Biochemistry, Biophysics, and Structural Biology Commons^™

Diversification Of Muroid Rodents Driven By The Late Miocene Global Cooling, Nelish Pradhan

Graduate College Dissertations and Theses

Late Miocene, 8 to 6 million years ago (Ma), climatic changes brought about dramatic floral and faunal changes. Cooler and drier climates that prevailed in the Late Miocene led to expansion of grasslands and retreat of forests at a global scale. Palaeogeographic studies suggest a global vegetation change causing an abrupt increase in C4 plant biomass while C3 biomass decreased between 8 and 6 Ma. Subsequent cycles of cooler and drier climatic conditions during the Mid-Pliocene (3.5–3 Ma) and Pleistocene (2.8–2.5; 1.8–1.6; 1.0–0.8 Ma) also caused forests to retreat into isolated refugia which played an important role in events that …

Detection Of The Lung Environment By Multi-Drug Resistant Gram-Negative Bacterial Pathogens, Graham Geier Willsey

Graduate College Dissertations and Theses

Nosocomial lung infections are a growing concern in the United States, with more than 300,000 cases reported annually. More than 30 % of which are caused by the Gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae. Similarly, Gram-negative bacteria establish chronic infections in individuals with cystic fibrosis (CF) that are difficult or impossible to eradicate.P. aeruginosa has historically been one of the most prevalent pathogens of adults with CF. However, as antipseudomonal therapy has improved, more antibiotic resistant species have taken hold, including Stenotrophomonas maltophilia, which now colonizes more than 10 % of individuals with CF. Regardless of …

Evidence For The Involvement Of Runx1 And Runx2 In Maintenance Of The Breast Cancer Stem Cell Phenotype, Mark Fitzgerald

Graduate College Dissertations and Theses

In the United States, metastatic breast cancer kills approximately 40,000 women and 400 men annually, and approximately 200,000 new cases of breast cancer are diagnosed each year. Worldwide, breast cancer is the leading cause of cancer deaths among women. Despite advances in the detection and treatment of metastatic breast cancer, mortality rates from this disease remain high because the fact is that once metastatic, it is virtually incurable. It is widely accepted that a major reason breast cancer continues to exhibit recurrence after remission is that current therapies are insufficient for targeting and eliminating therapy-resistant cancer cells. Emerging research has …

Single Molecule Imaging Reveals Tau Structure And Function On The Microtubule Surface, Jamie Stern

Graduate College Dissertations and Theses

Neurons are among the most highly polarized cells in the human body. This polarization allows the neuron to participate in the transfer of chemical and electrical signals which are crucial to the survival of the organism. As part of polarization, each neuron develops a dendritic arbor and an axon. To ensure the survival of the cell, materials synthesized in the cell body must be trafficked through the axon for delivery throughout ultimately ending at the synaptic termini. The bulk of this cargo transport is microtubule-based fast axonal transport which is molecular motor mediated and tightly regulated though many pathways. Motor …

Selenium In Thioredoxin Reductase: Resistance To Oxidative Inactivation, Oxidation States, And Reversibility Of Chemical Reactions, Drew Barber

Graduate College Dissertations and Theses

Selenium is a required trace element which was originally discovered by the Swedish chemist Jons Jacob Berzelius in 1817. It was initially believed to be a toxin as it was identified as being the cause of hoof maladies and excessive hair loss in horses that feed upon plants with high selenium content. It wasn’t until 1957 that the potential contributions of selenium to physiology were first demonstrated. Selenium is now known to play a critical role in the maintenance of human health. Interestingly, unlike other trace metals/semi-metals, selenium is directly incorporated into proteins in the form of the amino acid …

Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network, Andrew Thomas Lombardo

Graduate College Dissertations and Theses

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a …

Secondary Functions And Novel Inhibitors Of Aminoacyl-Trna Synthetases, Patrick Wiencek

