Life Sciences Commons^™

Uvrd1 Helicase Of Mycobacterium Tuberculosis That Can Be Activated By Multiple Unique Mechanisms, Ankita Chadda

Arts & Sciences Electronic Theses and Dissertations

Abstract Mycobacterium tuberculosis (Mtb) causes tuberculosis disease in humans and is one of the leading causes of death worldwide due to infectious agents. During infection, Mtb is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that causes DNA damage. UvrD-like helicases are involved in DNA repair and use energy from ATP hydrolysis to translocate on single stranded DNA (ssDNA) or unwind double-stranded DNA (dsDNA) to remove the damaged DNA strand. Previous studies on UvrD-like helicases have shown that they exist in a monomer-dimer equilibrium and unwind only as dimers in the absence of accessory …

A Universal Mechanism Of G Protein Inhibition, Tyson Daniel Todd

Arts & Sciences Electronic Theses and Dissertations

G protein coupled receptors transduce a truly staggering number of diverse extracellular signals including chemical messengers, physical force, and even photons into specific cellular responses through their coupling to heterotrimeric G proteins. G proteins amplify the originating signal through their binding to downstream effectors, activating a complex network of overlapping responses that allow the cell to respond perfectly to that specific stimulus. It is critical to the cell that this process is carried out faithfully in order to respond to the myriad environmental cues and avoid injury, exhaustion, and death for the individual cell or the development of pathology if …

A Structural Perspective On Neutralizing Antibodies To Flaviviruses And Coronaviruses, John Michael Errico

Arts & Sciences Electronic Theses and Dissertations

The presence of neutralizing antibodies typically correlates strongly with protection from infection, particularly for RNA viruses; elicitation of neutralizing antibody responses is thus the primary goal of most vaccines, and monoclonal antibodies (mAbs) can be leveraged as potent therapeutics. Thus, understanding the properties of individual antibodies that comprise polyclonal antibody responses and their epitopes is essential for countermeasure design. In this dissertation, I explore the epitopes and functional properties of monoclonal antibodies targeting the structural proteins of two families of RNA viruses, flaviviruses and coronaviruses.

Flaviviruses are globally distributed arthropod-borne positive-sense RNA viruses transmitted primarily by either mosquitos or ticks. …

Molecular Characterization Of Integrase-Rna Interactions And Their Role In The Replication Of Hiv-1 And Other Retroviruses, Christian Shema Mugisha

Arts & Sciences Electronic Theses and Dissertations

HIV-1 integrase (IN) enzyme has an emerging non-catalytic role in particle maturation, whichinvolves its binding to the viral genome in virions. Allosteric integrase inhibitors (ALLINIs) and class II integrase substitutions inhibit the binding of IN to the viral genome and cause formation of eccentric non-infectious HIV-1 particles. These viruses are characterized by the mislocalization of the viral ribonucleoprotein complexes between the translucent conical CA lattice and the viral lipid envelope. We have previously demonstrated that IN binding to the viral genome is mediated by basic residues within the C-terminal domain of IN. In the first chapter, we show how basic …

Regulatory Effects Of The E. Coli Recbcd Nuclease Domain On Dna Unwinding Kinetics, Nicole Fazio

Arts & Sciences Electronic Theses and Dissertations

I have examined the effects of deleting the nuclease domain of the E. coli helicase RecBCD on the rates of ATP-independent DNA melting, single stranded (ss) DNA translocation, and double stranded (ds) DNA unwinding by RecBCD. The canonical role of the nuclease domain is DNA degradation, but the removal of this domain showed unexpected effects on other RecBCD activities including DNA binding, melting, and unwinding. This thesis presents a mechanistic study of DNA unwinding by RecBCD and a RecBCD variant with the nuclease domain deleted (RecBΔnucCD). I examined the rates of ssDNA translocation and dsDNA unwinding by RecBCD and RecBΔnucCD …

Dissecting The Molecular Mechanism Of Familial Cardiomyopathies, Sarah Ruth Clippinger Schulte

Arts & Sciences Electronic Theses and Dissertations

Familial cardiomyopathies, including hypertrophic (HCM), restrictive (RCM) and dilated cardiomyopathy (DCM), are the leading cause of sudden cardiac death in young people. These diseases, which are characterized by altered cardiac contractility and remodeling of the heart, can lead to heart failure. These diseases are primarily caused by point mutations in sarcomeric proteins that generate or regulate heart contraction, such as troponin T. In the heart, the troponin complex together with tropomyosin lie along the actin filament and regulate myosin’s ability to bind actin and produce force. Here I show how mutations in troponin T affect contractility at the molecular level …

