UMD Theses and Dissertations
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Item THE STANDALONE REGULATOR ROFA OF STREPTOCOCCUS PYOGENES EXHIBITS CHARACTERISTICS OF A PRD-CONTAINING VIRULENCE REGULATOR(2024) Hart, Meaghan Taylor; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Streptococcus pyogenes (Group A Streptococcus; GAS) is a human pathogen estimated to cause nearly 790 million cases of disease annually at diverse tissue sites. To successfully infect these sites, GAS must detect nutrient availability and adapt accordingly. One mechanism employed to detect and import carbohydrates is the phosphoenolpyruvate transferase system (PTS), which mediates both carbohydrate uptake and metabolic gene regulation. Gene regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several stand-alone regulators that contain PRDs, with corresponding regulons encoding both metabolic genes and important virulence factors. These regulators form a family called PRD-Containing Virulence Regulators (PCVRs). RofA is a putative member of this family and is known to regulate the expression of genes important for virulence. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate appropriate virulence gene expression. In this dissertation, the RofA regulon was determined in strain 5448, a representative strain of the globally disseminated M1T1 serotype. The pilus and capsule operons were consistently dysregulated across growth in the absence of RofA. This correlated with increased capsule production and decreased adherence to primary keratinocytes. Purified RofA-His was phosphorylated in vitro by the general PTS components EI and HPr, and phosphorylated species of RofA-FLAG were detected in vivo late in stationary phase in a glucose-dependent manner. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar detection and utilization with GAS virulence gene regulation. Additionally, a bioluminescent construct was generated for allelic exchange into any S. pyogenes strain. Allelic exchange of this construct into WT 5448 yielded strains that were highly bioluminescent, grew to a similar density as WT, and survived as well as WT when challenged with human neutrophils. This tool could be used to study the contribution of specific proteins on in vivo virulence in a non-invasive manner, including RofA and RofA phosphorylation.Item Metabolic Profiling of Brain Microvascular Endothelial Cells: Investigating the Role of Sex, Stress, APOE Genotype, and Exercise in Alzheimer's Disease Risk(2024) Weber, Callie; Clyne, Alisa M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Alzheimer’s disease (AD) is the 7th leading cause of death in the United States, yet there are still no effective treatments to prevent or slow the progression of the disease. AD develops from a combination of genetic and lifestyle risk factors including female sex, elevated stress hormone exposure, the apolipoprotein (APOE) ε4 genotype, and a sedentary lifestyle. In order to better identify the manifestations of AD, it is vital to understand how each of these risk factors impact brain health and lead to neurological dysfunction associated with AD. Brain microvascular endothelial cells (BMEC) line the blood vessels of the brain and have specialized tight junctions designed to strictly regulate nutrient and waste transfer between the blood and the brain. Two of the early indicators of AD development are breakdown of the tight junctions and whole brain glucose hypometabolism. Since BMEC form the first line of defense for the brain against neurotoxic compounds in the blood and are responsible for glucose transport to the rest of the brain, the overarching goal of this thesis is to understand how female sex, elevates stress hormone exposure, the APOE ε4 genotype, and a sedentary lifestyle induce breakdown of tight junction proteins and glucose hypometabolism in BMEC. I first demonstrate that female sex exacerbates endothelial dysfunction in response to high levels of a stress hormone, Angiotensin II (AngII). Specifically, I show that in response to AngII, female endothelial cells increase oxidative stress and inflammatory responses while male endothelial cells do not. Next, I used CRISPR/Cas9 to generate a set of induced pluripotent stem cells (iPSC) homozygous for the APOE ε3 and ε4 genotype and differentiated them into BMEC (hiBMEC). Using the hiBMEC I showed the APOE ε4 genotype induces barrier deficiencies that are partially mediated through reduced levels of protein deacetylase Sirtuin 1 (SIRT1), and that the APOE ε4 genotype causes glucose hypometabolism through decreased insulin signaling. Finally, by adding serum from sedentary and exercise trained individuals to genotype-matched hiBMEC, I show that APOE ε3 and ε4 hiBMEC have divergent responses to treatment with serum from sedentary and exercise trained individuals. Treatment with exercise trained serum increases SIRT1 and glycolytic enzymes compared to sedentary serum, while exercise trained serum decreases SIRT1 and glycolytic enzymes in APOE ε4 hiBMEC compared to sedentary