Biochemistry, Biophysics, and Structural Biology Commons^™

Intrinsically Disordered Proteins And Their Role In Biomolecular Condensates, Danielle Latham

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Proteins are biomacromolecules responsible for the functions of life. While classically proteins are thought to be well structured in order to perform a specific function, 50% of proteins within Eukaryotic cells contain intrinsically disordered regions (IDRs), regions with no well-defined structure. IDRs are often used for cell signaling, responding to external factors such as temperature changes or the presence of small molecules. To understand how IDRs can function without structure, it is important to understand the dynamics of such systems. Understanding IDR intramolecular and intermolecular interactions will shed light on IDR dynamics. Intramolecular interactions are first explored using fluorescence spectroscopy …

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 …

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

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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

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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, …

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 …

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 …

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

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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 …

Acetate Metabolism In The Fungal Pathogen Cryptococcus Neoformans, Oly Ahmed

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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 …

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 …

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

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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

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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. …

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

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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 …

Heat Stress Response And Excystation In Entamoeba Histolytica, Irem Bastuzel

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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 …

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

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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 …

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

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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 …

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

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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, …

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

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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 …

Tackling Adverse Environment—Molecular Mechanism Of Plant Stress Response And Biotechnology Tool Development, Ning Yuan

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Abiotic and biotic stresses such as drought, salt, nutrition starvation, and pathogen infection are major factors threatening our agricultural production. With the rapidly increasing population and limited arable land area, genetic engineering of crops for new products with more stable and higher yield than conventional cultivars under adverse environment provides a powerful new tool for use in developing novel GMOs (Genetically Modified Organisms) to feed the large population in the immediate future. To develop novel GMOs with enhanced performance under adverse conditions, we need first to understand molecular mechanisms underlying plant stress response. To better understand how signaling transduction pathway …

Development And Validation Of A Novel In Vitro Model For The Assessment Of Heterocellular Interactions Mediated By Connexin43, Emily Ongstad

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The injury border zone (IBZ), a region of transitional tissue between intact myocardium and the ischemic area, is often the site of lethal reentrant arrhythmia generation in post-myocardial infarction (MI) patients. Disruption to normal connexin43 (Cx43) localization at the intercalated disc (ID), separation of myocytes by activated fibroblasts and deposited scar tissue are thought to be factors that render the IBZ a pro-arrhythmic substrate, though there is a current need to better understand these changes so directed therapies can be developed. There are no clinically available therapies focused on the mechanistic changes in the IBZ. Additionally, generation of new compounds …

Arsenic Inhibits P19 Stem Cell Differentiation By Altering Microrna Expression And Repressing The Sonic Hedgehog Signaling Pathway, Jui Tung Liu

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Arsenic is a naturally-occurring toxicant that exists in bedrock and can be leached into ground water. Humans can be exposed to arsenic via contaminated drinking water, fruit, rice or crops. Epidemiological studies have shown that arsenic is a developmental toxicant, and in utero exposure reduces IQ scores, verbal learning ability, decreases long term memory, and increases the likelihood of dying from a neurological disorder. Arsenic can also reduce birth weight, weight gain, and muscle function after an in utero exposure. Although the mechanism behind these physiological changes is not known, in vitro studies have shown that arsenic can reduce muscle …

Development, Validation, And Application Of Analytical Methods For Characterizing Adsorbed Protein Orientation, Conformation, And Bioactivity, Aby Thyparambil

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The structure and bioactivity of adsorbed proteins are tightly interrelated and play a key role in their interaction with the surrounding environment. These factors are of critical importance in many biotechnological applications. However, because the bioactive state of an adsorbed protein is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary …

Phosphotransacetylase And Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase: Two Eukaryotic Partners Of Acetate Kinase, Tonya Taylor

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Although acetate is a predominant metabolite produced by many eukaryotic microbes, far less attention has been given to acetate metabolism in eukaryotes than in bacteria and archaea. Acetate kinase (Ack), which catalyzes the reversible phosphorylation of acetate from ATP, is a key enzyme in bacterial acetate metabolism. Ack primarily partners with phosphotransacetylase (Pta), which catalyzes the generation of acetyl phosphate from acetyl-CoA, but can also partner with xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which produces acetyl phosphate from either xylulose 5-phosphate or fructose 6-phosphate. The Ack-Pta pathway, found primarily in bacteria, is also present in lower eukaryotes such as the green …

Specificity And Catalytic Mechanism Of Dna Glycosylases In Udg Superfamily, Bo Xia

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DNA can be damaged by several kinds of endogenous and exogenous reactive nitrogen species. Under nitosative stress, uracil (U), hypoxanthine (I), xanthine (X) and oxanine (O) are four major deaminated DNA bases derived from cytosine (C), adenine (A) and guanine (G) respectively. To repair this type of DNA damage, several different repair pathways are involved.