C. Elegans Response To Cadmium Toxicity, Brian James Earley

Arts & Sciences Electronic Theses and Dissertations

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. Residing in the same group of the periodic table, cadmium and zinc share chemical characteristics that are important for their industrial uses in electroplating, batteries, pigments, and metal alloys. The similarities of ionic cadmium and zinc have significant repercussions on biological systems. While it has long been clear that cadmium is toxic to biological systems, the mechanisms of cadmium toxicity remain poorly understood. In contrast, mechanisms of zinc homeostasis have been elucidated in growing detail. In C. elegans high zinc homeostasis is regulated …

Method Development For Enhancing Sensitivity Of Dynamic Nuclear Polarization Nuclear Magnetic Resonance Spectroscopy For Structural Studies Of Pkc-Drug Interactions, Patrick Terrence Judge

Arts & Sciences Electronic Theses and Dissertations

To perform the most relevant structural studies on biological systems, experiments need to be carried out when the target proteins are in their endogenous cellular environment. Nuclear magnetic resonance (NMR) is well-suited to probe the structure and dynamics of a wide variety of systems, including biologically relevant proteins. However, NMR suffers from an inherent lack of sensitivity. Dynamic nuclear polarization (DNP) NMR is a powerful technique that is used to enhance NMR sensitivity by transferring the greater polarization of exogenously doped electron spins to nuclear spins of interest though the use of a high-power microwave source. Solid effect radicals offer …

The Regulation Of Plasmodium Falciparum Metabolism By Haloacid Dehalogenase Proteins, Philip Frasse

Arts & Sciences Electronic Theses and Dissertations

Malaria is an enormous financial and public health burden for much of the world, infecting over 200 million and killing over 400,000 people every year. While much progress has been made combating malaria in the past few decades, those advances have slowed in recent years, partially due to the emergence of resistance to all known antimalarials used to date. To achieve the goal of eliminating malaria as a major global health problem, new therapeutics need to be developed, targeting novel categories of parasite biology. One poorly understood area of parasite biology is the regulation of various metabolic pathways. We have …

Chemical Damage To Mrna And Its Impact On Ribosome Quality-Control And Stress-Response Pathways In Eukaryotic Cells, Liewei Yan

Arts & Sciences Electronic Theses and Dissertations

Ribosome often faces defective adducts that disrupt its movement along the mRNA template. These adducts are primarily caused by chemical damage to mRNA and are highly detrimental to the decoding process on the ribosome. Hence, unless dealt with, chemical damage to RNA has been hypothesized to lead to the production of toxic protein products. Even more detrimental is the ability of damaged mRNA to drastically affect ribosome homeostasis through stalling. This in turn would lead to greatly diminished translation capacity of cells. Therefore, the inability of cells to recognize and resolve translational-stalling events is detrimental to proteostasis and could even …

The Role Of Excited States In Determining Β-Lactamase Function And Bacterial Fitness, Catherine Rae Knoverek

Arts & Sciences Electronic Theses and Dissertations

Proteins are macromolecular machines that play a role in nearly every biological process. They are dynamic molecules which adopt many different conformations as they fold into their 3D structures, interact with their binding partners, and perform their functions. The most probable (lowest energy) protein conformation is referred to as the ground state, and this is often assumed to be the state determined by experimental methods such as x-ray crystallography. However, proteins also adopt higher energy excited states which can have significant probabilities. As these excited states are notoriously difficult to find and study, it is unclear if excited states contribute …

Rna Polymerase Binding Protein A (Rbpa) Regulation Of Mycobacteria Transcription And Sensitivity To Fidaxomicin, Jerome Prusa

Arts & Sciences Electronic Theses and Dissertations

Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB) and remains one of the deadliest microorganisms on the planet. The effort to eradicate M. tuberculosis would benefit from the development of novel therapeutics, which requires a detailed understanding of M. tuberculosis physiology. Like all living organisms, M. tuberculosis gene expression requires transcription. Transcription in the phylum Actinobacteria, which includes mycobacteria, is unique because it includes RNA Polymerase Binding Protein A (RbpA) that is essential in both M. tuberculosis and the nonpathogenic model organism Mycobacterium smegmatis. RbpA increases the housekeeping A and housekeeping like B interactions with the RNA …

