UMD Theses and Dissertations
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Item Metabolic Acid Transport in Human Retinal Pigment Epithelium(2010) Adijanto, Jeffrey; Wang, Nam S; Miller, Sheldon S; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)At the back of our eyes, photoreceptors capture light and convert it into electrical signals that we perceive in our brain as vision. Photoreceptor function is energy expensive, even more so than many other processes in the body. Furthermore, photoreceptor metabolism increases in the dark and releases more metabolic by-products (CO2, lactic acid, and water) into the photoreceptor extracellular space (SRS). The retinal pigment epithelium (RPE) maintains photoreceptor health by transporting these metabolic acids from the SRS to the choroidal blood supply. By using native and cultured fetal human RPE, we show that the apical membrane is significantly more permeable to CO2 than the basolateral membrane. This feature traps CO2 in the cell and drives carbonic anhydrase (CA)-mediated hydration of CO2 into HCO3, which is subsequently transported out of the basolateral membrane by a Na-linked HCO3 co-transporter (NBC). This process increases net steady-state fluid absorption, thus maintaining retinal adhesion to the RPE. Oxidative metabolism generates significantly more ATP than glycolysis, but photoreceptors derive 50% of their total ATP consumed from glycolysis due to the low oxygen level at the photoreceptor inner segment. Furthermore, lactic acid production and release into the SRS almost doubles in the dark. We show that the RPE transports lactic acid from the SRS via a proton-linked monocarboxylate transporter (MCT1), and this process activates pHi-regulatory mechanisms at the RPE apical membrane: Na/H exchanger (NHE) and Na-linked HCO3 transporters (NBC1 & NBC3). These mechanisms also facilitate MCT1-mediated lactic acid transport by preventing buildup of a proton-gradient across the RPE apical membrane. We show that an increase in SRS CO2 or lactic acid level causes RPE cell swelling. The RPE alleviates swell-induced osmotic stress by activating apical membrane K-channel (Kir 7.1) and basolateral membrane Cl -channel (ClC-2), which drives KCl (and fluid) out of the cell to decrease cell volume. In this study, we identified the cellular mechanisms in RPE that prevent acidosis and fluid accumulation in the SRS caused by increased photoreceptor metabolism in the dark. These homeostatic processes maintain the close anatomical relationship between photoreceptors and RPE, thus protecting photoreceptor health and preserving visual function.Item Physiological Determinants of Candidate Physical Ability Test in Firefighters(2009) Sheaff, Andrew; Hurley, Ben; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The purpose of this study was to examine the relative importance of physiological characteristics during firefighting performance, as assessed by the Candidate Physical Ability Test (CPAT). Participants included professional and volunteer firefighters, ages 18-39 (n=33). Muscle strength, muscle endurance, muscle power, body composition, aerobic capacity, anaerobic fitness, and the cardiovascular response to stairclimbing were assessed to determine the physiological characteristics of the participants. To quantify firefighting performance, the CPAT was administered by members of the fire service. Absolute and relative mean power during Wingate anaerobic cycling test (WAnT), relative peak power during WAnT, and absolute maximal oxygen uptake (VO2max) were significantly higher in those who passed the CPAT (n=18), compared to those who failed (n=15) (P < 0.01). Absolute and relative mean power during WAnT, fatigue index during WAnT, absolute VO2max, upper body strength, and the heart rate response to stairclimbing were all significantly related to CPAT performance time (all P < 0.01). However, absolute VO2max and anaerobic fatigue resistance during WAnT combined were the best predictors of total CPAT performance (Adj. R2 = 0.817; P < 0.001). Performance on the ceiling breach and pull was the only individual CPAT task that could not be significantly predicted by the physiological characteristics assessed. Rate-pressure product during the stairclimb was not related to CPAT performance. In conclusion, measures of anaerobic and aerobic fitness best predict overall CPAT performance, as well as individual task performance. Remedial programs aimed at improving firefighting performance should target anaerobic and aerobic fitness qualities.Item THE EFFECT OF THROMBIN ON ENDOTHELIAL PROGENITOR CELLS WITH EXERCISE AND EXERCISE TRAINING.