Life Sciences Commons^™

The Development And Characterization Of Nanobodies Specific To Protein Tyrosine Phosphatase 4a3 (Ptp4a3/Prl-3) To Dissect And Target Its Role In Cancer., Caroline Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, …

Investigating The Relationship Between Metabolic Reprogramming And Peripheral Cd4+ T-Cell Inflammation In Human Type 2 Diabetes Pathogenesis, Gabriella Kalantar

Theses and Dissertations--Microbiology, Immunology, and Molecular Genetics

Chronic, low-grade systemic inflammation rises in obesity and promotes type 2 diabetes (T2D). Circulating immune cells are key indicators of obesity and T2D pathogenesis. T cells outnumber monocytes, in blood, suggesting that T cells might fuel peripheral inflammation in obesity/T2D. Our lab’s work supports this idea by identification of a Th17 cytokine profile in T2D from T-cell stimulated peripheral blood mononuclear cells. Work described herein further supported this work by demonstrating that T cells dominate peripheral inflammation over monocytes across the spectrum of obesity and glycemic control. Our lab has also recently shown that inflammation changes during prediabetes (preT2D), identified …

Characterization Of The Function And Regulation Of The Hmpv Phosphoprotein, Rachel Thompson

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus (NNSV) that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. Despite the initial identification of HMPV in 2001, there are currently no FDA approved antivirals or vaccines available. Therefore, understanding the mechanism of HMPV replication is critical for the identification of novel therapeutic targets. A key feature in the replication cycle of HMPV and other NNSVs is the formation of membrane-less, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). Recent work on NNSV IBs suggests they display characteristics of biomolecular condensates formed …

Developing A Biocatalytic Toolbox To Aid In Understanding Nucleoside Antibiotics, Jasmine Brianna Woods

Theses and Dissertations--Pharmacy

Antibiotic resistance happens when bacteria develop the ability to survive medications that normally terminate them. Instead, these super germs are able to survive in the body and produce a community of antibiotic resistance germs which can cause human fatalities. It is important to discover and develop new compounds and molecules that will improve this clinical obstacle. This research focused on analyzing the biosynthesis that incorporates distinctive chemical characteristic of various nucleoside antibiotics, ß-hydroxy amino acids and α-methyl-amino acids. ß-hydroxy amino acids and α-methyl-amino acids are considered an important class of industrially useful compounds, particularly for pharmaceutical development, and are found …

The Impact Of Plant Secondary Metabolites On Auxin And Cytokinin Signaling, Timothy E. Shull

Theses and Dissertations--Plant and Soil Sciences

Secondary metabolites are a broad class of specialized compounds that mediate plant-environment interactions and mitigate stress. It is increasingly clear that many phenylalanine-derived secondary metabolites are nearly indispensable for plant survival and that plants adjust their growth according to their secondary metabolic outputs. Consequently, many phenylalanine-derived secondary metabolites have influence over hormone activity. For instance, multiple phenylpropanoid intermediates and catecholamines alter the sensitivity of plants to the central hormone auxin, which in concert with cytokinin directs most aspects of plant growth and development. This dissertation reviews previous research on the influence of phenylpropanoid intermediates and catecholamines on plants, with a …

Apoe Genotype And Cerebral Glucose Metabolism: A Multi-Omics Approach, Holden C. Williams

Theses and Dissertations--Physiology

Apolipoprotein E (APOE) is encoded by the APOE gene, present in humans as three main isoforms (E2, E3, and E4). E4 carriers face up to a 15-fold increased risk for developing late-onset Alzheimer’s disease (AD), while E2 carriers are protected. Understanding the risk conferred by E4 has been an extensive research focus for nearly three decades, but the exact mechanism has yet to be proven. Many studies have demonstrated attenuated roles of E4 in classical hallmarks of AD, notably amyloid processing and neurofibrillary formation, which normally present later in disease progression. How APOE influences hallmarks that present much earlier are …

A Multidisciplinary Characterization Of The Enzymology And Biology Of Reversible Glucan Phosphorylation In Toxoplasma Gondii , Robert Murphy