My dissertation work mainly focused on the uracil-DNA glycosylase (UDG) superfamily, which includes several groups of enzymes that recognize the damaged DNA bases and initiate the base excision repair (BER) pathway, one of the most important repair pathways to deal with deaminated DNA bases. Chapter …

Allosteric Regulation Of Bacterial And Fungal Xylulose 5-Phosphate/ Fructose 6-Phosphate Phosphoketolases (Xfps), Katie Glenn

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Acetate is excreted as a metabolic end product in many microbes. Acetate production has primarily been studied in bacteria and archaea but is known to occur in eukaryotic organisms as well. For example, acetate is one of the most abundant metabolites excreted by the fungal pathogen Cryptococcus neoformans during cryptococcosis suggesting that acetate production may be important during pathogenesis. One possible pathway for acetate production in C. neoformans involves the enzymes xylulose 5-phosphate/ fructose 6-phosphate phosphoketolase (Xfp), which can generate acetyl phosphate from either fructose 6-phosphate (F6P) or xylulose 5-phosphate (X5P), and acetate kinase (Ack), which can then convert acetyl …

Prolactin And Isplatin Combination Treatment Inhibit Tumorspheres Formation And Tumor Growth In Mice, Eric Hingleung Lee

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Cancer stem cells (CSCs) are defined as a small population of tumor initiating cells that are responsible for the initiation, development, progression, and recurrence of cancer. The chemo and radiation resistance of CSCs remains one of the major obstacles in conventional anti-cancer therapies. One of the reasons that conventional chemotherapeutics are not effective in targeting CSCs is that CSCs are usually in a non-proliferative or dormant state. In this perspective, targeting CSCs by inducing its proliferation and differentiation and simultaneously applying chemotherapeutics may be an alternative approach. The current study investigates the effect of prolactin (PRL), a hormone intimately involved …

Biochemical Investigation Into Initiation Of Fatty Acid Synthesis In The African Trypanosomes, Sunayan Ray

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My doctoral studies focused on studying FA metabolism in the deadly protozoan parasite T. brucei. In my dissertation, I will be addressing various aspects of the regulation of TbACC, which catalyzes the first committed step in FA synthesis. In the second chapter, I hypothesized that TbACC is regulated in response to environmental lipids. I examined changes in TbACC RNA, protein, and activity in response to different levels of environmental lipids in both BF and PF cells. I also delineated the mechanisms by which TbACC expression and activity is regulated by phosphorylation in response to altered lipid environments. In the third …

Role Of Phosphoinositide-Based Signaling In Virulence In Entamoeba Histolytica, Amrita Koushik

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Entamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. Upon entering the human host, cellular processes such as adhesion, phagocytosis, motility and secretion play a vital role in its propagation and pathogenicity. In other systems, each of these cellular processes is preceded by activation of signal transduction pathways, which often involve membrane phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Little is known about phosphoinositide signaling in E. histolytica pathogenicity. In this study, we demonstrated that PI(4,5)P₂ is localized to cholesterol-rich microdomains, lipid rafts, of the E. histolytica membrane and to the trailing edge …

Regulation Of The Lipid Raft Localization Of The Gal/Galnac Lectin, An Adhesin On The Surface Of The Human Protozoan Parasite, Entamoeba Histolytica, Amanda Goldston

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Lipid rafts, sterol- and sphingolipid-rich membrane microdomains, have been shown to control virulence in a variety of parasites including Entamoeba histolytica, an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components, and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts, Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. Exposure to bonafide Gal/GalNAc lectin ligands is associated with …

Lignin Modification In Arabidopsis And Populus For Studies Of Gene Function And Improving Lignin Degradation, Yi Xu

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Lignin is one of the most abundant biopolymers in plants and plays an important role in plant structure and stress defense. Lignin is also considered to be a hallmark of vascular plants because of its crucial role in plant terrestrialization. However, lignin is an undesired component in the pulp and paper industry, bioethanol production, and forage digestibility. Thus, understanding the functions and the evolution of lignin biosynthesis genes can not only advance our knowledge of the evolution of land-adaptation for vascular plants but also help guide the effort to exploit the potential for genetic manipulation of lignin for desirable traits …

An Investigation Of Trypanosoma Brucei Hexokinases: Localization, Oligomerization, And Inhibition, April Joice

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Trypanosoma brucei is the causative agent of African sleeping sickness in humans and nagana in livestock. The parasite inhabits multiple environmental niches including the bloodstream of the mammalian host and the mid-gut of the tsetse fly vector. While in the bloodstream of its mammalian host, the organism depends solely on glycolysis for production of ATP. My studies focus on the first enzyme in glycolysis, hexokinase.
T. brucei has two hexokinases, TbHK1 and TbHK2 that are 98.5% identical at the nucleotide level. The hexokinases are expressed in the glycosomes of both procyclic form and bloodstream form parasites. Glycosomes are peroxisome-like organelles …