(2009) Lockard, Michael Morley; Hagberg, James; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)BACKGROUND: Circulating number of endothelial progenitor cells (EPCs) and EPC colony forming units (EPC-CFUs) have emerged as valuable predictors of cardiovascular health. Improvements in EPC number and EPC-CFUs have been associated with exercise and exercise training, although the mechanism for these improvements has not been identified. PURPOSE: This study examined the association of exercise-induced thrombin production with changes in circulating EPC number and EPC-CFUs as well as investigated the effects of exercise and thrombin supplementation on EPC differentiation and proliferation in vitro through gene expression analysis. METHODS: Subjects included healthy male Masters athletes (n = 12) and sedentary matched controls (n = 11) aged 55 - 80 years. Circulating EPC number, EPC-CFUs, plasma prothrombin fragment (F1+2) concentration and plasma thrombin-antithrombin III (TAT) concentration were measured at rest and after 30 minutes of vigorous treadmill exercise. Gene expression was assessed on resting EPC samples treated with 0U, 1U, 5U, or 10U of thrombin, as well as on EPC samples obtained after exercise. Gene expression analysis was performed for cell cycle genes cyclin A2, cyclin D1, and p27, and for cell surface markers VE-Cadherin and VEGFR2. To investigate the association of long-term exercise training, all outcomes were compared between Masters athletes and sedentary controls. RESULTS: Plasma concentrations of F1+2 and TAT increased significantly after exercise, however, EPC number and EPC-CFUs did not change. Changes in plasma F1+2 concentration with exercise correlated with changes in EPC number and EPC-CFUs in Masters athletes and with EPC number in control subjects Expression of the cell cycle genes cyclin A2 and cyclin D1 increased with thrombin treatment, while expression of the cell cycle inhibitor p27 decreased, peaking at 5U of thrombin. No change in VE-Cadherin or VEGFR2 expression was observed in control subjects, however, expression increased at 1U thrombin treatment in Masters athletes. Similarly, EPCs isolated after exercise demonstrated increased expression in cyclin A2 and cyclin D1, decreased in p27, and showed no change in VE-Cadherin or VEGFR2. CONCLUSION: Elevated thrombin production during vigorous exercise may play an important role in the regulation of EPCs, specifically cellular proliferation through changes in expression of cell cycle genes.Item Bioenergetic responses of Chesapeake Bay white perch to nursery conditions of temperature, salinity, and dissolved oxygen(2009) Hanks, Deanna McQuarrie; Secor, David H.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Changes in the physical and chemical structure of estuaries affect the habitat availability for anadromous species. White perch, an estuarine species, are among the most abundant and important fishes in the Chesapeake Bay. Here, I evaluate nursery quality for juvenile white perch by measuring metabolic and growth responses over a range of environmental conditions such as salinity, temperature, and dissolved oxygen. Rearing white perch in 10-d trials varying in temperature, salinity and dissolved oxygen conditions, I estimated growth rates, feeding rates, gross growth efficiency, and routine metabolism. Juveniles experienced higher feeding and growth rates in warmer, more oxygenated waters. In hypoxic environments (<40% saturation), metabolic rates increased as much as 4-fold while growth decreased 3-fold and feeding decreased 2-fold. My results indicate that while white perch are well suited to the saline and thermal conditions present in the Bay, nursery habitat value can be substantially curtailed by hypoxia.Item Physical activity or estradiol supplementation can prevent the metabolic syndrome in ovariectomized mice(2008-12-08) Wohlers, Lindsay Michelle; Spangenburg, Espen E; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss of ovarian hormones (ie. menopause) results in a significant increase in risk for developing the metabolic syndrome. Thus, the purpose of this study is to investigate the effectiveness of exercise in preventing the metabolic syndrome in a model of menopause. Female C57/BL6 mice were divided into three groups, ovariectomized (OVX), SHAM, and ovariectomized supplemented with 17β-estradiol (OVX+E2), with mice placed in either voluntary running wheel cages or standard cages for eight weeks. OVX animals ran significantly less than SHAM animals, with the OVX+E2 group exhibiting a partial recovery in distance. Visceral fat mass and blood glucose levels were elevated in the OVX sedentary group as compared to SHAM and OVX+E2 groups. Voluntary wheel running and 17-β estradiol supplementation attenuated increases in visceral fat mass, blood glucose levels, and ERK1/2 phosphorylation in OVX animals, thus preventing symptoms of the metabolic syndrome which accompany loss of ovarian hormones.Item Bioenergetic, reproductive, and population-level effects of dissolved copper and