Theses and Dissertations--Molecular and Cellular Biochemistry

Toxoplasma gondii is an opportunistic, protozoan parasite of all warm-blooded animals, infecting roughly one-third of humans worldwide. Humans acquire infections by consuming T. gondii tissue cysts in undercooked meat or from oocysts shed in cat feces. Encysted parasites convert into rapidly growing tachyzoites that disseminate throughout the body, defining the acute phase of infection. Under host immune pressure, tachyzoites convert into bradyzoites that populate tissue cysts found in CNS or muscle tissue and persist for the lifetime of the host, defining the chronic phase of infection. Tissue cysts are responsible for transmission via carnivory, but also possess the ability to …

Peripheral And Central Glucose Flux In Type I Diabetes, Jelena Anna Juras

Theses and Dissertations--Neuroscience

Diabetes is a complex metabolic disorder, of which high blood glucose concentration is the primary hallmark. Type I diabetes mellitus (T1DM) is characterized by the lack of insulin production, due to a poorly understood autoinflammatory cascade. In the words of historian Barnett “Diabetes may no longer be a death sentence, but for more and more people in the 21st century, it will become a life sentence”, making it the focal point of many research groups. It is estimated that around 20 million individuals worldwide live with T1DM.

Effects of long-term chronically elevated blood glucose are not only seen in micro/macro-vascular …

Building Tools For Improved Modulation Of The Human Gabaa Receptor, A Central Nervous System Target For The Treatment Of Anxiety, Garrett Edward Zinck

Theses and Dissertations--Pharmacy

In the U.S., anxiety is recognized as an increasing range of mentally and physically debilitating psychiatric health disorders with significant economic repercussions. Over the last 20 years, several novel anti-anxiety therapies have entered the drug development pipeline, but none have made it to market.

The work in this dissertation focused on structurally modifying valerenic acid (VA), a structurally unique carboxylated sesquiterpene acid found in Valeriana officinalis. VA is putatively reported to have allosteric modulatory activity of the human GABA_A receptor, a ligand-gated ion channel responsible for attenuating neurotransmissions. Structural modeling of VA’s GABA_A receptor interaction suggests that …

Synthesis Of 6,6- And 7,7-Difluoro-1-Acetamidopyrrolizidines And Their Oxidation Catalyzed By The Nonheme Fe Oxygenase Lolo, Nabin Panth

Theses and Dissertations--Chemistry

One of the remarkable steps in loline alkaloid biosynthesis is the installation of an ether bridge between two unactivated C atoms in 1-exo-acetamidopyrrolizidine (AcAP). LolO, a 2-oxoglutarate-dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of AcAP and the resulting alcohol's cycloetherification to give N-acetylnornoline (NANL). The mechanism of hydroxylation is well understood, but the mechanism of the oxacyclization is not. I synthesized difluorinated analogs of AcAP in an attempt to further understand the mechanism of the unusual cycloetherification step.

I prepared 6,6-F₂-AcAP in eight steps from N,O-protected 4-oxoproline. The key step was a Dieckmann …

Structural Basis Of Bacterial Flagellin For Naip5 Binding And Nlrc4 Inflammasome Activation And The Mechanism Of Flagellin Induced Release Of Cytokines In Vivo, Jian Cui

Theses and Dissertations--Chemistry

The bacterial flagellum is a whip-like structure that protrudes from the cell membrane and is one of the most complex and dynamic biological molecular machines that propels bacteria to swim toward beneficial environments and the sites of infection. It is composed of a basal body, a hook, and a long filament. The flagellar filament contains thousands of copies of the protein flagellin (FliC) monomer arranged helically and ending with a filament cap composed of oligomer protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases …

Antibiotic Permeation In Gram-Negative Bacteria And Contribution Of Inflammasome Activation And Pyroptosis In Pathogenesis Of Salmonella Systemic Infection, Ankit Pandeya

Theses and Dissertations--Chemistry

Antibiotic resistance is one of the major global issues in the field of public health and medicine. Good antibiotic candidates need to be selectively toxic, inhibit cellular target, and effectively penetrate and accumulate in bacterial cells. The last factor is a formidable barrier in the development of antimicrobials effective in Gram-negative bacteria, due to the presence of two layers of cell envelope. The first half of my thesis focuses on understanding the permeation of small molecules through this formidable cell envelope, distribution inside the cell of Gram-negative bacteria, and design of novel methods to make small molecules effectively cross the …

