UMD Theses and Dissertations
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Item ANALYSIS OF OBJECTIVES AND CONSTRAINTS TOWARDS PREDICTIVE MODELING OF COMPLEX METABOLISM(2020) Boruah, Navadeep; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The central theme of this dissertation is predictive modeling of metabolism in complex biological systems with genome-scale stoichiometric metabolic models to (i) gain nontrivial insight on cellular metabolism and (ii) provide justifications for hitherto unexplained metabolic phenomena. The crux of high-quality predictive modeling with genome-scale stoichiometric metabolic models is appropriate selection of (i) a biologically relevant objective function and (ii) a set of constraints based on experimental data. However, in many complex systems, like a plant tissue with its wide array of specialized cells, a biological objective is not always apparent. Additionally, generation of experimental data to develop biochemically relevant constraints can be nearly impossible in systems that cannot be cultured under a controlled environment for the duration of an experiment. Such limitations necessitate careful reformulation of the biological question, development of novel methods and data analysis strategies. Here, we push the boundaries of predictive modeling by demonstrating its first application in deciphering hitherto unexplained metabolic phenomena and in developing novel hypotheses on metabolism. Towards achieving this goal, we developed several novel approaches and employed them in diverse biological systems. Firstly, we investigated the selection of carbohydrate degrading pathway employed by Saccharophagus degradans, an aerobic cellulosic marine bacterium. Flux balance analyses of its growth in nutrient rich hypoxic marine environment predicted that the selection of carbohydrate degrading pathway is possibly influenced by inorganic nutrient availability. Secondly, multi-tissue genome-scale metabolic modeling of Populus trichoparpa, a perennial woody tree, and analyses with a novel strategy based on multiple biologically relevant metrics provided a metabolic justification for the predominance of glutamine as the predominant nitrogen transport amino acid for internal nitrogen recycling. Thirdly, predictive modeling of maize grain filling predicted amino acid fermentation as a mechanism for expending excess reductant cofactors for continual starch synthesis in the hypoxic environment of endosperm. Finally, we developed bilevel optimization framework to integrate publicly available transcriptome datasets with metabolic networks. This framework predicted accumulation of specific classes of maize endosperm storage protein at distinct stages of grain filling. We anticipate that the employment of these aforementioned approaches in other biological systems will lead to the generation of a wide array of nontrivial hypothesis on cellular metabolism and to develop targeted experiments to validate them.Item Investigating the role(s) of mammalian heme transport by HRG1(2019) Pek, Rini; Hamza, Iqbal; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The recycling of hemoglobin from damaged or senescent red blood cells (RBCs) contributes almost 90% of daily body iron requirements in humans for bone marrow erythropoiesis. Previously, our cell biological studies have shown that HRG1, a four transmembrane protein first discovered in C. elegans, facilitates the transport of heme within reticuloendothelial system (RES) macrophages during the turnover of RBCs, a process termed erythrophagocytosis. HRG1 transports heme from the phagolysosomes into the cytosol where heme is degraded to liberate iron for erythropoiesis. Here we show that mice deficient for HRG1 are defective in heme- iron recycling by RES macrophages, resulting in over ten-fold excess heme accumulation as visible dark pigments within lysosomal compartments that are 10- 100 times larger than normal. The sequestered heme is tolerated by macrophages through polymerization into crystallized hemozoin, a phenomenon typically observed in blood-feeding parasites as a detoxification method to protect against heme toxicity. HRG1-/- mice display ineffective bone marrow erythropoiesis which results in a reduction in hematocrit and extramedullary erythropoiesis in the spleen. Under iron- deficient conditions HRG1-/- mice are unable to utilize hemozoin as an iron source to sustain erythropoiesis, causing severe anemia and lethality. Our studies establish that polymerizing cytotoxic heme into hemozoin is a previously-unanticipated heme tolerance pathway in mammals.Item Absorption and metabolism of 3-MCPD 1-monopalmitate in rats(2017) Gao, Boyan; Yu, Liangli (Lucy); Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fatty acid esters of 3-monochloropropane 1,2-diol (3-MCPD esters) are a group of potential chemical toxicants. Their toxic effects primarily include nephrotoxicity and hepatotoxicity. To understand the toxic mechanisms of 3-MCPD esters, one of the key points is to advance the understanding of their metabolic mechanisms in vivo. This dissertation investigated 1) the absorption and kinetics of 3-MCPD 1-monopalmitate in rats, 2) the possible metabolites of 3- MCPD 1-monopalmitate after oral administration to rats, and 3) the possible metabolic pathways of 3-MCPD 1-monopalmitate in vivo. The greatest concentration of 3-MCPD 1-monopalmitate in the plasma was 873.72 ng/mL (Cmax) at about 1.67 hours (Tmax) after oral administration. The concentration of 3-MCPD 1-monopalmitate reduced to half after 3.42 hours (t1/2). No 3-MCPD 1-monopalmitate could be detected after 4 hours, which was its mean resident time (MRT). The area under curve (AUC) for 3-MCPD 1-monopalmitate in rat plasma was 1676.15 h.ng/mL, which represented the maximum amount of 3-MCPD 1-monopalmitate absorbed into plasma under the testing conditions. Beside, 39 possible metabolites were tentatively identified in the liver, kidney, testis, brain, plasma and urine samples at 6, 12, 24 and 48 hours after oral administration of 3-MCPD 1-monopalmitate to rats. In addition, five major metabolic pathways of 3-MCPD esters were derivate to evaluate their metabolic conditions in vivo. These