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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, …

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 …

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 …

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 …

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 …

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. …

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 …

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 …

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, …

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 …

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 …

Stromal-Epithelial Interactions Modulate Cross Talk Between Prolactin Receptor And Her2/Neu In Breast Cancer, Cong Xu

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The tumor microenvironment is a crucial factor in breast tumorigenesis. Tumor epithelial cells maintain 3D structure in tumor stroma and they interact with soluble factors secreted by stromal cells such as cancer associated fibroblasts (CAFs) or directly with the extracellular matrix (ECM). Recent studies have shown that the hormone prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been demonstrated not only to be able to directly inhibit PRLR activation but also …

Spidroin N-Terminal Domain: A Ph Sensor In The Spider Silk Assembly Process, William Gaines

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Spider silks are protein-based fibers with remarkable mechanical qualities. Perhaps even more impressive is the spinning process in which the spider silk proteins (spidroins) are assembled from a highly soluble storage state into a well-ordered and insoluble fiber. Indeed, the ordered arrangement of spidroins, which is endowed by the spinning process, is the basis of fiber strength. However, the forces driving fiber assembly and the mechanisms by which spidroins respond those forces are only poorly understood. Spidroins have a tripartite domain architecture consisting of a large and repetitive central domain flanked by small, non-repetitive N- and C-terminal domains. Both terminal …

The Role Of Car And Pxr In Toxicant Sensitivity, Linda Mota

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The Constitutive Androstane Receptor (CAR) and the Pregnane X Receptor (PXR) are nuclear receptors of significant importance in the regulation of enzymes that metabolize, detoxify and eliminate compounds from the body. In this study we assessed the protective role of CAR and PXR in the basal and inducible regulation of Cytrochrome P450s (CYPs), and the potential of CAR and PXR to help protect individuals from the organophosphate, parathion and the plasticizer, nonylphenol, putatively due to improved metabolism and elimination. Knockout models of these receptors were used to model susceptible populations such as children that are known to have lower CAR …

Genomic, Proteomic And Metabolomic Approaches To Study Drought Responses In Aquilegia, David Henry

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Global population is expected to increase 30% by 2040, which will result in an increased need for crop production to feed the growing population. Combined with projected increased drought conditions worldwide, plant genetic research is necessary to gain a deeper knowledge of the molecular factors involved in plant drought response in order to engineer crop species with improved drought tolerance. Aquilegia has been recently developed as a model species for gene exploration based on its ability to thrive in a wide variety of environments including arid locations. An attractive asset of Aquilegia is its evolutionary position, equidistant between rice and …

Development Of Novel Prolactin And Growth Hormone Receptor Agonists And Antagonists, John Langenheim

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Potential indications for human prolactin (hPRL) and human growth hormone (hGH) are the support and sustainment of lactation and the enhancement or reconstitution of the immune system when under dysregulation. A prolactin receptor (PRLR) antagonist, hPRL-G129R (G129R), and a PRLR and growth hormone receptor (GHR) antagonist, hGH-G120R (G120R), have been developed which may be useful for the treatment of breast cancer since PRLR is elevated in a majority of human breast tumors and makes cancer cells highly sensitive to the mitogenic and anti-apoptotic activity of hPRL and hGH.

As a single agent, G129R is more cytostatic than cytotoxic to human …

The Role Of Prolactin And Its Antagonist In Her2/Neu Tumorigenesis, Michele Scotti

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Purpose: To study the role of prolactin (PRL) and its antagonist, G129R, in HER2/Neu tumorigenesis. Specifically, to investigate the interaction between the oncogene HER2 and the PRL receptor (PRLR) signaling pathways for designing effective combinational therapeutics for breast cancer.
Experimental Design: The combination effects of G129R and an anti-HER2 antibody, Herceptin, were tested against HER2-overexpressing human breast cancer cell lines, T-47D and BT-474, using cell based assays and xenografts established in athymic mice. Furthermore, four different bitransgenic mouse lines co-expressing the murine version of HER2 and PRL or G129R were generated. The mammary tumor incidence, characterization of mammary gland development, …