Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item HIPPOCAMPAL GLUCOSE TRANSPORT AND OXIDATION IN RESPONSE TO DISRUPTED BLOOD FLOW IN AN AGING RAT MODEL OF HEART FAILURE(2023) Pena, Gabriel Santiago; Smith, J. Carson; Kuzmiak-Glancy, Sarah; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The primary objective of this dissertation was to investigate, in a rodent model of cardiovascular disease promoted by transverse aortic constriction (TAC), whether cerebral hypoperfusion stemming from chronic high pulsatile blood flow, and cerebral hypoperfusion stemming from low cerebral blood flow differentially affected hippocampal glucose transport and hippocampal mitochondrial function. We first, characterized the changes in right and left carotid hemodynamics and diameter in response to TAC and in a SHAM control group at three different time points (20-, 30-, and 40 weeks) post-surgery. Then, right, and left hippocampal mitochondrial content and substrate oxidation were investigated, and protein expression of glucose transporters and mitochondrial quality control markers were quantified. In this study, both the SHAM and TAC conditions included male and female rats to address possible sex differences. We report that all time points within TAC, right carotid blood flow velocities and pulsatility were greater than the left, but did not worsen over time. No differences in mitochondrial content were found within TAC nor between TAC and SHAM, but within TAC animals there were impairments in right hippocampal coupled and uncoupled respiration when compared to the left. When compared to the SHAM controls, right and left hippocampi of TAC animals had higher protein expression of mitochondrial quality control markers, but no differences in glucose transporter expression were found. Thus, while both high blood flow and/or pulsatility as well as low cerebral blood flow may lead to brain hypoperfusion, the metabolic consequences of the two may not be the same. The results from this dissertation contribute to the expanding literature characterizing the intersection between cardiovascular disease and neurodegeneration.Item Regulation of Mitochondrial Metabolism and Lipogenesis(2021) Surugihalli, Chaitra; Sunny, Nishanth E; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Non-alcoholic fatty liver disease is one of the most common liver disorders with a global prevalence of over 25%. Fatty liver is the most common comorbidity of insulin resistance, obesity, and type 2 diabetes mellitus. During fatty liver, nutrient overload and the associated hyperinsulinemia results in elevated circulating free fatty acids and lipid accumulation in the liver. High rates of lipid accumulation in the liver is partly attributed to high rates of lipogenesis from carbohydrates, together with dysfunctional lipid oxidation. Further, these dysfunctional metabolic networks will induce oxidative stress and inflammation. Thus, understanding the metabolic mechanisms contributing towards the etiology of fatty liver and its associated morbidities is of major interest towards developing prevention and management strategies. This dissertation utilizes a combination of in-vivo (chicken and mice) and in-vitro (isolated mitochondria) systems with stable isotope-based methodologies to study metabolic regulation.Chicken embryos utilize yolk lipids (>45%), deriving over 90% of their energy through lipid oxidation for development. However, during the last few days of incubation and immediately after hatch, there is a substantial induction of lipogenesis. Despite the hepatic lipid overload, the synergistic remodeling of hepatic metabolic networks during embryonic-to-neonatal development blunted inflammatory onset, prevented accumulation of lipotoxic intermediates, and reduced reactive oxygen species production. Elevated plasma branched-chain amino acids (BCAAs) are a characteristic of insulin resistance and are relevant in predicting T2DM. Defects in BCAA degradation networks are also evident in several tissues during insulin resistance and associated co-morbidities. Furthermore, alterations in BCAA metabolism are associated with changes in lipogenesis and mitochondrial oxidative networks. We utilize a combination of isolated mitochondrial systems and stable isotope tracers in diet-induced mouse models of fatty liver, to determine its impact on mitochondrial metabolism and lipogenesis. In summary, the dissertation highlights i) the importance of the natural but dynamic remodeling of hepatic mitochondrial metabolism and lipogenesis during the efficient embryonic-to-neonatal transition in chicken ii) the significance of BCAAs as important regulators of hepatic mitochondrial lipid metabolism. Thus, these studies provide a platform to modulate hepatic metabolic networks and utilize the embryonic-to-neonatal transition phase and dietary intervention of BCAAs as management strategies to alleviate metabolic dysfunctions.Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.Item BIOPHYSICAL STUDIES OF THE MECHANISM OF CERAMIDE CHANNEL DESTABLIZATION BY BCL-XL IN APOPTOSIS AND THE USE OF RECTIFICATION TO PROBE THE STRUCTURE AND DYNAMICS OF A NOVEL ESCHERICHIA COLI CHANNEL(2015) Chang, Kai-Ti; Colombini, Marco; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ceramide forms a novel type of channel in the mitochondrial outer membrane and these channels are involved the release of intermembrane space proteins from mitochondria, a decision-making step in the apoptotic process. An antiapoptotic protein, Bcl-xL, regulates the apoptotic process and inhibits the formation of ceramide channels. However, there is no precedent to indicate how a protein regulates a lipid channel. We investigated the mechanism of this regulation and identified the hydrophobic groove of the Bcl-xL as the binding site by which Bcl-xL binds to the channel. This was demonstrated by using a combination of experimental and modeling methods, including site-directed mutagenesis, a fluorescence quenching assay, a mitochondrial outer membrane permeability assay, and molecular dynamic simulations. Interestingly, the hydrophobic groove serves to inhibit another channel former, Bax. We found that the binding sites for Bax and ceramide on Bcl-xL are distinct but overlapping. We used that fact to generate mutants that have differential abilities to inhibit one or the other of these channels. These are useful because although ceramide is important in apoptosis, it is still controversial that whether ceramide channels result in apoptosis in vivo. To probe the relative importance of these two channels in apoptosis, Bcl-xL mutant proteins were expressed in Bcl-xL deficient cells. Weakening the inhibitory potency of Bcl-xL on either Bax or ceramide channels resulted in cells being more sensitive to the induction of apoptosis. This is the first evidence for the role of ceramide channels in the apoptotic process in vivo. In a separate investigation, a novel voltage-gated channel unit was found in E. coli extracts. The unit is consistent with three channels forming the functional triplet. These channels are highly voltage gated and highly cooperative. Those results indicated that one of the channels is oriented in an antiparallel fashion compared to the rest. This arrangement is very rare in protein channels. Rectification of the current flowing through the channels was used to identify the orientation of the channels to provide evidence for or against the antiparallel hypothesis. The results favor the antiparallel hypothesis but also reveal an unexpected asymmetry in the transmembrane electrostatics.Item VISUALIZATION OF CERAMIDE CHANNELS BY TRANSMISSION ELECTRON MICROSCOPY(2011) SAMANTA, SOUMYA; COLOMBINI, MARCO; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40 nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.