Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
9 results
Search Results
Item MECHANISTIC STUDY AND THE DESIGN OF IRON-CATALYZED MULTI-COMPONENT CROSS-COUPLING REACTION(2021) Lee, Wes; Gutierrez, Osvaldo; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cross-coupling reactions (CCRs) are one of the most versatile methods for the formation of C-C bonds. Traditionally, palladium and nickel are broadly used as the catalyst in this type of transformation. However, due to the low cost, low toxicity, and high natural abundance, iron has become an alternative metal catalyst for CCRs. The first iron-catalyzed asymmetric cross-coupling reaction was reported by Nakamura in 2015 but the mechanism remained unknown. Since then, our lab has been working on 1) the elucidation of the mechanism using quantum mechanical calculations and experimental probes; and 2) the rational design and development of new types of iron-catalyzed cross-coupling reactions. Quantum mechanical calculations were applied to study the mechanism (Chapter 1). With multiple possible pathways computed and extensive conformational search, we determined that the lowest energy pathway proceeds via radical formation by Fe(I), radical addition to Fe(II), and reductive elimination from Fe(III) to form the desired cross-coupled product. With the mechanism in hand, we then designed and developed many new types of iron-catalyzed CCRs (Chapter 2-5), that included an intra- and inter-molecular dicarbofunctionalization of vinyl cyclopropanes, a three-component difunctionalization of unactivated alkenes, and a multicomponent radical cascade/annulation reaction. Finally, in Chapter 6, we introduced the [1.1.1]propellane as the σ-type radical acceptor in the three-component difunctionalization of iron-catalyzed cross-coupling reaction. These reactions showcases the potential of iron-catalyzed CCRs and expanded the toolbox for organic synthesis.Item HYDROLOGIC DRIVERS OF SOIL ORGANIC CARBON STORAGE AND STABILITY IN FRESHWATER MINERAL WETLANDS(2019) Kottkamp, Anna Isabel; Palmer, Margaret; Tully, Katherine; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mineral wetlands comprise most of historic wetland loss, yet few studies focus on mineral wetland soil organic carbon (SOC). We explore SOC across continuous hydrologic gradients within and among seasonally flooded mineral wetlands. First, we quantify SOC stabilization (e.g., organo-mineral associations and aggregates) across a wetland–upland gradient. Second, we examine relationships between hydrologic regime and SOC stocks among wetlands. From wetland–upland, saturation was highly variable in the transition zone. Organo-mineral associations peaked in the transition zone while large macroaggregate SOC declined from wetland–upland. Across wetlands, indicators of drying (e.g., minimum water level and summertime recession rate) were more related to SOC than inundation duration. From wetland basin–upland, SOC stocks were significantly related to both mean water level and relative elevation. We highlight relationships between SOC and the dynamic hydrology of wetlands, emphasizing the need for research on how changing hydrologic regime may influence mineral wetland SOC.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 FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH(2017) Zhang, Jianbing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.Item MRP5/ABCC5, A CONSERVED ABC TRANSPORTER, REGULATES METAZOAN HEME HOMEOSTASIS(2014) Korolnek, Tamara; Hamza, Iqbal; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hemes are metalloporphyrins used by nearly all organisms as cofactors for proteins involved in respiration, binding and sensing gases, and as catalysts for various reactions. Despite extensive knowledge about heme biosynthesis and catabolism, the pathways for transporting heme between cells and within cells remain poorly understood. C. elegans serves as a unique animal model for uncovering these pathways, as it is unable to synthesize its own heme and depends on the uptake of dietary heme for growth and reproduction. Functional RNAi screens implicated mrp-5 as a potential heme transporter in C. elegans. This gene encodes a membrane-bound ABC transporter that localizes to the basolateral intestinal membrane and is required for worm growth and reproduction. Depletion of mrp-5 activates heme deprivation signals within the worm, protects worms from toxicity associated with a toxic heme analog, and results in worms accumulating the fluorescent heme analog, zinc mesoporphyrin IX, in intestinal cells. Taken together, these results indicate a defect in heme export from the intestine when MRP-5 activity is lost. Functional assays in yeast support the hypothesis that MRP-5 is capable of exporting heme across cell membranes, and that this function is conserved in the human ortholog. Knockdown of mrp5 in zebrafish embryos results in developmental defects and