during which the GABAA receptor antagonist, bicuculline, was infused systemically or directly into the nucleus of the solitary tract (NTS). Acute breathing trials were then performed to measure resting ventilation and ventilatory chemoreflexes. Systemic administration of bicuculline caused reductions in breathing during acute hypoxia and acute hypercapnia in the control but not the CH animals. Continuous infusion of bicuculline in to the NTS caused a reduction in the acute hypoxic ventilatory response in animals exposed to CH but not in the control animals. The results indicate that exposure to CH alters the GABAA-mediated regulation of acute ventilatory chemoreflexes both in the NTS and elsewhere in the brain.