results can greatly enhance the understanding about the absorption, distribution and metabolism conditions of 3-MCPD esters in vivo, and promote further research about the biological actions of 3-MCPD esters.Item EXAMINING THE EFFECTS OF EXERCISE ANCESTRY ON TWO GENERATIONS OF MOUSE OFFSPRING(2014) Guth, Lisa Marie; Roth, Stephen M; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation research is comprised of three projects examining the effect of voluntary parental exercise on health-related phenotypes in two generations of mouse offspring. We developed a novel model of exercise ancestry where C57BL/6 mice (F0) were exposed to voluntary exercise (EX) or a sedentary (SED) lifestyle and were bred with like-exposed mates to produce first-generation (F1) offspring; F1 offspring were bred with like-exposed offspring to produce second-generation offspring (F2). F0 mice exercised before breeding and continuously through gestation and lactation; all offspring remained sedentary after weaning, thus F0 exercise exposure was the only distinguishing factor between offspring. The first project examined whole body and tissue masses, glucose tolerance, and skeletal muscle gene expression in two generations of 8-week old offspring of exercised vs. sedentary parents. F1 EX females were lighter with less fat mass compared to F1 SED females. F2 EX females had lower baseline blood glucose and impaired glucose tolerance. Further, skeletal muscle lipogenic gene expression was downregulated in females with exercise ancestry, while it was upregulated in males with exercise ancestry. The second study further examined these phenotypes in two generations of adult (28 week) offspring. Parental exercise did not influence offspring body mass or glucose tolerance in 28 week-old offspring, though F1 EX females had higher baseline glucose. Additionally, while some differences in skeletal muscle gene expression were observed, the effect of parental exercise on offspring was blunted at 28 compared to 8 weeks of age. The third study further examined the effects of parental exercise in skeletal muscle as well as adipose and hepatic tissue with regards to metabolite content and gene expression. Exercise ancestry did not affect offspring skeletal muscle or liver triglyceride or glycogen content. Further, there were no effects of exercise ancestry on gene expression levels of glycogen- or triglyceride-related enzymes in skeletal muscle, liver, or adipose tissue. Overall, these studies suggest no adverse effects of parental exercise on metabolic health in multiple generations of mouse offspring.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 High Sucrose, Fructose, and Glucose Diets and Glucocorticoid Dysregulation in Rats(2009) London, Edra; Castonguay, Thomas W; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Approximately two-thirds of U.S. adults are overweight or obese and the prevalence of overweight in children has tripled since 1980. Intake of added sugars has also increased. The etiology of obesity remains unclear and the role of glucocorticoids in obesity is one area of ambiguity. The enzyme 11beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1) interconverts active and inactive glucocorticoid, thereby regulating intracellular glucocorticoids. Dysregulation of 11beta-HSD-1 in liver and adipose is characteristic of human and animal models of obesity. Hexose-6-phosphate dehydrogenase (H6PDH) is colocalized with 11beta-HSD-1 and determines the set point for 11beta-HSD-1 oxidoreductase activity. In a long-term (10 wk) study, rats given ad libitum access to 16% sucrose solution, chow, and water were fatter than controls, had increased 11beta-HSD-1 mRNA in adipose, suppressed 11beta-HSD-1 mRNA in liver, and increased H6PDH mRNA in both tissues. The primary research questions were as follows: Can high sugar diets induce glucocorticoid dysregulation in the absence of excess adiposity? Does sugar type matter? Energy intake, weight gain, and parameters of lipid and carbohydrate metabolism were measured. Rats were randomly assigned to either ad libitum access to chow and water only (control), or in addition to ad libitum access to either 16% sucrose, fructose, or glucose solution (n=16/gp). After 24h and 1 wk, eight rats per group were randomly selected for sacrifice. Daily caloric intakes among sugar-fed groups did not differ and were higher than the mean intake of the control group. Within 24h, fructose induced increased 11beta-HSD-1 message in mesenteric adipose and liver. Plasma TG and insulin were acutely increased in groups with fructose-containing diets only. All high sugar diets induced suppressed hepatic 11beta-HSD-1 mRNA and protein after 1 wk. Upregulation of H6PDH mRNA observed in response to long-term high sucrose diets may result from increased adiposity and not solely diet. High sugar diets, irrespective of sugar type, initiate glucocorticoid dysregulation in the absence of phenotypic changes associated with obesity. Sucrose, fructose, and glucose have distinct metabolic and endocrine responses. Fructose has the unique ability to induce glucocorticoid dysregulation in liver and adipose in 24h.Item Regulation of glutamine utilization during T cell activation(2007-08-13) Carr, Erikka Lynnette; Frauwirth, Kenneth; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Activation of a naïve T cell is a highly energetic event, which requires an increase in metabolism. Upon stimulation, T cells increase in size, rapidly proliferate and differentiate, all of which lead to a high demand for energetic and biosynthetic precursors. Even though amino acids are the basic building blocks of protein biosynthesis, the role of amino acid metabolism in this process has not been well characterized. We have found that glutamine in particular is required for both proliferative as well as effector function. We have evidence that glutamine regulates ERK signaling and that ERK in turn may also regulate glutamine transport. These data indicate that glutamine may play a significant role in T cell signaling and that a better understanding of glutamine utilization in T cells may reveal novel targets for immunomodulatory and/or anti-leukemia therapy.