decreased blood formation, indicating that this transporter likely regulates heme homeostasis in vertebrates. Loss of Mrp5 in mammalian cells leads to decreased heme transport into the secretory pathway as measured by activity of a Golgi-targeted heme-dependent enzyme. Furthermore, macrophages from mice lacking Mrp5 are unable to activate a number of cellular responses when undergoing erythrophagocytosis, the process whereby the heme-iron in senescent red bloods is recycled. Altogether, our results implicate MRP-5 as a key heme transporter in C. elegans, and point to an evolutionarily conserved role for MRP5 proteins in regulating heme homeostasis.Item Investigation into the Potential Toxicity of Zero-Valent Iron Nanoparticles to a Trichloroethylene-Degrading Groundwater Microbial Community(2013) Zabetakis, Kara Margaret; Torrents, Alba; Yarwood, Stephanie A; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The microbiological impact of zero-valent iron remediation of groundwater was investigated by exposing a trichloroethylene-degrading anaerobic microbial community to bare and coated iron nanoparticles. Changes in population numbers and metabolic activity were analyzed using qPCR and were compared to those of a blank, negative, and positive control to assess for microbial toxicity. Additionally, these results were compared to those of samples exposed to an equal concentration of iron filings in an attempt to discern the source of toxicity. Statistical analysis revealed that the three iron treatments were equally toxic to total Bacteria and Archaea populations, as compared with the controls. Therefore, toxicity appears to result either from the release of iron ions and the generation of reactive oxygen species, or from alteration of the redox system and the disruption of microbial metabolisms. There does not appear to be a unique nanoparticle-based toxicity.Item FUNCTIONAL INSIGHTS INTO HRG-1-MEDIATED HEME TRANSPORT(2012) Yuan, Xiaojing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor involved in various biological processes including oxygen transport, xenobiotic detoxification, oxidative metabolism, gas sensing, circadian rhythm, signal transduction, microRNA processing and thyroid hormone synthesis. Heme is also an essential nutrient for parasites and is the major dietary iron source for humans. Despite our extensive understanding of the mechanisms of heme synthesis and degradation in eukaryotes, little is known as to how heme is transported and trafficked in eukaryotes. Recently, CeHRG-1 and CeHRG-4 were identified as the first bona fide heme importers/transporters using the heme auxotroph, Caenorhabditis elegans. To gain mechanistic insights into the heme transport function of HRG-1-related proteins, we conducted a structure-function analysis of CeHRG-1 and CeHRG-4 by exploiting yeast mutants that are genetically defective in heme synthesis. Our studies reveal that HRG-1-related proteins transport heme across membranes through the coordinated actions of strategically placed amino acids that are topologically conserved in both, the worm and human proteins. To further dissect the functional elements that dictate their intracellular localization, we generated a series of chimeras by swapping the amino and carboxy terminal segments of CeHRG-1 and CeHRG-4. Our analysis in yeast and mammalian cells demonstrate that the C-terminal domains are essential for membrane localization of the protein, while the N-terminal domains are important for proper function, and plausibly multimerization of HRG-1-related proteins. Currently, there are no pharmacological means to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We, for the first time, developed and executed a high-throughput screen of 233,360 compounds, to identify potential antagonists of HRG-1-related proteins by utilizing parasite heme transporters as the primary screening bait. Subsequent study in parasites will provide novel drug candidates against helminths that infect humans, livestock, and plants, as well as against genetic disorders of heme and iron metabolism in humans. Taken together, results from our studies will significantly advance novel functional and therapeutic insights into HRG-1 mediated heme transport in health and disease.Item THE DIVALENT CATION TRANSPORTER NRAMP IN PARASITE PERKINSUS MARINUS: GENOMIC, MOLECULAR, STRUCTURAL, FUNCTIONAL AND EVOLUTIONARY ASPECTS(2010) Lin, Zhuoer; Vasta, Gerardo R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Perkinsus marinus, the causative agent of Dermo disease in eastern oyster Crassostrea virginica has been a great hurdle for oyster population restoration. Iron was shown to be an essential element for P. marinus growth and virulence, but iron uptake pathways have not been elucidated. Natural Resistance-associated Macrophage Protein (Nramp), an iron transporter, was considered to be a potential virulence factor in intracellular pathogens. One