Biochemistry, Biophysics, and Structural Biology Commons^™

Biochemical And Kinetic Analysis Of Phosphofructokinase In The Eukaryotic Human Pathogen Entamoeba Histolytica, Jin Cho

All Dissertations

Entamoeba histolytica is a water- and food-borne intestinal parasite that causes amoebiasis and liver abscess in ~100 million people each year leading to ~100,000 deaths. This amitochondriate parasite lacks many metabolic pathways including the tricarboxylic acid cycle and oxidative phosphorylation, and cannot synthesize purines, pyrimidines, or most amino acids. As a result, E. histolytica is presumed to rely on its modified pyrophosphate (PP_i)-dependent glycolytic pathway for ATP production during growth on glucose. This pathway relies on a PP_i-dependent rather than ATP-dependent phosphofructokinase (PFK) and thus has a net production of three ATP per glucose. However, in …

Biochemical Analyses Of Udgx-A Crosslinking Uracil-Dna Glycosylase, Chuan Liang

All Dissertations

DNA base damage is common due to exposure to various endogenous and exogenous factors. To repair the base lesions, such as uracil from cytosine deamination, enzymes from the uracil-DNA glycosylase (UDG) superfamily are critical, which can recognize the damaged base and initiate the base excision repair (BER) pathway. There used to be six families of proteins identified in the UDG superfamily until a new member, UDGX, was found in Mycobacterium smegmatis, which is a unique DNA-crosslinking UDG. In this dissertation work, a series of biochemical analyses of the newly found UDGX are performed, including the analyses of structures, functions, …

Characterization Of The Effects Of The Pyrazolopyrimidine Inhibitor Grassofermata (Nav-2729) In The Eukaryotic Pathogen Trypanosoma Brucei, Kristina Marie Parman

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The protozoan pathogen, Trypanosoma brucei, is the causative agent of sleeping sickness in humans and nagana in livestock in sub-Saharan Africa. T. brucei cycles between tsetse fly and mammalian hosts, and it is adapted to survive in diverse host tissues. Variant Surface Glycoprotein (VSG) plays a key role in immune evasion in the mammalian host. The VSG membrane anchor requires two myristates, 14-carbon saturated fatty acids (FAs) that are scarce in the host. T. brucei can synthesize FAs de novo, but also readily takes up exogenous FAs, despite lacking homologs to fatty acid uptake proteins found in other …

Fatty Acids And Parasitism: Towards A Better Understanding Of Lipid Metabolism In Trypanosoma Brucei, Joshua Saliutama

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Trypanosoma brucei is an extracellular eukaryotic parasite that causes sleeping sickness in humans and cattle. As an extracellular parasite, T. brucei relies on the host’s nutrients to satisfy its growth requirements. The parasite is unusual because it does not uptake most of the host’s lipid species. Instead, T. brucei prefers to perform de novo synthesis of most lipid species. One of the lipid species that T. brucei can both uptake and synthesize is fatty acids. In my thesis work, I investigated the dynamics of fatty acid uptake, metabolism, and utilization of T. brucei. My work starts by determining the …

Comparing Development Of Drug Resistance By Cryptococcus Neoformans To Chemically Distinct Azole Anti-Fungal Compounds, Lindsey Burke

Honors College Theses

Cryptococcus neoformans is a fungus that primarily infects humans who have weakened immune systems. An azole drug, Fluconazole, is commonly administered against C. neoformans in regions were cryptococcosis is most prevalent, most notably Sub-Saharan Africa. However, C. neoformans can gain resistance to Fluconazole through becoming an aneuploid. To better understand the basis of resistance, we employed a disk diffusion assay and investigated several chemically-distinct azole compounds with anticryptococcal properties for their effectiveness against C. neoformans and to identify potential differences in the capacity of the fungus to become resistant to each of the tested compounds. Different C. neoformans strains were …

Investigating The Role Of Cnag_05113 In The Carnitine Biosynthesis Pathway In Cryptococcus Neoformans., Jasmine Meltzer, Rodrigo Catalan-Hurtado, Perry Kezh, Kerry Smith