Copper-Mediated Regulation Of A Traditional Iron Uptake System In Uropathogenic Escherichia Coli., George Lwanga Katumba

Arts & Sciences Electronic Theses and Dissertations

Transition metals constitute an important part of the host-pathogen interface. Iron is an essential nutrient that functions as a cofactor for numerous bacterial and host proteins, as either a ligand for oxygen in carrier proteins or an enzyme catalytic site due to its natural redox properties. As part of the innate immune response, infected hosts sequester iron from pathogens to limit their growth, a phenomenon known as nutritional immunity. On the other hand, copper ions are deployed at infection sites as a potent antimicrobial agent to kill bacteria. The ability to survive within multiple, often harsh, microenvironments is fundamental to …

Potentiation Of Tmem16a Currents By Clca1 In Cystic Fibrosis Airway, Kayla Berry

Arts & Sciences Electronic Theses and Dissertations

In the airway, proper activity of the anion channel cystic fibrosis transmembrane conductance regulator (CFTR) contributes to innate immune defense by maintaining a hydratedand alkaline mucus layer through the conductance of chloride and bicarbonate ions. This allows potentially pathogenic microorganisms to be trapped, quickly killed, and cleared via mucociliary clearance, thus preventing microbial colonization of the lungs. In cystic fibrosis (CF), this activity is impaired, resulting in repeated pulmonary infections that damage the lung and, if severe and prolonged, may lead to premature death without lung transplantation. Available therapies remain focused on targeted rescue of the CFTR mutation. However, given …

Deep Learning Deleterious Small Molecule Biochemistry From The Lab To The Clinic, Matthew Matlock

Arts & Sciences Electronic Theses and Dissertations

Small molecules are key tools in biology and medicine. In biology, small molecules are used to probe biological systems and gain insight into their structure and function. In medicine, this role is further refined to reverse the biological conditions that contribute to human disease. Developing new small molecules into biological probes or drugs can be a daunting scientific task. These projects often begin with many thousands of potential candidates, which are progressively screened and eliminated from consideration by high-throughput experimental assays. Those molecules that emerge from this process as candidate drugs are also subject to extensive in vitro, in vivo, …

Protein Synthesis Adaptation To The Au-Rich Transcriptome Of Plasmodium Falciparum, Jessey Lee Erath

Arts & Sciences Electronic Theses and Dissertations

The process of protein synthesis whereby a messenger RNA is decoded into an amino acid chainis conserved among the domains. Fastidious protein synthesis is necessary for organism survival. However, exceptions negatively affecting the mRNA translation cycle – inadvertently or by design – may occur. Polyadenosine tracts are one such motif causing ribosomal stalling and frameshifting in almost all organisms tested thus far; save Plasmodium spp. Thus, with ~60% of their protein-coding genome harboring polyadenosine tracts, the elucidation of such paradigm-breaking adaptations enabling Plasmodium spp. to translate this typically problematic motif without issue is salient from both basic science and clinical …

Mass Spectrometry-Based Strategies In Protein Higher Order Structure Analysis: Fundamentals And Applications In Protein-Ligand Interactions, Xiaoran Liu

Arts & Sciences Electronic Theses and Dissertations

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which …

Measuring And Manipulating Tension-Dependent Behavior Of Mechanosensitive Ion Channels, Angela M. Schlegel

Arts & Sciences Electronic Theses and Dissertations

Mechanical forces play critical roles throughout the lives of all organisms and, as such, diverse arrays of mechanotransduction systems have evolved to detect and initiate responses to force. Many mechanotransduction systems consist of mechanosensitive (MS) ion channels, membrane pores that open in response to sufficient mechanical force. My dissertation focuses on both the study and application of force-dependent conformational changes of MS channels. I tested whether charged pore-lining residues R326 and D327 of the Arabidopsis thaliana mitochondrial MS channel MSL1 function in inward rectification or gating kinetics. Mutating these residues showed no effect on MSL1 rectification; however, these residues are …

Understanding The Molecular Mechanisms Of Photoferrotrophy And Phototrophic Extracellular Electron Uptake, Dinesh Gupta