serum. The work described in this thesis gives a fundamental, mechanistic understanding to the roles of female sex, stress hormone exposure, the APOE ε4 genotype, and a sedentary lifestyle in BMEC dysfunction and hypometabolism, giving insight into how these factors contribute to AD development and progression.Item COMPUTATIONAL ANALYSIS OF METABOLIC NETWORKS AND ISOTOPE TRACER EXPERIMENTS FOR METABOLIC FLUX EVALUATION IN COMPLEX SYSTEMS(2021) Lugar, Daniel James; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metabolic engineering endeavors seek to develop microorganisms as feedstocks for biofuels and commodity chemicals. Towards this, quantifying metabolic fluxes is an important step for characterizing an organism’s metabolism and designing effective engineering strategies. Metabolic fluxes are quantified using sophisticated techniques, namely flux balance analysis (FBA), an in silico technique, and isotope-assisted metabolic flux analysis (MFA), a hybrid experimental and computational technique. FBA uses a network’s stoichiometry with linear programming techniques to generate in silico flux predictions for genome-scale networks. MFA uses measurements from stable isotope (typically 13C) tracer experiments to estimate fluxes of central carbon metabolism. In MFA, fluxes are parameters to a model developed from the network’s carbon atom rearrangements, which is fit to isotope labeling data, typically acquired using mass spectrometry.We developed novel mathematical and computational techniques for quantifying and analyzing flux predictions obtained using MFA and FBA. FBA applications typically generate flux predictions for networks with on the order of 1000 [O(1000)] reactions and metabolites. We developed a network reduction algorithm that uses matrix algebra to reduce a large network and flux prediction to a smaller representation. From this reduced representation, a researcher may quickly gain holistic insights from the FBA model. In isotopically nonstationary MFA, time-series labeling measurements are acquired on the approach to steady state. A model consisting of a large system of typically O(1000) ordinary differential equations is fit to the measurements to estimate fluxes and pool sizes. For detailed networks, the number of parameters may be large. We developed a computationally effective framework for solving this problem having robust convergence and efficient scalability to large networks. In this approach, we formulate the problem as an equality-constrained nonlinear program (NLP), solved efficiently using a solver implemented on an algebraic modeling language. Finally, we apply this approach to a detailed model of Phaeodactylum tricornutum photoautotrophic and mixotrophic (on acetate) metabolism. Using the flux estimates, we characterized this organism’s metabolism under disparate growth conditions, which may inform future endeavors to engineer P. tricornutum as a chemical feedstock.Item AN INTEGRATIVE EXPERIMENTAL AND COMPUTATIONAL FRAMEWORK FOR THE GENOME-SCALE FLUX ANALYSIS OF ANTIBIOTIC RESISTANCE(2020) Mack, Sean; Dwyer, Daniel J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The prevalence of antibiotic-resistant bacterial pathogens demands the development of novel therapeutic approaches. An attractive target is metabolism, where the impact of antibiotics and resistance remains poorly understood. Numerous omics-driven studies have identified a metabolic response due to antibiotic stress and suggested that metabolism adjusts to accommodate the genetic burden of resistance. Arising from these data is the hypothesis that context-specific modification of metabolism is a key component of antibiotic resistance and stress. Further exploration of the relationship between metabolism, antibiotic stress, and resistance is clearly needed. To elucidate metabolic signatures of antibiotic resistance, we analyzed the metabolic behaviors of wild-type and resistant strains of Escherichia coli through a combined transcriptomic and fluxomic analysis. Specifically, we compared wild-type E. coli to isogenic strains expressing integrated copies of tetRA and dhfr resistance genes, respectively under normal and antibiotic stress conditions. From comprehensive genome-scale (GS) flux predictions, we observed a resistance-associated metabolic phenotype as well as mechanism- and target-specific metabolic shifts. Furthermore, we identified a distinct metabolic response to antibiotic stress in both resistant strains. To improve our computational framework, we developed NetRed, NetRed-MFA, and NetFlow, each designed to reduce complexity of GS flux analysis. Through lossless reduction of genome-scale models (GSMs), NetRed generated a comprehensive minimal model for aerobic and anaerobic growth in E. coli and rapidly elucidated the mechanism driving artemisinin production in yeast. NetRed-MFA extended the original algorithm by incorporating full carbon mapping to generate reduced models for 13C metabolic flux analysis. NetFlow leveraged GS carbon mapping to isolate the major carbon flows