Honors College Theses

Cryptococcus neoformans, the leading cause of fungal meningitis, is a fungal pathogen that causes severe infection of the central nervous system in patients with compromised immune systems, typically caused by HIV/AIDS. C. neoformans infections are present in developed countries including the United States, but most fatalities occur in sub-Saharan Africa where antiretroviral therapy, the treatment for HIV/AIDS, is less accessible. Current treatments for severe cryptococcal infections are extensive and outdated. There is a critical need for an improved understanding of the fungus and new targeted therapies. Our goal is to identify metabolic pathways important to the survival of C. …

Biochemical Analysis Of Dna Glycosylase In Dragonfish (Scleropages Formosus, Sfo) Tdg, Jenna Perry

Honors College Theses

TDG, which is a member of the uracil DNA glycosylase superfamily, plays a critical role in the active demethylation process in mammals by removing oxidized derivatives of 5-methylcytosine (mC). While human TDG has been extensively studied and found to possess 5-formylcytosine and 5-carboxylcytosine DNA glycosylase activity, little is known regarding this demethylation process in vertebrates other than the human species. Previous research proposed a catalytic sequence in motif 1 of zebrafish TDG1 and TDG2 that modulates DNA glycosylase activity based on mutational, kinetic, and modeling analyses of these enzymes. Building on this work, researchers discovered that the Sfo TDG is …

Inhibitors Of Human Eno2 Are Potent Anti-Trypanosomal Agents, Danielle Lavigne

Honors College Theses

Kinetoplastid parasite infections remain a global health burden. Here, we have characterized inhibitors of an essential Trypanosoma brucei glycolytic enzyme, enolase (TbENO). We anticipate TbENO inhibitors will be potent anti-trypanosomals, as T. brucei relies on glycolysis for ATP production in the blood of infected mammals. Additionally, the phosphonate enolase inhibitors being considered are well-tolerated in mammals, suggesting their potential in developing novel therapeutics for kinetoplastid infections. TbENO was cloned into the bacterial expression vector, pQE-30, and the heterologously-expressed protein was purified by nickel affinity and assayed in a coupled enzyme assay. Enzyme activity paralleled the abundance …

The Role Of Fto, A Human Rna Demethylase In Perennial Grass Development And Abiotic Stress Responses, Andrew Fiorentino, Qian Hu, Xiaotong Chen, Zhaohui Chen

Honors College Theses

The integration of the human fat mass and obesity associated (FTO) gene into turfgrass is a novel approach at improving cell proliferation and abiotic stress resistance. The FTO protein is an RNA demethylase responsible for epigenetic regulation of the genome. In related rice, the gene is associated with increased crop yield, tiller number, and aerial biomass. It is proposed to work via demethylation of repeat RNA associated with chromatin remodeling, causing widespread transcriptional activation. In this study, the feasibility of using FTO for plant trait modification in perennial grasses is being investigated. Potentially transformed embryogenic calli of creeping bentgrass with …

Methyltransferase, Glucose Adaptation, And Import Complex In Trypanosoma Brucei, Emily Knight

All Dissertations

Trypanosoma brucei is a kinetoplastid parasite responsible for human African trypanosomiasis (HAT) and nagana, a livestock wasting disease, which both endemic to sub-Saharan Africa. Unique to kinetoplastids are the specialized peroxisomes, named glycosomes, which compartmentalize the first several steps of glycolysis and gluconeogenesis, nucleotide sugar biosynthesis, and many other metabolic processes. Kinetoplastids are unique in that they have a single mitochondrion. In this work, I present the first study into SET domain proteins in any kinetoplastid parasites. We have characterized a predicted SET domain protein, TbSETD3, that localizes to the mitochondrion and a depletion of the protein results in growth …

New Dna Repair And Demethylation Functions In Uracil Dna Glycosylase Superfamily, Chenyan Chang

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Uracil-DNA glycosylase (UDG) superfamily, which consists of several groups of enzymes that recognize the damaged DNA bases and initiate the base excision repair (BER) pathway, is most important in dealing with DNA deamination and other base modifications. Thymine DNA glycosylase (TDG), which belongs to family 2 in the UDG superfamily, is able to specifically recognize and cleave the 5-methylcytosine (mC) oxidative derivatives including 5-formylcytosine (fC), 5-carboxylcytosine (caC), 5-hydromethyluracil (hmU) caused by active demethylation or DNA damage. My dissertation work is mainly focused on the fC and caC glycosylase activity within UDG superfamily. Chapter 1 is a general introduction to the …

Glycolytic Inhibitors As Leads For Drug Discovery In The Pathogenic Free-Living Amoebae, Jillian Milanes