The Role Of Charge On Dna Packaging And Integrity Within Reconstituted Peptide-Dna Assemblies, Ehigbai Oikeh

Theses and Dissertations--Chemistry

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20^th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, …

Development Of Fluorescence Based Approaches To Understand Astrocyte Biology In The Context Of Nicotine And Nicotinic Receptor Activity, Surya P. Aryal

Theses and Dissertations--Chemistry

Smoking and tobacco use (STU) is a major global health problem and worldwide more than six million people die due to tobacco related diseases each year. Although majority of smokers try to quit smoking several times in their life, traditional therapeutic approaches, which focus only on neuronal cells, have a very low success rate. Understanding the effect of nicotine on glial cells, synaptic communication and blood vasculature in the brain can provide further insights on the neurobiology of substance abuse and can potentially help to design better therapeutic approaches. Glial cells are non-excitable cells in the brain which do not …

An Empirical Pipeline For Personalized Diagnosis Of Lafora Disease Mutations, M. Kathryn Brewer, Maria Machio-Castello, Rosa Viana, Jeremiah L. Wayne, Andrea Kuchtová, Zoe R. Simmons, Sarah Sternbach, Sheng Li, Maria Adelaida García-Gimeno, Jose M. Serratosa, Pascual Sanz, Craig W. Vander Kooi, Matthew S. Gentry

Molecular and Cellular Biochemistry Faculty Publications

Lafora disease (LD) is a fatal childhood dementia characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing the functional consequences of laforin missense mutations in vitro using complementary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes that were supported by clinical …

Toward The Discovery Of Biological Functions Associated With The Mechanosensor Mtl1p Of Saccharomyces Cerevisiae Via Integrative Multi-Omics Analysis, Nelson Martínez-Matías, Nataliya Chorna, Sahily González-Crespo, Lilliam Villanueva, Ingrid Montes-Rodríguez, Loyda M. Melendez-Aponte, Abiel Roche-Lima, Kelvin Carrasquillo-Carrión, Ednalise Santiago-Cartagena, Brian C. Rymond, Mohan Babu, Igor Stagljar, José R. Rodríguez-Medina

Biology Faculty Publications

Functional analysis of the Mtl1 protein in Saccharomyces cerevisiae has revealed that this transmembrane sensor endows yeast cells with resistance to oxidative stress through a signaling mechanism called the cell wall integrity pathway (CWI). We observed upregulation of multiple heat shock proteins (HSPs), proteins associated with the formation of stress granules, and the phosphatase subunit of trehalose 6-phosphate synthase which suggests that mtl1Δ strains undergo intrinsic activation of a non-lethal heat stress response. Furthermore, quantitative global proteomic analysis conducted on TMT-labeled proteins combined with metabolome analysis revealed that mtl1Δ strains exhibit decreased levels of metabolites of carboxylic acid metabolism, decreased …

Arginase 1 Insufficiency Precipitates Amyloid-Β Deposition And Hastens Behavioral Impairment In A Mouse Model Of Amyloidosis, Chao Ma, Jerry B. Hunt, Maj-Linda B. Selenica, Awa Sanneh, Leslie A. Sandusky-Beltran, Mallory Watler, Rana Daas, Andrii Kovalenko, Huimin Liang, Devon Placides, Chuanhai Cao, Xiaoyang Lin, Michael B. Orr, Bei Zhang, John C. Gensel, David J. Feola, Marcia N. Gordon, Dave Morgan, Paula C. Bickford, Daniel C. Lee

Sanders-Brown Center on Aging Faculty Publications

Alzheimer’s disease (AD) includes several hallmarks comprised of amyloid-β (Aβ) deposition, tau neuropathology, inflammation, and memory impairment. Brain metabolism becomes uncoupled due to aging and other AD risk factors, which ultimately lead to impaired protein clearance and aggregation. Increasing evidence indicates a role of arginine metabolism in AD, where arginases are key enzymes in neurons and glia capable of depleting arginine and producing ornithine and polyamines. However, currently, it remains unknown if the reduction of arginase 1 (Arg1) in myeloid cell impacts amyloidosis. Herein, we produced haploinsufficiency of Arg1 by the hemizygous deletion in myeloid cells using Arg1 …