Nramp homolog (PmNramp1) was reported in P. marinus previously. Two other PmNramp isotypes (PmNramp2 and PmNramp3) were identified through genome mining followed by molecular characterization. The three PmNramp isotypes with distinct gene structures were transcribed in parasite trophozoites cultured in defined medium. Transcripts of a number of P. marinus genes, including PmNramp isotypes, superoxide dismutases (PmSOD), ascorbate peroxidase (PmAPX) and heat shock proteins (PmHSP70 and PmHSP90) were trans-spliced with a trans-splicing leader (SL) highly similar to dinoflagellate SL. No change in transcription level of those genes was detected by real-time quantitative reverse transcription PCR (qRT-PCR), under iron/manganese overload, iron depletion and host hemolymph exposure, indicating a constitutive polycistronic transcription in the parasite. Functional study by yeast complementation assays suggested iron uptake activity by PmNramp1. Prediction of PmNramp1 topology by homologous modeling indicated that PmNramp1 was an integral protein with 12 transmembrane segments (TMS). The central position of the Nramp-specific triplets Asp-Pro-Gly (TMS1) and Met-Pro-His (TMS6) in a three-dimensional (3D) arrangement formed with TMS3 and TMS8 provided the mechanistic basis for iron acquisition via PmNramp1. Site-directed mutagenesis of the residues on the triplets in PmNramp1 caused the lost of complementation activity as iron transporter in yeast. A chimeric protein with PmNramp1 N- and C-termini but PmNramp3 core structure from TMS1 to TMS12 complemented yeast growth, suggesting PmNramp3 an iron transporter. Phylogeny data implied that all the three PmNramp isotypes were archetype Nramp. Protein sequence divergence among PmNramp isotypes was not related to diversification of critical functional elements, which remained constrained by purifying selection. This result was consistent with the function of both PmNramp1 and PmNramp3 as iron transporters in yeast, despite their different evolutionary rate and substitution patterns. Subcellular localization of PmNramp isotypes in P. marinus trophozoites are in progress. PmNramp3 was shown to localize on cell peripheral when the parasite proliferates by binary fission. The data were consistent with the previous observation that iron is important for P. marinus growth. As the first functional study of Nramp homolog in protozoan parasites, the work in the dissertation may serve as the reference for research in other protozoan Nramp and iron transporters.Item THE ROLE OF ORGANIC MATTER IN THE DISSOLVED PHASE SPECIATION AND SOLID PHASE PARTITIONING OF MERCURY(2006-01-24) Miller, Carrie Lynn; Mason, Robert; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The interaction of mercury (Hg) and methylmercury (MeHg) with organic matter is extremely important in the dissolved phase speciation and solid phase partitioning of Hg and MeHg in aquatic systems. This study shows, that under oxic conditions Hg and MeHg will likely associated with Fe oxides through an indirect association with organic matter, while under sulfidic conditions, solid phase Fe sulfide will dominate the complexation of Hg to the solid phase. As a result of the association of Hg with Fe solids, which undergo dynamic changes at redox interfaces in aquatic systems, the distribution of Hg on particles is likely changing at redox boundries, areas that have been shown as active zones of methylation. Redox zones are also going to be important in controlling the mobility of MeHg from the site of production to areas in aquatic systems in which uptake by biota occurs. Although the dissolved phase speciation of Hg has been shown as an important factor in Hg methylation, as a result of the diffusive uptake of neutral Hg-sulfide into bacterial cells, this speciation had previously not been measured. Hg forms stronger bonds with reduced sulfide relative to dissolved organic matter (DOM), therefore, it was not previously thought that DOM was important in the speciation of Hg under sulfidic conditions. Using modified octanol-water partitioning extractions and centrifugal ultrafiltration, the speciation of Hg in sulfidic natural samples and laboratory solutions was examined. It was shown that the concentration of neutral Hg-sulfide complexes are lower than predicted by thermodynamic models, as a result of an interaction of these species with DOM. It is proposed that the interaction of Hg with DOM is not a complexation, but rather, a partitioning of neutral Hg-sulfide complexes into hydrophobic portion of the DOM. Thermodynamic constants were calculated for this interaction and applied to model the speciation of Hg in natural samples. The concentration of neutral Hg-sulfide is lower than models previously predicted, as a result of the DOM interaction. Since the concentration of neutral Hg-sulfide affects methylation, DOM could impact the rate of Hg methylation in aquatic systems.