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The free-living amoeba, Naegleria fowleri, can cause a rare yet usually lethal infection of the brain called primary amebic meningoencephalitis. Because of poor diagnostics and limited treatment options, the mortality rate associated with the disease is >97%. Due to our finding that glucose is critical for trophozoite growth in culture, we have been interested in exploiting amoebae glucose metabolism to identify new potential drug targets. We have characterized the first enzyme of the glycolytic pathway, glucokinase (Glck), from N. fowleri and two other pathogenic free-living amoeba, Acanthamoeba castellanii and Balamuthia mandrillaris. We have assessed their biochemical properties and …

Function Of Septin Proteins In Cryptococcus Neoformans In Cell Wall And Plasma Membrane Integrity And Homeostasis, Emma Hatchell

Honors College Theses

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that causes meningitis in immunocompromised patients. This lethal fungus is sometimes referred to as the “sugar coated killer” due to its polysaccharide capsule. It is estimated that 152,000 cases of cryptococcal infection occur each year and result in 112,000 deaths. The ability of C. neoformans to adapt to host temperature is a main factor responsible for virulence. Septins are conserved filament-forming GTPases that are confirmed to be involved in cytokinesis and morphogenesis and have been implicated in heat stress response and virulence of C. neoformans. C. neoformans genome encodes four septins, Cdc3, …

Acetate Metabolism In The Fungal Pathogen Cryptococcus Neoformans, Oly Ahmed

All Dissertations

Cryptococcus neoformans is an environmental basidiomycetous fungus with a worldwide distribution and a wide range of habitats. Inhalation of the desiccated yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections in immunocompromised individuals, and in severe cases causes lethal meningitis following hematogenous dissemination. During infection, depending on the tissue and disease state, the invading fungi experience a range of nutrient microenvironments within the host body. As a result, rapid metabolic adaptations geared towards efficient utilization of carbon sources alternative to glucose become one of the prime determinants of survival and growth for the pathogen. Incidentally, cryptococcal infection …

The Role Of Fatty Acid Metabolism In The Pathogenesis Of Trypanosoma Brucei, Nava Poudyal

All Dissertations

Trypanosoma brucei is the protozoan parasite that causes African Sleeping Sickness in humans and nagana, a wasting disease in cattle. T. brucei completes its life cycle in two hosts, mammals and the tsetse fly insect vector. Due to the geographical restriction of the tsetse fly, the disease is endemic in sub-Saharan Africa. Both the insect and mammalian forms of the parasite need fatty acids to anchor their surface proteins. We worked on three projects on fatty acid metabolism and its role in immune evasion strategies of T. brucei. First, we assessed the role of T. brucei surface proteins in …

The Role Of Vsmc Mir-33a Expression On Apoa-I Mediated Cholesterol Efflux And Macrophage-Like Cell Transdifferentiation, Ikechukwu Esobi

All Dissertations

Atherosclerosis is a condition caused by cholesterol accumulating in arterial intimal cells and is a disease that kills more people in the United States and globally than any other disease. Atherosclerosis is commonly recognized to arise from arterial intimal macrophage cholesterol accumulation, but cell lineage tracing technology has shown that a large majority of cholesterol-laden intimal cells found in atherosclerotic arteries are actually vascular smooth muscle cells that have switched phenotypes to a macrophage-like cell. This vascular smooth muscle cell to macrophage-like cell phenotypic switch is known as transdifferentiation and can be triggered by vascular smooth muscle cell cholesterol accumulation. …

The Effects Of Tubulin Post-Translational Modifications On The Flagellar Motility Of Trypanosoma Brucei, Katherine Wentworth

All Theses

Trypanosoma brucei is a parasitic kinetoplastid that causes African trypanosomiasis and is transmitted to a mammalian host by the tsetse fly (Glossina spp.). T. brucei relies on its flagellar motility to carry out its morphological changes from the procyclic form (predominant in the fly vector) to the bloodstream form (infectious form in mammals) and navigate the bloodstream of its host. The driving structure within the flagellum is the axoneme, which is composed of microtubules and dynein motor proteins. The tubulin code hypothesis suggests that cells regulate microtubule motor protein activity through post-translational modifications (PTMs) of alpha and beta …

Heat Stress Response And Excystation In Entamoeba Histolytica, Irem Bastuzel

All Dissertations

Entamoeba histolytica is a water- and food-borne intestinal protozoan parasite that causes amoebiasis and liver abscess and is responsible for symptomatic disease in approximately 100 million people each year leading to ~ 100,000 deaths. The most common disease transmission follows the oral-fecal route, but it can also be transmitted by mechanical vectors such as animals carrying the amoeba from contaminated sources to water systems. In rare cases, disease transmission has been recorded in some patients in which men-to-men sexual practices were preferred.