Substrate Trafficking Within The Type Vii Secretion Systems Of Pathogenic Mycobacteria, Zachary A. Williamson

Theses and Dissertations--Molecular and Cellular Biochemistry

Tuberculosis (TB), primarily caused by infection of Mycobacterium tuberculosis (Mtb) in the lungs, is the deadliest infectious bacterial disease killing 1.5 million people annually. A major determinant of virulence is active secretion through three specialized type VII secretion (ESX) systems; ESX-1, ESX-3, and ESX-5. A large group of substrates exported by the ESX systems is the PE (Proline-Glutamine) and PPE (Proline-Proline-Glutamate) families of proteins, which are highly expanded in the pathogenic species of Mycobacteria and encompass over 7% of Mtb’s genome coding capacity. PE and PPE proteins interact together to form PE-PPE heterodimers, and are secreted through …

Clpxp Degradation System In Escherichia Coli, A Study Of Its Energy Sources And Its Applications In Managing The Expression Levels Of Targeted Membrane And Soluble Proteins, Thilini Abeywansha

Theses and Dissertations--Chemistry

ClpXP is an Escherichia coli protease that carries out energy-dependent intracellular proteolysis. In recent years, this system has been widely studied due to its importance as protein regulatory machinery and a virulence factor. Protein substrates of ClpXP contain degrons with a specific protein sequence. SsrA tag is one of the five degrons known to designate proteins for ClpXP degradation. SsrA is an 11 amino acid peptide added to the C-terminus of nascent polypeptide chains translated from aberrant messenger RNAs lacking stop codons via a process called trans-translation.

ClpXP was known to targets only cytosolic proteins with degrons until recently, …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Entry And Replication Of Negative-Strand Rna Viruses, Kerri Boggs

Theses and Dissertations--Molecular and Cellular Biochemistry

Hendra virus (HeV) and human metapneumovirus (HMPV) are negative-sense, singled-stranded RNA viruses. The paramyxovirus HeV is classified as a biosafety level 4 pathogen due to its high fatality rate and the lack of a human vaccine or antiviral treatment. HMPV is a widespread pneumovirus that causes respiratory tract infections which are particularly dangerous for young children, immunocompromised individuals, and the elderly. Like HeV, no vaccines or therapies are available to combat HMPV infections. These viruses fuse their lipid envelopes with a cell to initiate infection. Blocking cell entry is a promising approach for antiviral development, and many vaccines are designed …

Reversible Glucan Phosphorylation In The Red Alga, Cyanidioschyzon Merolae, Corey Owen Brizzee

Theses and Dissertations--Molecular and Cellular Biochemistry

Starch and glycogen are an essential component for the majority of species and have been developed to maintain homeostasis in response to environmental changes. Water-soluble glycogen is an excellent source of quick, short-term energy in response to energy demands. In contrast, plants and algae have developed the macromolecule starch that is elegantly suitable for their dependence on external circumstances. Semi-crystalline starch is water-insoluble and inaccessible to most amylolytic enzymes, thus plants and algae have developed a coordinated system so that these enzymes can gain access to the denser starch energy cache. Starch-like semi-crystalline polysaccharides are also found in red algae, …

Developing Synthetic Strategies For Multifaceted Applications Of Stable Gold-Based Complexes, Randall Tyler Mertens

Theses and Dissertations--Chemistry

Development of stable gold-based complexes has been a rapidly advancing field due to the popularity of gold complexes, particularly for use in biomedical research and catalytic transformations. Given that auranofin, a gold(I) complex with FDA approval for the treatment of rheumatoid arthritis is used in the clinic, the development of stable gold-based molecules of clinical relevance is urgently needed. Herein are reported, synthetic strategies used for the development of new classes of gold(I) and gold(III) complexes for advancement in mitochondrial modulation for use as chemotherapeutics as well as application to gold catalysis due to the unique geometry of complexes presented …

Machine Learning And Bioinformatic Insights Into Key Enzymes For A Bio-Based Circular Economy, Japheth E. Gado