Graduate College Dissertations and Theses

The aminoacyl-tRNA synthetases are a family of enzymes involved in the process of translation, more specifically, ligating amino acids to their cognate tRNA molecules. Recent evidence suggests that aminoacyl-tRNA synthetases are capable of aminoacylating proteins, some of which are involved in the autophagy pathway. Here, we test the conditions under which E. coli and human threonyl-tRNA synthetases, as well as hisidyl-tRNA synthetase aminoacylate themselves. These reactions are ATP dependent, stimulated by Mg2+, and are inhibited by increasing cognate tRNA concentrations. These data represent the foundation for future aminoacylation experiments, specifically delving into the relationship between the autophagy pathway and the …

The A-Site In The Pkg Iα Regulatory Domain Controls Both Cgmp- And Oxidative-Dependent Activation, Jessica Lynne Sheehe

Graduate College Dissertations and Theses

The type Iα cGMP-dependent protein kinase (PKG Iα) is an essential regulator of vascular tone and systemic blood pressure. Located in the smooth muscle of resistance vessels, PKG Iα stimulates vasodilation through the phosphorylation of multiple intracellular substrates. Its primary regulator is the small molecule, 3',5'-cyclic guanosine monophosphate (cGMP); however, the Iα isoform can also be activated by oxidation. Despite the established physiological importance of PKG Iα, the structural underpinnings of these two activation mechanisms are largely unknown.

The work presented in this dissertation demonstrates the importance of the cGMP-binding domain A (CBD-A) in regulating both of these mechanisms of …

Methylation Controlled J Protein Is A Master Regulator Of Mitochondrial Metabolism, Devin Pierre Champagne

Graduate College Dissertations and Theses

Methylation controlled J protein (MCJ) is a negative regulator of mitochondrial metabolism that has a substantial impact on overall cell metabolism and function. MCJ is highly expressed by naïve CD8+ T cells, however its role in their immune effector functions was unknown. In this dissertation, it will be demonstrated that MCJ restricts the mitochondrial metabolism of CD8+ T cells, in part by reducing respiratory supercomplex formation. MCJ deficiency enhances the immune effector functions and memory responses of CD8+ T cells in a mitochondrial ATP dependent manner. As a consequence, protection to influenza virus infection is substantially improved. Reduced expression of …

21st Century Approaches To Addressing Childhood Diarrhea In Low And Middle-Income Countries: Zinc As A Cornerstone Of New Prevention Strategies, Elizabeth Ross Colgate

Graduate College Dissertations and Theses

During the 20th century, significant strides were made in curtailing the burden of childhood diarrhea, including advances in vaccine research, the advent of antibiotics, improved water and sanitation, and expanded access to health information across the globe. Despite this progress, today diarrhea ranks second only to pneumonia as a leading cause of mortality in children under five years, with a disproportionate burden of 90% of diarrheal deaths in South Asia and Sub-Saharan Africa. Additionally, substantial morbidity due to diarrhea persists in young children, with more than 45 million disability-adjusted life years (DALYs) lost due to diarrhea in 2015. Long-term consequences …

Biochemical Characterization Of The Dimerization Domain Of Purine-Rich Element Binding Protein B: An Essential Subdomain Mediating The Repression Of Smooth Muscle Alpha Actin Gene Expression, Lauren Ferris

Graduate College Dissertations and Theses

A number of physiologic processes require the expression of smooth muscle alpha actin (SMαA) to mediate cellular contraction. Stable expression of SMαA in differentiated vascular smooth muscle cells is associated with a contractile phenotype that is essential for regulation of blood flow and pressure. The transient expression of SMαA in myofibroblasts during wound repair facilitates wound closure. Hence, it is no surprise that dysregulation of SMαA gene expression in both cell types can have pathological consequences. Indeed, aberrant SMαA gene regulation has been implicated in diseases such as atherosclerosis and fibrosis. Therefore, a better understanding of the molecular mechanisms that …

Rumen Microbial Ecology And Rumen-Derived Fatty Acids: Determinants Of And Relationship To Dairy Cow Production Performance, Laura Marie Cersosimo

Graduate College Dissertations and Theses

Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge …

Molecular Mechanisms Of Mrna Transport By A Class V Myosin And Cytoplasmic Dynein, Thomas Edward Sladewski

Graduate College Dissertations and Theses

mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. Using a single molecule in vitro approach, we provide insight into the mechanisms by which localizing mRNAs are carried by molecular motors on cytoskeletal tracks to their destination.