The life cycle of E. histolytica starts through ingestion of infectious cysts, which are non-dividing, quadri-nucleated structures surrounded …

Improved Molecular Detection Tools For The Invasive Crop Pest Helicoverpa Armigera (Hübner), Mitchell D. Rich

All Theses

Helicoverpa armigera is a major crop pest native to Europe, Asia, Australia, and Africa. H. armigera has recently invaded South America and has caused billions of dollars in agricultural losses. It is difficult to differentiate H. armigera from H. zea, a closely-related species native to North and South America. A few genetic tests have been previously developed to detect H. armigera DNA in pooled samples of moth legs. In this study, an improved qPCR melt curve assay with higher detection sensitivity and a field-based recombinase polymerase amplification (RPA) assay were developed for specific detection of H. armigera DNA …

Investigating The Biochemical Properties Of A Novel Mutation, A194v, In Human Rad51, Briana Vollbeer

All Theses

DNA double-strand breaks (DSB) are one of the most serious DNA lesions because improper repair of a DSB can lead to loss of heterozygosity, aneuploidy, and cancer. One of the primary pathways to repair DSBs is homologous recombination (HR). HR resects the DNA around the DSB and then uses homologous DNA as a template to restore the broken sequence. RAD51 has a vital function in this pathway by forming a nucleoprotein filament on a resected end of the DSB. The nucleoprotein filament searches for homology within the homologous DNA. Once homology is located, strand invasion followed by strand exchange occurs. …

Characterization Of The Wee1 Homologues And The Investigation Of Factors Promoting Cellular Enlargement In Cryptococcus Neoformans, Rodney J. Colón Reyes

All Dissertations

Cryptococcus neoformans is an opportunistic fungal pathogen, infecting mainly immunocompromised individuals. As the main cause of cryptococcosis, it is responsible for over 180,000 deaths every year. As an environmental yeast, it has unique adaptations that allow it to proliferate in the human host. Among these adaptations its capacity to transition to an extreme phenotype known as Titan cells is of special interest to researchers. With sizes above 10 um and able to reach 70 um or more in cell size. This size is accompanied with a large vacuole, larger polysaccharide capsule, and an increased resistance to fluconazole (FLC). FLC is …

Modeling Electrostatics In Molecular Biology And Its Relevance With Molecular Mechanisms Of Diseases, Mahesh Koirala

All Dissertations

Electrostatics plays an essential role in molecular biology. Modeling electrostatics in molecular biology is complicated due to the water phase, mobile ions, and irregularly shaped inhomogeneous biological macromolecules. This dissertation presents the popular DelPhi package that solves PBE and delivers the electrostatic potential distribution of biomolecules. We used the newly developed DelPhiForce steered Molecular Dynamics (DFMD) approach to model the binding of barstar to barnase and demonstrated that the first-principles method could also model the binding. This dissertation also reflects the use of existing computational approaches to model the effects of Single Amino Acid Variations (SAVs) to reveal molecular mechanisms …

Optimization Of Modular, Long-Range, Ultra-Fast Optical Tweezers With Fluorescence Capabilities For Single-Molecule And Single-Cell Based Biophysical Measurements, Subash C. Godar

All Dissertations

An Optical tweezer is a tightly focused laser beam that applies and senses precise and localized optical force to a dielectric microsphere and offers a unique and effective tool for manipulating the single cell or cell components, including nucleotides and dynein motor proteins. Here, I used highly stabilized optomechanical components and ultra-sensitive detection modules to significantly improve the measurement capabilities over a wide range of temporal and spatial scales. I combined the optical tweezer-based force spectroscopy technique with fluorescence microscopy to develop an integrated high-resolution force-fluorescence system capable of measuring displacements at sub-nanometer, forces at sub-piconewton over a temporal range …

Floating Treatment Wetlands For Brackish Waters: Plant Selection And Nutrient Uptake Potential., Andrea Landaverde