Arts & Sciences Electronic Theses and Dissertations

Several anoxygenic phototrophs grow by utilizing soluble iron or insoluble mixed-valence iron minerals (such as rust) as electron donors to fix carbon dioxide using light energy, a process called photoferrotrophy. Photoferrotrophs can also use electron donors such as poised electrodes that serve as proxies for rust via phototrophic extracellular electron uptake (EEU). Despite the recognition that these two related microbial processes contribute to various biogeochemical cycles such as iron and carbon, the electron uptake mechanisms underlying photoferrotrophy and phototrophic EEU are poorly understood. To address the key knowledge gaps in our understanding of these microbial metabolisms, here we characterized Rhodopseudomonas …

Understanding And Exploiting Protein Allostery And Dynamics Using Molecular Simulations, Sukrit Singh

Arts & Sciences Electronic Theses and Dissertations

Protein conformational landscapes contain much of the functionally relevant information that is useful for understanding biological processes at the chemical scale. Understanding and mapping out these conformational landscapescan provide valuable insight into protein behaviors and biological phenomena, and has relevance to the process of therapeutic design.

While structural biology methods have been transformative in studying protein dynamics, they are limited by technicallimitations and have inherent resolution limits. Molecular dynamics (MD) simulations are a powerful tool for exploring conformational landscapes, and provide atomic-scale information that is useful in understanding protein behaviors. With recent advances in generating datasets of large timescale simulations …

Prodrug Activation In Staphylococci And The Implications For Antimicrobial Development, Justin J. Miller

Arts & Sciences Electronic Theses and Dissertations

Antibiotic resistance is an increasing concern for global health care, with some estimates suggesting that 10 million people will die from antibiotic resistant infections in the year 2050. Fueling this prospect, few antimicrobials are being actively developed and recently commercial entities have fled from the development of new anti-infectives. New antimicrobials and drug development strategies are urgently needed to revitalize this critical pipeline. While many putative antibiotics demonstrate promising in vitro potency, they routinely fail in vivo due to poor drug-like properties (e.g. oral bioavailability, serum-half life, toxicity) resulting in overly expensive drug development pipelines. Fortunately, drug-like properties can be …

Molecular Strategies To Overcome Antibiotic Resistance, Luting Fang

Arts & Sciences Electronic Theses and Dissertations

Antibiotics have greatly reduced the number of deaths caused by infectious diseases for the last 70 years, but as a result of overuse, antimicrobial resistance emerged. Resistance is the major reason why most traditional antibiotics currently have reduced clinical efficacy. However, there is still a drive to overcome this resistance in a variety of ways. There are two main avenues of research to overcome resistance; to discover and develop new drug candidates acting on new targets and pathways, and to determine the mechanisms of antibiotic resistance in order to combat it through multi-drug therapies, bringing back the effectiveness of antibiotics …

Thermodynamics And Conformational Heterogeneity Of Recbcd Binding To Dna Ends, Linxuan Hao

Arts & Sciences Electronic Theses and Dissertations

E. coli RecBCD is crucial in initiating repair of double stranded (ds) DNA breaks. It is a heterotrimeric helicase and nuclease complex possessing two ATPase motors, RecB and RecD, and a regulatory subunit without ATPase activity, RecC. The RecB subunit also contains a 30kDa nuclease domain (RecBNuc) that, according to published structural data, is situated over 60Å away from the site of dsDNA binding. Surprisingly, we have shown in previous studies that deletion of RecBNuc to form RecBΔNucCD affects its dsDNA unwinding properties. The mechanism by which RecBNuc influences RecBCD dsDNA unwinding is unclear. In this thesis, equilibrium binding techniques, …

Bumpy Road Ahead: Overcoming Dna Replication Obstacles One Barrier At A Time, Melanie Anne Sparks

Arts & Sciences Electronic Theses and Dissertations

DNA replication must occur efficiently and timely every cell cycle to protect the integrity of the genome. Stalled or slowed replication forks lead to replication stress that can cause replication fork collapse, and potentially genome instability. Scattered throughout the genome are tightly bound proteins, such as transcription factors, that are necessary for cell function and survival. These proteins have the potential to impede timely DNA replication. Furthermore, genomic DNA is packaged around histone octamers into structures called nucleosomes that both compact the DNA and provide an additional layer of information and regulation termed epigenetics. Thus, DNA replication is not only …

Development And Application Of Mass Spectrometry-Based Approaches For Protein Higher Order Structure Analysis And Protein-Protein Interaction Characterization, Mengru Zhang