Budding yeast serves as a model system for studying the mechanisms of mRNA transport because localizing mRNAs are moved on actin tracks in the cell by a single class V myosin motor, Myo4p. Molecular motors that specialize in cargo transport are generally double-headed so that they can "walk" for many microns without dissociating, a feature known as processivity. …

The Role Of Inflammasomes In Asbestos-Induced Mesothelial To Fibroblastic Transition, Joyce K. Thompson

Graduate College Dissertations and Theses

Malignant Mesothelioma (MM) is a fatal disease with a low median survival between 8 to 12 months after diagnosis. MM has a long latency period (10-60 years), is causally related to asbestos exposure, and is refractory to all available modes of therapy. Despite the causal association between asbestos exposure and MM however, the mechanisms by which asbestos induces this deadly disease remain unclear. Chronic inflammation due to the presence of asbestos fibers is believed to play an important role in all aspects of MM pathogenesis, from development to progression and resistance. Chronic inflammation has been shown to promote dysregulated wound …

Myo2 Motor Function In The Contractile Ring And The Regulation Of Fission Yeast Cytokinesis, Luther Woodrow Pollard

Graduate College Dissertations and Theses

Animals, fungi, and amoebas require an actomyosin contractile ring at the division site to perform cytokinesis. The contractile ring initiates and guides the invagination of the plasma membrane as it forms new barriers between the nuclei at the cell equator. Defects in the contractile ring can result in misdirected, delayed, or premature cytokinesis, which leads to abnormal chromosome numbers. Aneuploidies resulting from failed cytokinesis sometimes lead to aggressive forms of cancer. This dissertation was motivated by the goal of better understanding the properties of the contractile ring and how it drives cytokinesis.

Actomyosin is initially recruited to the cell equator …

Target Identification Strategies For Mmv Malaria Box Inhibitors Of Toxoplasma Gondii Growth, Jenna Elizabeth Foderaro

Graduate College Dissertations and Theses

Small molecule screening is commonly used to discover lead compounds for drug development, but it can also be a powerful way to identify chemical probes for studying biological mechanisms. Our lab uses small molecules to study the mechanisms by which the protozoan parasite Toxoplasma gondii infects and replicates within its hosts. In this work, we employed a fluorescence-based assay to screen the Medicines for Malaria Venture (MMV) Open Access Malaria box for compounds that affect T. gondii growth. The box contains 400 previously identified small-molecule inhibitors of the related parasite, Plasmodium falciparum. We identified 79 hits, including a 2,4-diaminoquinazoline (MMV006169; …

Understanding The Role Of Runx2 In A Breast Cancer Progression Cell Model, Alexandra Ojemann

Graduate College Dissertations and Theses

Runx2 is a transcription factor required for bone formation and osteoblastic differentiation during normal development and is implicated in metastatic disease during breast cancer progression. Runx2 is highly expressed in many metastatic breast cancers and breast cancer cell lines Knockdown of Runx2 in various breast cancer cell lines restores epithelial characteristics and reduces proliferation, migration, and invasion. However, the role of Runx2 in breast cancer progression from early to late stages is not well understood. The MCF10A derived breast cancer progression model provides the opportunity to study the role of Runx2 in a series of cell lines that progress from …

Changes In Threonyl-Trna Synthetase Expression And Secretion In Response To Endoplasmic Reticulum Stress By Monensin In Ovarian Cancer Cells, Jared Louis Hammer

Graduate College Dissertations and Theses

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum …

Silica Nanoparticles For The Delivery Of Dna And Rnai In Cancer Treatment, Michael Aaron Vrolijk

Graduate College Dissertations and Theses

DNA and interfering RNA (RNAi) – short interfering RNA (siRNA) and micro RNA (miRNA) – are promising new cancer therapies, especially for drug resistant lines. However, they require a delivery system in vivo to prevent degradation and off target effects. Silica based nanoparticles, both solid and mesoporous, are a promising option due to their biocompatibility, ease of preparation and morphology control, reproducibility, and facile addition of functional groups including targeting ligands.

