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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 …

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, …

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 …

Rnai-Mediated Gene Silencing In The Exotic Redbay Ambrosia Beetle, Xyleborus Glabratus, And Insect-Fungal Interactions Within The Laurel Wilt Complex, Morgan Christine Knutsen

Theses and Dissertations--Entomology

Laurel wilt disease (LWD) is a lethal vascular disease impacting lauraceous hosts caused by Harringtonia lauricola, the fungal symbiont of the redbay ambrosia beetle (Xyleborus glabratus Eichoff) (RAB) (Coleoptera: Curculionidae). LWD has caused tree mortality throughout the southeastern United States and is continuing to spread into new regions. Current management methods have not been successful in preventing spread, warranting investigation into innovative techniques including RNA interference (RNAi).

Elongation factor-1 alpha (ef1a) and actin (act) were established as stably expressed reference genes after exposing beetles to different photoperiod, temperature, and dsRNA exposure. After RAB oral …

Development And Biological Evaluation Of Selective Small-Molecule Inhibitors Of The Human Cytochrome P450 1b1, Austin Hachey

Theses and Dissertations--Chemistry

The human cytochrome P450 1B1 (CYP1B1) is an emerging target for small- molecule therapeutics. Several solid tumors overexpress CYP1B1 to the degree that it has been referred to as a universal tumor antigen. Conversely, its expression is low in healthy tissues. CYP1B1 may drive tumorigenesis through promoting the formation of reactive toxins from environmental pollutants or from endogenous hormone substrates. Additionally, the expression of CYP1B1 in tumors is associated with resistance to several common chemotherapies and with poor prognoses in cancer patients. However, inhibiting CYP1B1 with small molecules has been demonstrated in cellular and murine model systems to reverse this …

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 …

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 …

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, …

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 …

The Tumor Suppressor Par-4 Regulates Hypertrophic Obesity, Nathalia Araujo

Theses and Dissertations--Toxicology and Cancer Biology

Prostate Apoptosis Response-4 (Par-4) is a tumor suppressor ubiquitously expressed in all tissues and able to selectively induce apoptosis in cancer cells. Although well established in the context of cancer, relatively little is known about the function of Par-4 in the healthy and non-tumorigenic context. Observations from our lab showed that Par-4 knockout mouse lines were obese and displayed adipocyte hypertrophy under a normal chow diet when compared to Par-4 wild-type mice. These Par-4 knockout mice exhibited hepatic steatosis and hyperinsulinemia as secondary consequences of obesity. Par-4 knockout mice displayed increased intestinal dietary fat absorption and its subsequent storage in …

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 …

Abc Transporters In Glioblastoma: Anticancer Drug Transport And Transporter Regulation At The Blood-Brain Barrier, Julia A. Schulz

Theses and Dissertations--Pharmacy

Glioblastoma is one of the deadliest cancers, with a median survival of only one year. Even after aggressive treatment consisting of surgical resection, radiation, and chemotherapy, most glioblastoma patients suffer from tumor recurrence within 6-9 months. One reason for treatment failure of anticancer drugs is the blood-brain barrier that protects the brain by impeding xenobiotic uptake from the blood. To this end, efflux transporters at the human blood-brain barrier, such as P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2), prevent many compounds, including anticancer drugs, from entering the brain. Thus far, approaches to deliver anticancer drugs across the blood-brain barrier …

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 …

Leveraging Chemical And Computational Biology To Probe The Cellulose Synthase Complex, B. Kirtley Amos

Theses and Dissertations--Plant and Soil Sciences

Cellular expansion in plants is a complex process driven by the constraint of internal cellular turgor pressure by an expansible cell wall. The main structural element of the cell wall is cellulose. Cellulose is vital to plant fitness and the protein complex that creates it is an excellent target for small molecule inhibition to create herbicides. In the following thesis many small molecules (SMs) from a diverse library were screened in search of new cellulose biosynthesis inhibitors (CBI). Loss of cellular expansion was the primary phenotype used to search for putative CBIs. As such, this was approached in a forward …

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: …

Epigenetic Regulation Of Hormone Action In The Red Flour Beetle, Tribolium Castaneum (Herbst), Smitha George