All Theses

Brackish water bodies in coastal regions provide critical ecosystem services that support human and environmental health. Anthropogenic activities such as agricultural and industrial activities, construction, urban settlements, and tourism contribute to increased inputs of nitrogen (N) and phosphorus (P) in brackish coastal ecosystems. Excess nutrients can lead to impaired water quality and affect marine organisms. Floating treatment wetlands (FTWs) are a vegetated-base technology used to remove contaminants from water column, that has been mainly studied and applied in freshwater systems. Application of FTWs in brackish systems requires further investigation, as high salinity in brackish waters could result in toxicity to …

Characterization Of A Potential Glucose Transporter In Trypanosoma Brucei, Matthew Morgan

All Theses

Trypanosoma brucei, the African trypanosome, is an organism heavily dependent on glucose for ATP production during the infectious stage of its life cycle. Here, we have explored the role of an uncharacterized protein designated “novel glucose transporter” (NGT) as a potential glucose transporter. Sequence analyses suggests that NGT shares similarities (either at the primary sequence level or structurally) with Trypanosome Hexose Transporters 1 (TbTHT1), and human GLUT3, both of which are membrane sugar transporters. NGT was localized by fluorescence microscopy to subcellular structures consistent with lysosomes. Silencing NGT expression with RNA interference in parasites resulted in a growth defect …

An Investigation Into The Roles Of Aldose Reductase And Acetate Kinase In The Metabolism Of Entamoeba Histolytica, Matthew B. Angel

All Dissertations

Entamoeba histolytica is an amoebic parasite that infects an estimated 90 million people worldwide and causes approximately 100,000 deaths per year. As the causative agent of amoebic dysentery, this food- and water-borne pathogen represents a significant public health burden worldwide, particularly in areas with poor sanitation. While treatments for amoebiasis exist, they are often limited in their effectiveness. Thus, efforts to better understand the biology and physiology of this organism are vital to the development of novel treatments for this disease.

E. histolytica lacks the enzymes for many common metabolic pathways such as the citric acid cycle and oxidative phosphorylation …

Supertertiary Structural Dynamics Modulate Function In Postsynaptic Density Protein 95, George L. Hamilton Iii

All Dissertations

Proteins, RNA, and DNA serve as the primary sub-cellular machinery that give rise to the necessary functions of life. The long-standing paradigm has been that the structures of biomolecules, or the arrangement of the subunits that make up a biomolecule, determine biological function. However, biomolecules are not static objects. Instead, they often undergo structural rearrangements that are crucial to enabling and regulating their functions. In my thesis I present several studies of the interplay between the structures, dynamics, and functions of biomolecules that combine experimental fluorescence spectroscopy and computational methods to probe these systems at the single-molecule level. In particular, …

Toxoplasma Gondii Requires Its Plant-Like Heme Biosynthesis Pathway For Infection, Amy Bergmann, Kathleen Floyd, Melanie Key, Carly Dameron, Kerrick C. Rees, L. Brock Thornton, Daniel C. Whitehead, Iqbal Hamza, Zhicheng Dou

Publications

Heme, an iron-containing organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and sensing, to mitochondrial respiration, to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates. Intracellular pathogenic microbes switch routes for heme supply when heme availability fluctuates in their replicative environment throughout infection. Here, we show that Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived …

Trait Differentiation And Modular Toxin Expression In Palm-Pitvipers, Andrew J. Mason, Mark J. Margres, Jason L. Strickland, Darin R. Rokyta, Mahmood Sasa, Christopher L. Parkinson

Publications

Background

Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A2 (PLA2) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis …

Comparison Study Of Mold Growth Resistance Of Plastic Based Material Flooring (Pbm Flooring) And Ceramic Tile Flooring, Jyothi Rangineni, Jeremy Tzeng

Publications

Clemson University Department of Biological Sciences and Tile Council of North America Product Performance Testing Laboratory evaluated whether ceramic tile and Plastic Based Material (PBM¹) flooring support mold growth when exposed to fungal spores.

Mold grows in moisture-rich environments and requires only minimal sources of nutrition to support growth. It has long been identified to cause damage to buildings and construction materials and its presence in buildings has been connected to many major health concerns with various studies and reviews published on this matter.²

The method used to evaluate mold growth was ASTM G21-96 (2015).³ This …