After a brief introduction to cancer treatment and review of the current nanoparticle treatments undergoing clinical trials, this thesis details the many methods explored over the past ten years …

The Biochemical Characterization Of Human Histidyl-Trna Synthetase And Disease Associated Variants, Jamie Alyson Abbott

Graduate College Dissertations and Theses

Human histidyl-tRNA synthetase (HARS) is an aminoacyl-tRNA synthetase (AARS) that catalyzes the attachment of the amino acid histidine to histidyl-tRNA (tRNAHis) in a two-step reaction that is essential for protein translation. Currently, two human diseases, Usher Syndrome IIIB (USH3B) and an inherited peripheral neuropathy, Charcot Marie Tooth Syndrome (CMT), have been linked genetically to single point mutations in the HARS gene. The recessive HARS USH3B mutation encodes an Y454S substitution localized at the interface between the anticodon-binding domain and the catalytic domain of the opposing subunit. Patients with Usher Syndrome IIIB lose their sight and hearing during their second decade …

Functional And Mechanistic Consequences Of Dual Oxidase 1 Suppression In Lung Cancer, Andrew Charles Little

Graduate College Dissertations and Theses

The NADPH oxidase homolog, dual oxidase 1 (DUOX1), is an H2O2 producing transmembrane enzyme highly expressed in the airway epithelium. DUOX1-dependent redox signaling has been characterized to regulate many homeostatic processes in the lung epithelium, such as host defense, wound healing, and type II immune responses. Intriguingly, DUOX1 has been found to be suppressed in many epithelial cancers, including lung cancer, by hypermethylation of its promoter. Epigenetic silencing of DUOX1 in cancer is paradoxical to the understanding that tumors harbor elevated levels of reactive oxygen species (ROS), suggesting that DUOX1 may be a tumor suppressor.

Since DUOX1 loss occurs in …

Barcoding The Actin Track: Differential Regulation Of Myosin Motors By Tropomyosin, Joseph Emerson Clayton

Graduate College Dissertations and Theses

Myosins and tropomyosins represent two types of actin filament-associated proteins that often work together in contractile and motile processes in the cell. While the role of thin filament troponin-tropomyosin complexes in regulating striated muscle myosin II is well characterized, the role of tropomyosins in non-muscle myosin regulation is not well understood. Fission yeast has recently proved to be a useful model with which to study regulation of myosin motors by tropomyosin owing to its tractable genetics, well-defined actin cytoskeleton, and established actin biochemistry.

A hallmark of type V myosins is their processivity -- the ability to take multiple steps along …

Functional Consequences Of Ama1-Ron2 Interaction During Host Cell Invasion By Toxoplasma., Shruthi Krishnamurthy

Graduate College Dissertations and Theses

T.gondii is a model organism of the phylum Apicomplexa that infects one third of the human population. While the majority of infections are asymptomatic or manifest with mild flu-like symptoms, toxoplasmosis can be fatal in immunocompromised individuals and in the developing fetus. The lytic cycle of tachyzoite-stage parasites causes damage to the host by repeated rounds of host cell invasion, intracellular replication and lysis of the host cell upon egress.