Theses and Dissertations--Entomology

Hormones are the chemical communication signaling molecules released into the body fluid to stimulate target cells of multicellular organisms. Two major hormones, ecdysteroids (20-hydroxyecdysone, 20E) and juvenile hormones (JH), regulate a wide variety of physiological and developmental processes in insects. Therefore, hormones have been extensively studied and are attractive targets for the development of target-specific insect control methods. Recent studies suggest that epigenetics adds another layer of regulation to explain multiple functions of the same circulating hormone in different tissues and at various time points. In my dissertation, I focused on a major post-translational modification, ‘acetylation,’ to elucidate the epigenetic …

Pneumovirus Infections: Understanding Rsv And Hmpv Entry, Replication, And Spread, Jonathan T. Kinder

Theses and Dissertations--Molecular and Cellular Biochemistry

Pneumoviruses including human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are significant causes of respiratory tract infections globally. Children, elderly, and immunocompromised patients are at the greatest risk for developing severe infections, which can have devastating outcomes. Although these viruses are ubiquitous with significant impacts on human health, there are no antivirals or vaccines available. The only FDA approved therapy is a monoclonal antibody for RSV, given prophylactically during the infectious season, and this treatment is only available for high risk infants. The work presented in this thesis aims to increase our understanding of how these viruses enter, replicate, and …

Epigenetic Implications In Inorganic Arsenic-Mediated Carcinogenesis, Meredith Eckstein

Theses and Dissertations--Molecular and Cellular Biochemistry

Chronic, low dose exposure to inorganic arsenic (iAs) is a public health concern throughout the world, contributing to the development of many diseases, including lung cancer. Several mechanisms for iAs-mediated carcinogenesis have been proposed, of which the production of reactive oxygen species and formation of chromosomal aberrations are the most studied. Another equally important, yet less studied mechanism is dysregulation of epigenetic marks. “Epigenetics” refers to changes that occur on the DNA and chromatin that do not alter base pair identity, but alter compaction, expression, and regulation of specific DNA sequences. There are several types of epigenetic marks including histone …

Novel Post-Translational Modification And Function Of Fus: The Relevance To Amyotrophic Lateral Sclerosis, Alexandra Arenas

Theses and Dissertations--Toxicology and Cancer Biology

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in Sarcoma (FUS) is a ubiquitously expressed RNA binding protein implicated in familial ALS and frontotemporal dementia (FTD). FUS is ubiquitously expressed in cells and has a variety of functions in the nucleus and cytoplasm. FUS mutations in the nuclear localization sequence (NLS) causes mislocalization of FUS in the cytoplasm, where it can undergo liquid-liquid phase separation and become stress granules or protein inclusions. Although FUS inclusion bodies can be found in …

The Balancing Act Of Cytokinin In Environmental Stress Tolerance, Sumudu Sandeepani Karunadasa

Theses and Dissertations--Plant and Soil Sciences

Cytokinin, long known as a phytohormone that regulates plant growth and development, has been recently recognized as an important regulator of stress responses. However, our current knowledge about the mechanisms by which cytokinin regulates stress responses is fragmentary, as many of the studies in this field yielded conflicting results. Most of the work described here has focused on analyses of the molecular mechanisms of cytokinin-dependent regulation of growth and development under stress conditions, with an emphasis on the role of cytokinin-dependent regulation of protein synthesis in development and stress tolerance.

One of the important contributions of this study is the …

Characterization And Enzyme Engineering Of Laccases Towards Lignin Valorization In Aqueous Ionic Liquids, Joseph Stevens

Theses and Dissertations--Biosystems and Agricultural Engineering

Lignin is one of the most abundant polymers found in nature, making up 15 – 40% of the weight of terrestrial biomass. Due to the structural and monomeric heterogeneity of lignin, it is recalcitrant thermochemical and biological valorization methods. Converting lignin to value-added products via sustainable and cost-effective pathways will reduce waste and add value to future cellulosic biorefineries. Biological methods for lignin valorization (e.g. lignin degrading enzymes or microbes) is limited by low lignin solubility in biocompatible solvents, resulting in low product yield. Recent reports on biocatalysts for lignin valorization have focused on the lignolytic multicopper oxidase laccase, …