cadmium on the grass shrimp, Palaemonetes pugio(2008-08-03) Manyin, Teresa; Rowe, Christopher L; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The grass shrimp, Palaemonetes pugio, was exposed to dissolved copper or cadmium in a series of laboratory experiments to determine effects on bioenergetics, reproduction, and population growth. In 14-day exposures, adults were exposed to either copper or cadmium to quantify bioenergetic effects. Both metals caused a decline in oxygen consumption (lowest observed effects concentrations [LOECs] = 7.5 μg Cu sup 2+/L and 6.6 μg Cd sup 2+L) and growth rate (LOECs = 27 μg Cu sup 2+/L and 6.2 μg Cd sup 2+/L). Effects of copper on growth were more severe than those of cadmium, resulting in weight loss during the exposure. Reductions in oxygen consumption and growth, in combination with declines in reproduction observed in longer exposures, suggest that both copper and cadmium reduce energy allocation to respiration and production pathways. In eight-month exposures, P. pugio were exposed to either copper or cadmium for a full life cycle, allowing larvae to attain maturation and reproduce. While survival was little affected by exposure to cadmium, brood size and the percentage of ovigerous females were significantly reduced (LOECs = 1.5 and 2.5 μg Cd sup 2+/L, respectively). Population growth of P. pugio exposed to cadmium was projected using a stage-based matrix model and a z-transformed life cycle graph analysis. Both models projected a decrease in population growth rate (LOEC = 1.5 μg Cd sup 2+/L), although population growth remained positive. Decomposition analysis indicated that cadmium-induced declines in population growth could be attributed mainly to contributions from reproductive effects. In the eight-month exposure to copper, no lethal effects on larvae, juveniles, or adults were observed, but larval development was significantly delayed (LOEC = 9 μg Cu sup 2+/L). Upon reaching maturation, females exposed to copper were able to produce embryos, but the embryos did not hatch, precluding completion of the life cycle (LOEC = 9 μg Cu sup 2+/L). The results from subsequent experiments, which further examined reproductive effects, suggested that copper may inhibit larval recruitment via a combination of effects on hatching success, parental bioenergetics, and processes before or during spawning and/or fertilization. In conclusion, both copper and cadmium may have negative impacts on the sustainability of natural populations of P. pugio in contaminated habitats.Item The Effects of Finger Movement Conditions and Speed on Finger Interdependency(2008-09-02) Lieu, James; Shim, Jae K; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Various factors may cause non-task fingers to involuntary move during task finger movement. Finger interdependency is well documented, but only a few attempts have been made to quantify these peripheral and neurological factors, especially during different movement and frequency conditions. The purpose of the study was to quantify and differentiate the contribution of peripheral and neurological factors during different movement and speed conditions. This was examined at two different movement (active and passive) conditions and two movement speeds(0.25 Hz and 0.5 Hz) and compared to previous studies to confirm the proximity hypothesis. The results showed that finger interdependency was greater in voluntary than involuntary movement conditions. Finger interdependency was also greater during the slower speed(0.25 Hz) condition, and the proximity hypothesis was confirmed. Finger interdependency is larger during voluntary movement and at slower speeds, with the involvement of both peripheral and neurological factors.Item Towards the derivation of bovine embryonic stem cells(2008-01-28) Pant, Disha; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The ability of embryonic stem cells (ESCs) to self-renew and differentiate into a wide range of cell types has encouraged researchers to attempt to isolate ESCs from embryos of domestic species for the past two decades. Success has been limited. The aim of the current study was to investigate whether colonies derived from inner cell masses (ICMs) of bovine blastocysts expressed the same markers of pluripotency and candidate genes representing the various signaling pathways as those found in human or mouse ESCs. The ability of selected cytokines to maintain the major transcription factors associated with pluripotency (NANOG, POU5F1 and SOX2) in the ICM explants was also tested. The results of the study showed that the three major transcription factors (NANOG, POU5F1 and SOX2) were expressed initially in culture but were lost with continued culture and passaging. Markers of differentiation (BMP4, HNF4, NCAM, and CDX2) were also expressed in the initial days of culture. The