Invasion is a key step for the parasite to maintain its intracellular lifestyle. Apical Membrane Antigen 1 (AMA1) is an adhesin released from a unique set of secretory organelles called …

Novel Mechanisms Governing Autoregulation Of The Src Family Kinase Fyn And Its Crosstalk With Protein Kinase A, Marion Weir

Graduate College Dissertations and Theses

ABSTRACT

Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that …

Epicardial Cell Engraftment And Signaling Promote Cardiac Repair After Myocardial Infarction, Krithika Rao

Graduate College Dissertations and Theses

The epicardium is a single layer of epithelial (mesothelial) cells that covers the entire heart surface, but whose function in adult mammals is poorly understood. Defining the role of epicardial cells during homeostasis, growth and injury has potential to provide new treatment strategies for human diseases that result in heart failure, due to extensive loss of viable cardiac tissue. We hypothesized that epicardial cells contribute to repair as transplantable progenitor cells for cellular regeneration and as a source of secreted growth factors for cell protection after myocardial infarction.

Adult epicardial cells were prospectively isolated as uncommitted epithelial cells using epithelial-specific …

The Role Of Kinesin-2 In Navigating Microtubule Obstacles: Implications For The Regulation Of Axonal Transport, Gregory Hoeprich

Graduate College Dissertations and Theses

Neurons are specialized cells that transmit information through electrical and chemical signals using structural processes known as dendrites and axons. Dendrites receive information for the cell to interpret while the exceedingly long axon transmits the processed information to its target destination. To ensure the neuron properly carries out its extracellular functions, the orchestration of intracellular cargo (e.g. mitochondria) is critical. This is especially true in the axon, which can be up to a meter in length. There are many challenges involved in the spatial and temporal regulation of cargo over such vast cellular distances. In order to accomplish cargo transport …

The Role Of Src Kinase Activation In Lung Epithelial Alterations In Response To The A,B-Unsaturated Aldehyde Acrolein, Robert Bauer

Graduate College Dissertations and Theses

Cigarette smoke (CS) exposure is the leading cause of preventable death in the United States contributing to over 480,000 deaths a year with over 300 billion dollars in CS related costs spent per year. While the dangers of CS exposure have been studied and characterized for decades being largely attributed to reactive oxygen species and oxidative stress, increasing evidence suggests that reactive aldehydes in CS, specifically the α,β-unsaturated aldehyde acrolein, are responsible for many of the negative pathologies associated CS exposure. Previous work has shown that acrolein can bind directly to a number of cellular proteins containing redox sensitive cysteine …

Mechanisms And Dynamics Of Oxidative Dna Damage Repair In Nucleosomes, Wendy J. Cannan

Graduate College Dissertations and Theses

DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of …

Defining Platelet-Derived Components Regulating The Prothrombinase Enzyme Complex, Francis Ayombil

Graduate College Dissertations and Theses

At sites of vascular injury, the critical blood clotting enzyme thrombin is generated from prothrombin via Prothrombinase, a macromolecular, Ca2+-dependent enzymatic complex consisting of the serine protease factor Xa and the non-enzymatic cofactor factor Va, assembled on the membranes of activated platelets. Platelets regulate thrombin formation by providing specific binding sites for the components of Prothrombinase and by releasing a platelet-derived factor V/Va molecule that is more procoagulant than its plasma counterpart and partially resistant to proteolytic inactivation. This dissertation identifies and characterizes the subpopulation of platelet-derived factor V/Va that is responsible for the observed protease resistance, and the mechanism …

Dna Glycosylases Remove Oxidized Base Damages From G-Quadruplex Dna Structures, Jia Zhou

Graduate College Dissertations and Theses

The G-quadruplex DNA is a four-stranded DNA structure that is highly susceptible to oxidation due to its G-rich sequence and its structure. Oxidative DNA base damages can be mutagenic or lethal to cells if they are left unrepaired. The base excision repair (BER) pathway is the predominant pathway for repair of oxidized DNA bases. DNA glycosylases are the first enzymes in BER and are responsible for removing base lesions from DNA. How DNA glycosylases remove base lesions from duplex and single-stranded DNA has been intensively studied, while how they act on G-quadruplex DNA remains to be explored.

In Chapter II …