The Functional Role Of Rna Binding Protein Rbms3 As A Tumor Promoter In Triple-Negative Breast Cancer Cells, Yuting Zhou

Theses and Dissertations--Molecular and Cellular Biochemistry

RBMS3 belongs to the family of c-myc gene single-strand binding proteins (MSSPs) that play important roles in transcriptional regulation. Here, we show that RBMS3 functions as a tumor promoter in triple-negative breast cancer (TNBC), a highly aggressive BC subtype. Analysis of RBMS3 expression shows that RBMS3 is upregulated at both mRNA and protein levels in TNBC cells. Functionally, overexpression of RBMS3 increases cell migration, invasion and cancer stem cell (CSC) behaviors. Moreover, RBMS3 induces expression of epithelial-mesenchymal transition (EMT) and CSC markers. Conversely, loss of RBMS3 in TNBC BT549 cells inhibits cell proliferation, migration and mesenchymal phenotype. Correlation analysis shows …

Mechanisms Of Trinucleotide Repeat Instability During Dna Synthesis, Kara Y. Chan

Theses and Dissertations--Toxicology and Cancer Biology

Genomic instability, in the form of gene mutations, insertions/deletions, and gene amplifications, is one of the hallmarks in many types of cancers and other inheritable genetic disorders. Trinucleotide repeat (TNR) disorders, such as Huntington’s disease (HD) and Myotonic dystrophy (DM) can be inherited and repeats may be extended through subsequent generations. However, it is not clear how the CAG repeats expand through generations in HD. Two possible repeat expansion mechanisms include: 1) polymerase mediated repeat extension; 2) persistent TNR hairpin structure formation persisting in the genome resulting in expansion after subsequent cell division. Recent in vitro studies suggested that a …

Automatic 13C Chemical Shift Reference Correction Of Protein Nmr Spectral Data Using Data Mining And Bayesian Statistical Modeling, Xi Chen

Theses and Dissertations--Molecular and Cellular Biochemistry

Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins. However, due to the intrinsic properties of NMR experiments, results from the NMR instruments require a refencing step before the down-the-line analysis. Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR. There is no available method that can rereference carbon chemical shifts from protein NMR without secondary experimental information such as structure or resonance assignment.

To solve this problem, we …

Biochemical Approaches For The Diagnosis And Treatment Of Lafora Disease, Mary Kathryn Brewer

Theses and Dissertations--Molecular and Cellular Biochemistry

Glycogen is the sole carbohydrate storage molecule found in mammalian cells and plays an important role in cellular metabolism in nearly all tissues, including the brain. Defects in glycogen metabolism underlie the glycogen storage diseases (GSDs), genetic disorders with variable clinical phenotypes depending on the mutation type and affected gene(s). Lafora disease (LD) is a fatal form of progressive myoclonus epilepsy and a non-classical GSD. LD typically manifests in adolescence with tonic-clonic seizures, myoclonus, and a rapid, insidious progression. Patients experience increasingly severe and frequent epileptic episodes, loss of speech and muscular control, disinhibited dementia, and severe cognitive decline; death …

Toward An Enzyme-Coupled, Bioorthogonal Platform For Methyltransferases: Probing The Specificity Of Methionine Adenosyltransferases, Tyler D. Huber

Theses and Dissertations--Pharmacy

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to …

Mutations Of Fus Cause Aggregation Of Rna Binding Proteins, Disruptions In Protein Synthesis, And Dysregulation Of Nonsense Mediated Decay, Marisa Elizabeth Kamelgarn

Theses and Dissertations--Toxicology and Cancer Biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the …

Host Restriction Factors In The Replication Of Tombusviruses: From Rna Helicases To Nucleocytoplasmic Shuttling, Cheng-Yu Wu

Theses and Dissertations--Plant Pathology

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication.

Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment.

In addition, the features rendering the abundant plant DEAD-box helicases either …

Functional Characterization Of Scaffold Protein Shoc2, Hyein Jang

Theses and Dissertations--Molecular and Cellular Biochemistry

Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear.

The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this …