Arts & Sciences Electronic Theses and Dissertations

Proteins, one of the most fundamental biomolecules, adopt unique higher order structures (HOS) to enable diverse biological functions. Deciphering protein HOS is crucial to gain deeper insights of their working mechanisms and to develop biotherapeutics. Mass spectrometry (MS)-based approaches evolved rapidly in the past 30 years and are now playing critical roles in protein HOS characterization. One of those approaches is MS-based footprinting whose principle is to map the solvent accessible surface area (SASA) to deliver structural information. Protein footprinting can be achieved by reversible labeling, e.g., hydrogen-deuterium exchange (HDX), and by irreversible labeling using radical-based reagents or other targeted …

Modeling Her2 Mutations In Colorectal Cancer Using A Her2 Transgenic Mouse Model And Gastrointestinal Organoids, Elisa Murray

Arts & Sciences Electronic Theses and Dissertations

Amplification or mutations in members of the epidermal growth factor receptor family, such as HER2, have been identified in several human diseases. In particular, mutations in the intracellular kinase domain have been identified in breast, colon, and lung cancers. The Cancer Genome Atlas has identified HER2 mutations or gene amplification in seven percent of colon cancer patients. These mutations are well known to promote enhanced cell growth and transformation of colon cancer cell lines. Previous studies have found HER2 mutations to confer anchorage independent growth and activation of downstream signaling pathways such as MAPK. Although HER2 mutations have been extensively …

A Mechanistic And Genomic Analysis Of Molluscum Contagiosum Virus Immune Evasion, Ian Benjamin Harvey

Arts & Sciences Electronic Theses and Dissertations

Molluscum contagiosum virus (MCV) is a common human-specific poxvirus with a proclivity for

infecting children and the immune-compromised. A characteristic MCV infection is restricted to

the epidermal layers of the skin and can persist for weeks to years in an otherwise healthy

individual. The high clinical burden of MCV is at odds with our limited knowledge regarding how

it successfully evades the human immune response, which is in part due to the lack of an animal

model or cell line to propagate the virus. Through this dissertation, we have uncovered and

characterized a novel mechanism by which MC80, a protein …

Post-Lysosomal Cholesterol Trafficking: Novel Tools And Insights, Mckenna Rae Feltes

Arts & Sciences Electronic Theses and Dissertations

Cholesterol is an essential mammalian lipid. It is a major component of cellular membranes, a precursor molecule for the synthesis of hormones and bile acids, and a regulator of protein function. Although cholesterol is synthesized, de novo, in the endoplasmic reticulum, cells principally meet cholesterol requirements through uptake of lipoprotein particles. Lipoprotein-derived cholesterol is transported to the lysosome where it is transferred from the soluble lysosomal protein, NPC2, to limiting-lysosomal membrane protein NPC1. Cholesterol is then re-distributed to other cellular membranes in order to fulfill organellar cholesterol requirements; however, the cellular machineries involved in coordinating this distribution are poorly characterized. …

The Enzymatic Function Of The Tir Domain: From Axon Degeneration To Innate Immunity, Kow Essuman

Arts & Sciences Electronic Theses and Dissertations

The Toll/Interleukin-1 Receptor (TIR) domain is an evolutionarily ancient protein domain conserved from bacteria to eukaryotes, and is an essential signaling component of innate immunity pathways. In animal innate immunity, TIR domains have primarily been described for their scaffolding function in assembling protein complexes in host defense. In plant immunity, TIR domains are key components of the intracellular Nucleotide Binding Leucine rich repeat (NLR) immune receptors that confer resistance to pathogens. These NLR receptors trigger cell death and an immune response upon activation, but their mechanism has remained elusive. In bacteria, TIR domain proteins have been suggested to function as …

Structural Mechanism Of Poxvirus Sabotage Of T-Cell Costimulation, Jabari Issa Elliott

Arts & Sciences Electronic Theses and Dissertations

Poxviruses are characterized by large double stranded DNA genomes that encode numerous proteins tailored for host immune response evasion. Our lab has been investigating a sequence-diverse family of secreted poxvirus proteins that appear to share a conserved beta-sandwich fold, but differ in their immunomodulatory functions. We have termed members of this superfamily Poxvirus Immune Evasion (PIE) proteins, and there appears to be at least 20 distinct subfamilies. As it turns out, cowpox virus (CPXV) encodes 10 PIE proteins, one of which, M2, can inhibit murine T cell activation through specific interactions with co-stimulatory ligands B7. 1 (CD80) and B7. 2 …