Theses and Dissertations--Chemical and Materials Engineering

The world is presently faced with a sustainability crisis; it is becoming increasingly difficult to meet the energy and material needs of a growing global population without depleting and polluting our planet. Greenhouse gases released from the continuous combustion of fossil fuels engender accelerated climate change, and plastic waste accumulates in the environment. There is need for a circular economy, where energy and materials are renewably derived from waste items, rather than by consuming limited resources. Deconstruction of the recalcitrant linkages in natural and synthetic polymers is crucial for a circular economy, as deconstructed monomers can be used to manufacture …

Interactions Of Post-Pks Enzymes Of The Mithramycin Biosynthetic Pathway, Ryan Wheeler

Theses and Dissertations--Pharmacy

Combinatorial biosynthesis is a powerful tool for generating new, more active drug analogues to combat disease. But in order for combinatorial biosynthesis to be employed to its full potential, a deep understanding of the enzymes that produce the parent molecule must be had. The goals of the work presented in this thesis are to characterize the reaction catalyzed by MtmW, the final enzyme in the mithramycin (MTM) biosynthetic pathway, and to discover the interaction between MtmW and MtmOIV.

MtmW is an aldol-ketoreductase responsible for reducing the most distal carbonyl on the MTM pentyl side chain. It forms an octamer that …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Polyglucosan Body Structure In Lafora Disease, M. Kathryn Brewer, Jean-Luc Putaux, Alberto Rondon, Annette M. Uittenbogaard, Mitchell A. Sullivan, Matthew S. Gentry

Lafora Epilepsy Cure Initiative Faculty Publications

Abnormal carbohydrate structures known as polyglucosan bodies (PGBs) are associated with neurodegenerative disorders, glycogen storage diseases (GSDs), and aging. A hallmark of the GSD Lafora disease (LD), a fatal childhood epilepsy caused by recessive mutations in the EPM2A or EPM2B genes, are cytoplasmic PGBs known as Lafora bodies (LBs). LBs result from aberrant glycogen metabolism and drive disease progression. They are abundant in brain, muscle and heart of LD patients and Epm2a^-/- and Epm2b^-/- mice. LBs and PGBs are histologically reminiscent of starch, semicrystalline carbohydrates synthesized for glucose storage in plants. In this study, we define LB architecture, …

Deconvolution Tools For Extracting Insight From Challenging Two-Flavin Systems, Dallas Michael Bell

Theses and Dissertations--Chemistry

Flavoproteins have long been explored for their ubiquity among a number of metabolic and energetic reactions. The flavin cofactor has the inherent benefit of distinct spectral changes associated with redox transitions; however, the double-edged sword is incurred as these distinct signatures overlap and take up much of the UV-vis spectral range. Therefore, it is crucial to create a method to demarcate the expressed redox transitions for studying these systems. The first portion of these studies discusses the creation of a program that deduces spectra for redox transitions in a single-flavin containing model protein: flavodoxin. The latter portions discuss the application …

Fluvial Sediment Organic Matter Degradation Identified With Elemental And Isotopic Fate During Laboratory Incubation, Brenden Riddle

Theses and Dissertations--Civil Engineering

Fluvial sediment is well recognized as a critical factor in both carbon and nutrient budgets within stream systems. However, we find very few studies of reactivity and isotope enrichment for stream water from agricultural and urban streams and the class of substrate known as fluvial sediment organic matter. This study investigated the hypothesis that fluvial sediment is subject to degradation even though many previous studies have considered this class of substrate generally inert. Therefore we qualify that elemental and isotopic signatures of fluvial sediment organic matter should be considered potentially non-conservative when used in tracer studies. Methods applied to this …

Plant-Soil Interactions Dominate Soil Microbial Respiration And Soil Organic Carbon Sequestration In A Subtropical Moist Evergreen Broadleaved Forest In China, Zhijie Yang

Theses and Dissertations--Plant and Soil Sciences

Tropical forest soils contain one-third of global soil carbon (C). The warm and moist climate in tropical forests leads to rapid soil organic carbon (SOC) decomposition, with the highest soil microbial respiration rates in the world, so even a slight change in soil C and microbial respiration could affect atmospheric carbon dioxide concentration. However, there remains a lack of understanding of the mechanisms driving microbial respiration in tropical forests, due to different climate and biophysical drivers compared to temperate or boreal forests. Furthermore, forest conversions (from natural forests to plantations) are most widespread in tropical regions, leading to a loss …