candidate genes representing the various signaling pathways were expressed in the initial days of culture as well as in subsequent passages. Noggin, a cytokine inhibiting the BMP4 pathway successfully up-regulated the relative expression of NANOG in the ICM explants with respect to controls. The results indicate that signaling pathways associated with regulating pluripotency are expressed in ICM explants and that with cytokine supplementation pluripotency may be maintained. An alternate approach in which differentiating cells in the primary colonies were selectively ablated to eradicate cells secreting pro-differentiation signals was tested. Bovine embryos that carried the hygromycin resistance gene driven by the NANOG promoter were generated by SCNT. Any pluripotent colonies generated from these embryos should survive in the presence of hygromycin. When cultured in the presence of Noggin and hygromycin, colonies were generated; however they failed to proliferate on passaging. This suggests that the culture conditions were not optimal for the NANOG promoter to remain active over extended culture.Item The influence of ECE-1 gene polymorphism and exercise training on plasma and urinary endothelin-1 levels in pre- and stage 1 hypertensives(2007-08-09) Attipoe, Selasi; Hagberg, James; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Endothelin-1 (ET-1) is a peptide that mediates blood pressure. ECE-1b-C-338A is a polymorphism in the ECE-1 gene that alters ET-1 synthesis. Thus, the goal of this study was to investigate the influence of the ECE-1b-C-338A polymorphism on plasma and urinary ET-1 levels, as well as on aerobic exercise training (AEX)-induced changes in these variables. Outcome variables were assessed before and after 24 weeks of AEX in hypertensive adults. Separate ANCOVAs were used to compare differences in outcome variables. Only AA homozygote men had higher plasma ET-1 levels. After AEX, there were no significant changes in the main outcome variables. Neither were these changes significantly different between genotype groups. Results suggest that this polymorphism is associated with plasma ET-1 levels in men but not with urinary ET-1 levels. Additionally, this polymorphism does not affect AEX-induced changes in plasma and urinary ET-1 levels.Item The Structural Basis for Function of the Escherichia coli Mechanosensitive Channel of Small Conductance, MscS(2007-04-26) Akitake, Bradley Chun-Yee; Sukharev, Sergei; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The 'small' mechanosensitive channel, MscS, resides in cytoplasmic membranes of most free-living bacteria. MscS is gated directly by membrane tension and functions as an osmolyte release valve in bacterial turgor regulation. In contrast to previously studied MscL, which is a strictly prokaryotic molecule, MscS homologs are found in eukaryotes increasing the value of this channel as a general model for gating by membrane stretch. Presented here are the results of three studies aimed at characterizing the structural basis for function of Escherichia coli MscS. In study one, we provide the first electrophysiological characterization of the wild-type channel in its native membrane free of other mechanosensitive channels. It is, to date, the most complete description the gating cycle specifying the kinetic scheme and dependencies of major rates on tension and voltage. Study two represents a collaborative effort to probe the strength of intersubunit contacts in the homo-heptameric MscS channel. In patch-clamp experiments we show that the dissociating effects of TFE alter MscS gating in a manner that provides significant insight into the mechanics of channel inactivation. In the final study our research group utilized a novel extrapolated motion technique to explore the conformational pathways of the MscS functional cycle. Guided by these new models, channel mutants were generated to alter helical propensity along the pore lining TM3 helix. Patch-clamp analysis revealed a vivid picture of the functioning MscS in which these TM3 domains provide a structural frame for the open channel. Dynamic collapse of these 'struts' at flexible points along TM3 modulates transitions from the open state to the inactivated and closed states. My contributions to these studies have allowed for (1) refinement of the MscS functional cycle including identification of a new desensitized state; (2) determination of the physical parameters and spatial scales of channel opening, closing and inactivation; and (3) identification of key hinge elements, residing in TM3, that along with membrane tension serve to modulate the functional cycle of MscS. These findings have led to a better understanding of the biophysical principles that underlie mechanotransduction and provide insights into the larger family of mechanically activated phenomena.