through a core network and GSM; from the GSM subnetwork, we identified a mechanistic relationship between a triple-knockout and increased lycopene production in E. coli. Our resistance work represents the first application of quantitative flux analysis to study the metabolism of resistant bacteria and should provide significant insight into the role of metabolic adaptation in antibiotic resistance. The developed tools each dramatically improve the interpretation of GS flux predictions and the mechanistic understanding of metabolic perturbations. Taken together, this dissertation describes a comprehensive framework for the prediction, comparison, and interpretation of altered metabolic states.Item O-GLCNACYLATION IS NOT INCREASED IN THE HYPOTHALAMUS OF RATS GIVEN 6 WEEK ACCESS TO SUCROSE SOLUTION DESPITE MARKERS OF METABOLIC DYSREGULATION(2018) Hudgins, Samantha Morgan; Castonguay, Thomas W; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The peptide hormone leptin acts globally to maintain various metabolic processes. Impaired response to leptin binding is referred to as leptin resistance and results in metabolic dysregulation. Leptin is essential in the prevention of weight gain through central signals to increase energy expenditure and reduce food intake. A sugar sensitive pathway, the hexosamine biosynthesis pathway (HBP), may be the cause of diet induced leptin resistance. The HBP glycosylates proteins by modifying fructose- 6-phosphate molecules from glycolysis. While high sugar diets have been linked to leptin resistance, O-GlcNAcylation of pathway proteins have not been examined. Approximately 8-week-old male rats were assigned to ad libitum access to diet and water or 30% sucrose solution, diet and water. On Day 5 rats were surgically fitted with a third ventricle cannula. On Day 41, diet and sugar solutions were removed for an overnight fast. On Day 42 each rat received a central injection of leptin or control solution and subsequently euthanized 30 minutes post injection. Body weight and body composition were not significantly different between treatment groups after 42 days. However, the Sucrose group exhibited signs of metabolic syndrome, evidenced by increased fasting serum triglycerides and glucose as well as decreased serum HDL. Analysis of hypothalamic O-GlcNAcylation revealed no significant difference between treatment groups. These data may be the result of variability of glucose utilization within the hypothalamus. These data support previous findings that 42-day access to a 30% sucrose solution yields evidence of metabolic syndrome in the absence of obesity as well as the absence of increased hypothalamic OGlcNAcylation. Future research should examine O-GlcNAcylation regionally within the hypothalamus. Analysis of protein specific O-GlcNAcylation was not achieved; however, a novel O-GlcNAcylation was observed in hypothalamic tissue at the Threonine 1808 residue of prolow-density lipoprotein receptor-related protein 1 isoform X1 (LRP-1), a protein that may play a crucial role in leptin signaling. These data give further evidence to support the use of 30% sucrose solution to model leptin resistance in Sprague Dawley rats, as well as provide a target protein for future analysis.Item CHARACTERIZING THE ROLE OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM ENZYME II LOCI IN THE PATHOGENESIS OF THE GROUP A STREPTOCOCCUS(2017) Sundar, Ganesh; McIver, Kevin S; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome to functionally characterize each EIIC. It was found that a few EIIs had a limited in uence on PTS sugar metabolism, whereas others were promiscuous. The mannose-speci c EII locus exhibited the most in uence on PTS sugar metabolism. Importantly, the mannose-speci c EII also acted to prevent the early onset of SLS-mediated hemolysis. These roles were not identical in two different M1T1 GAS strains, highlighting the versatility of the PTS to adapt to strain-speci c needs. This is further illustrated by the fructose-speci c EII, which is important for survival in whole human blood for MGAS5005, but not 5448. The mannose-speci c EII can transport glucose in other pathogens, but the route of glucose utilization is unknown in GAS. MGAS5005 mutants were generated in a non-PTS glucose transporter (GlcU) and a glucokinase (NagC) of an annotated non-PTS glucose metabolic pathway. Since ∆ptsI, ∆nagC, and ∆glcU all grow to some extent in glucose, it is evident that glucose can be metabolized both by PTS and non-PTS routes. . However, the route of glucose utilization affects overall pathogenesis, as ∆nagC survives like WT in whole human blood, whereas ptsI is unable to survive. Subcutaneous infection of mice with ∆nagC did not exhibit increased lesion size, although these lesions are more severe than MGAS5005 due to the early onset of hemolysis. Overall this suggests that the routes of glucose metabolism greatly in uence SLS-mediated hemolysis. These results highlight that PTS carbohydrate metabolism plays an important role for GAS pathogenesis in both the skin and whole human blood, through the actions of EIIs.Item Controlled Delivery of a Glutamate Receptor Modulator to Promote Regulatory T cells and Restrain Autoimmunity(2015) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Autoimmunity occurs when the immune system incorrectly recognizes and attacks self-molecules. Current therapies involve broad immunosuppressants that are not curative and leave patients immunocompromised. Dendritic cells (DCs) are a target for new therapies because DCs influence the differentiation of immune effector cells. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), a glutamate receptor enhancer, modulates DC cytokine profiles to polarize T cells toward regulatory phenotypes (TREG ) that are protective in multiple sclerosis (MS). However, PHCCC treatment is limited by poor solubility, a short half-life, and toxicity. We hypothesized that controlled delivery of PHCCC from nanoparticles would alter DC function with reduced treatment frequency. PHCCC nanoparticles attenuated DC activation and promoted TREGs while reducing toxicity 30-fold. In mouse models of MS, these particles delayed disease and reduced severity compared to an equivalent dosing schedule of soluble drug. This outcome demonstrates controlled delivery of metabolic modulators can promote tolerance, suggesting a new route to improve autoimmune therapy.Item METABOLIC CHANGES ASSOCIATED WITH ANDROGEN INDEPENDENT GROWTH IN A MOUSE MODEL OF PROSTATE CANCER(2014) Martin, Philip Lloyd; Samal, Siba; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)PTEN and TP53 loss are common molecular alterations in aggressive prostate cancer that progresses to castrate resistant prostate cancer (CRPC). PTEN/TP53 loss contributes to regulation of self-renewal and differentiation in prostate progenitor cells, the presumptive tumor and metastasis initiating cells for prostate cancer. TP53 plays an important role in regulating normal cellular metabolism, and loss of function is responsible for metabolic alterations in tumor cells, including increased aerobic glycolysis. We use a novel model of Pten/Tp53 deleted prostate cancer to investigate properties of tumor and metastasis initiating cells, and metabolic alterations that contribute to the evolution of CRPC. We employed a genetically engineered mouse model of Pten-/-Tp53-/- prostate cancer to develop an orthotopic model derived from a clonal cell line from the parental heterogeneous prostate carcinoma. We used histopathology and immunohistochemistry to characterize the orthotopic primary tumors and metastases. We performed metabolomic screening followed by focused analysis of HK II enzyme levels, activity, and cellular distribution in androgen replete and androgen deprived tumors. We also compared HK II levels in primary and metastatic human prostate cancer. Tumor heterogeneity was due to transformation of tumor and metastasis initiating cells with biphenotypic potential capable of basal and luminal differentiation. There was epithelial-to-mesenchymal transition (EMT) in cells of the luminal lineage. The model was capable of androgen independent growth, which influenced the differentiation of metastasis initiating cells. CRPC had increased reliance on glycolysis with increased cytoplasmic and mitochondrial-associated HK II. These metabolic adaptations afforded CRPC increased ability to withstand metabolic stress. HK II levels in human metastases were markedly increased compared to primary tumors. Pten/Tp53 loss in prostate cancer contributes to lineage plasticity in both tumor and metastasis initiating cells, contributing to heterogeneity observed in primary tumors and metastases. Increased glycolysis due to increased total and mitochondrial HK II is a metabolic adaptation that contributes to the evolution of aggressive disease, with progression to androgen independence, providing increased energy and carbon precursors for anabolic processes. Mitochondrial bound HK II blocks apoptosis and contributes to survival in the androgen deprived environment. Targeting this metabolic adaptation may provide improved treatment for this deadly disease.Item Breast Cancer Type 1 Susceptibility Protein is a Critical Regulator of Skeletal Muscle Lipid Metabolism(2013) Jackson, Kathryn Campbell; Spangenburg, Espen E; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation research consists of three investigations in an effort to determine how circulating estrogens affect skeletal muscle lipid metabolism. Loss of circulating estrogens results in significant increases in visceral fat mass and intramuscular lipids (IMCL). These increases in lipid storage are strongly associated with an elevated risk of developing type 2 diabetes. The first investigation examined how the loss of circulating estrogens alters skeletal muscle metabolic function. Ovariectomy (OVX) resulted in significantly higher visceral fat mass and fatty acid sarcolemmal transporter content, which corresponded with elevated IMCL. Skeletal muscle in the OVX group exhibited lower acyl carnitine species suggesting impaired lipid flux through the mitochondria. Lastly, mitochondrial oxygen consumption rates were impaired in OVX skeletal muscle fibers. The results from this study gave rise to a search to identify an estrogen- sensitive mechanism that regulated lipid transport into the mitochondria. Study two determined for the first time that the BRCA1 protein, which is encoded by an estrogen-sensitive gene, is present and functions as an integral regulator of lipid metabolism in skeletal muscle. Specifically, BRCA1 binds to acetyl CoA carboxylase in response to acute exercise. The in vitro induction of decreases in BRCA1 expression resulted in higher IMCL content, reduced mitochondrial oxygen consumption rates, and elevated reactive oxygen species production. Surprisingly, no differences in BRCA1 content were detected between males and females. In the final study, an inducible, skeletal-muscle specific, BRCA1 KO mouse was developed. Ablation of BRCA1 in skeletal muscle resulted in exercise intolerance and the development of kyphosis. Contrary to our hypothesis, loss of functional BRCA1 in skeletal muscle attenuated the negative metabolic consequences of chronic high fat diet exposure. Collectively, these data provide strong rationale that BRCA1 is an important regulator of skeletal muscle metabolic function and further provide evidence that BRCA1 function is critical in multiple tissues across the body.Item Investigation of chicken embryo metabolism and substrate utilization during later development(2013) Hu, Qiong; Bequette, Brian J; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The objective of this research was to determine metabolic adaptations and substrate utilization during chicken embryo development and to determine the influence of breeder age and egg size on embryonic growth and metabolism during development (embryonic day 11 to posthatch day 1). In Study One, for both small (n = 60, 53.2 ± 1.04 g) and large (n = 60, 69.0 ± 1.86 g) eggs from 26 versus 42 wk-old broiler breeders, glucose content in albumen decreased to negligible levels by embryonic day (e) 11 whereas mannose and fucose remained constant. Higher yolk glucose content was observed in small eggs from e17 onwards whereas proportions of yolk linoleic and linolenic acids were greater in larger eggs. Liver adenosine monophosphate protein kinase (AMPK), the central cellular energy-sensor, was higher in activity in embryos from large eggs, and AMPK activity was at its highest for both sizes of eggs on e14. These observations suggest that glucose was consumed in early development (before e11). Lower liver AMPK activity and higher yolk glucose at later stages in small eggs from young hens suggests that anaplerotic metabolism is enhanced to alleviate the relative nutrient deficiency. In Study Two, a gas chromatography-mass spectrometry-based metabolomic profiling approach was employed to investigate effects of hen age and egg size on embryo metabolism. Principal component analysis of liver and blood metabolites showed separate clusters on both e14 and e20 from 32 and 51 wk-old hens. The separate clusters featured branched-chain amino acid, glycine, serine and threonine metabolism. Clear separation of metabolites was not observed for embryos from small versus large eggs at any developmental age. Breeder age had a larger influence on embryo metabolism and growth. Three clusters corresponding to liver metabolites from e14, e17 and e20 embryos formed a sub-tree that merged with the cluster from posthatch day 1 chicks in the Hierarchical clustering analysis. This result confirmed that embryo metabolism adapted during later development. Embryos from 51 wk-old hens displayed predominant developmental changes in ketone, glycerolipid and glutathione metabolic pathways in the liver compared to 32 wk-old hens. Study Three aimed to quantify gluconeogenesis, and substrate utilization and partition in e14 and e19 embryos. A constant infusion protocol (8 h) was developed for delivery of [U-13C] glucose and [U-13C] glycerol into the chorio-allantoic fluid. Gluconeogenesis was higher in e19 compared to e14 embryos, consistent with the need for increased liver and muscle glycogen by e19 embryos in preparation for emergence. The contribution of glucose to non-essential amino acid (NEAA) synthesis was greater in e14 vs e19 embryos, indicating a higher demand for amino acids for tissue growth. Glycerol contributed very little (< 5%) to gluconeogenesis; thus the remainder must be ascribed to amino acids. Relatively more of acetyl-CoA flux was derived from fatty acid metabolism in e14 embryos compared to 3-carbon pool substrates. In summary, this thesis research established aspects of embryo metabolism and nutrient partition in developing embryos that previously were unknown. This included demonstration of a role of AMPK in development, the influence of breeder age on global embryo metabolism, and metabolic adaptations in gluconeogenesis, Krebs cycle activity and glycerol metabolism during the latter half of chicken embryo development.