Digital Commons Network^™

Mechanistic Analysis Of Four-Way Dna Junctions And Cytokine-Binding Aptamers For Therapeutic Interventions, Roaa S. Mahmoud

Theses and Dissertations

DNA is inherently dynamic and topologically diverse and can fold into many different structures. Besides the canonical Watson-Crick structure, other higher-order structures such as G-quadruplexes (G4), i-motifs (iM), and four-way DNA junctions are possible. Although these high-order DNA structures are known to form transiently, they are important due to the crucial roles they play in many cellular processes including DNA replication, recombination, and repair. Among these DNA structures, 4-way junctions (also known as Holliday junctions, HJ) which are formed during the repair of double-strand DNA breaks (DSBs) and interact with proteins have garnered significant attention due to their central role …

Protacs – A Novel And Rapidly Developing Field Of Targeted Protein Degradation, Hannah R. Gatley

Theses and Dissertations

There is a continued need for new technology and strategies for tackling cancer and other diseases, and within the current century a novel therapeutic strategy has emerged in the realm of targeted protein degradation called Proteolysis-Targeting Chimeras (PROTACs). This technology specifically targets and degrades disease-causing proteins via the ubiquitin-proteasome system, and has seen an explosion of research and intrigue in both academia and industry over the past two decades. The diversity of PROTAC classes based on the E3 ligase recruiting ligand and the target protein allows for a universal molecular structure that can be customized for a specific target and …

Treponema Denticola Synthesizes C-Di-Amp And Encodes The Cdaa-Type Diadenylate Cyclase Cdaa, Claire R. O'Brien

Theses and Dissertations

Periodontitis is a form of oral disease characterized by dysbiosis of the oral microbiome, leading to inflammation, bone resorption, and in severe cases, entire tooth loss, affecting 42% of adults in the US. One of the bacteria most associated with periodontal disease progression is Treponema denticola (Td), an oral spirochete which inhabits the mouth in small quantities during health but which can dominate the biofilms that form during periodontal disease. The ability of Td to survive in a disease environment and contribute to the progression of disease requires the use of robust signaling networks. Analysis of Td cultures …

The Type Iv Pilus Secretin Bfpb: Structural Analysis And Binding Interactions, Janay I. Little

Theses and Dissertations

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. The type IV pilus (T4P) of EPEC, known as the bundle-forming pilus (BFP), plays an important role in EPEC pathogenesis. T4Ps are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. One essential component of the BFP system is the secretin, BfpB. Secretins are a large family of integral outer membrane proteins found in T4Ps as well as type II and type III secretion systems, and filamentous phages. Details of the secretin structure have been limited to the overall …

The Role Of Irf-1 In Spontaneous Mouse Glioma, Aakash B. Vaidya

Theses and Dissertations

Glioblastoma Multiforme has been shown to be one of the deadliest primary brain cancers. One of the reasons why GBM is so deadly, is a unique immunosuppressive tumor microenvironment that promotes GBM growth and progression. Both astrocyte and microglia have been implicated in immunosuppression. In this study, we explored the role of Interferon Regulatory Factor 1 (IRF-1) in astrocytes and glioma cells on the growth of spontaneous glioma tumors. IRF-1 is regulated by the JAK/STAT pathway and induces expression of Programmed death ligand 1 (PD-L1). PD-L1 downregulates immune responses to glioma. We found that IRF-1 had no effect on spontaneous …

Protein Structure And Interaction: The Role Of Aromatic Residues In Protein Structure And Interactions Between Pyridoxine 5'-Phosphate Oxidase/Dopa Decarboxylase, Mohammed H. Al Mughram

Theses and Dissertations

Naturally developed proteins are capable of carrying out a wide variety of molecular functions due to their highly precise three-dimensional structures, which are determined by their genetically encoded sequences of amino acids. A thorough knowledge of protein structures and interactions at the atomic level will enable researchers to get a deep foundational understanding of the molecular interactions and enzymatic processes required for cells, resulting in more effective therapeutic interventions. This dissertation intends to use structural knowledge from solved protein structures for two distinct objectives.

In the first project, we conducted a bioinformatics structural analysis of experimental protein structures using our …

Targeting Bcl-2 Family Proteins In Therapy Induced Senescent Cancer Cell Models, Wade Cook

Theses and Dissertations

Non-small Cell Lung Cancer (NSCLC) originates from numerous different cell types in the lungs and is among the deadliest of cancers. Head and Neck Squamous Cell Carcinomas (HNSCC) are derived from the mucosal membranes of the oral cavity, pharynx, and larynx. Both NSCLC and HNSCC are predominately caused by tobacco smoke inhalation and as such mutations in the tumor suppressor gene TP53 are common. Since similarities exist in the root cause of NSCLC and HNSCC, they may also share similarities in treatment methods. Cisplatin is a platinum-based DNA damaging agent that has been used as a cancer chemotherapy for decades. …

Structure-Based Drug Discovery And Development Of Protein Structure Prediction Tools Using An Empirical Force Field, Noah B. Herrington

Theses and Dissertations

Traditional drug discovery has rapidly accelerated thanks to development of computational molecular modeling. The crucial component that these computational studies hinge upon is having a well-defined, and energetically favorable structure. Structures of proteins and ligands that meet these criteria are important for accurately simulating models used to study drug binding. To demonstrate the role of accurate structure simulation in the study of these events, this thesis presents, first, a story examining the problem of accurate structure modeling of ionizable residues within protein structures, specifically aspartic acid, glutamic acid, and histidine. I present our method, which uses the HINT force field …

Quantitative Pcr And Sanger Sequencing Of Mitochondrial Dna Recovered From Waterlogged Bone, Kailey Babcock

Theses and Dissertations

In forensic contexts, samples containing heavily fragmented DNA are commonly encountered. Compromised biological samples are especially prevalent in instances where human remains have been submerged in an aqueous environment for extended periods of time. Nuclear DNA is particularly vulnerable to the prolonged exposure to heat, moisture, and bacterial degradation that are prevalent in aquatic settings. Paired with the difficulty of recovering DNA from skeletal remains, which are often the only remaining component after the soft tissues have been stripped away, mitochondrial DNA (mtDNA) analysis serves as an invaluable alternative. In this multifaceted study, mtDNA analysis was performed on waterlogged bone …

Development And Evaluation Of A Combinatorial Rt-Qpcr Multiplex For Forensic Body Fluid Identification, Carolyn A. Lewis

Theses and Dissertations

Body fluid identification is essential in the forensic biology workflow that assists DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the sensitivity and specificity molecular techniques can achieve; therefore, molecular methods, such as microRNA and microbial signatures, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance. A disadvantage of RNA analysis in forensic …

Influence Of Metal Sleeves In The Accuracy Of Dental Implant Placement Using Guided Implant Surgery, Coleman Adams

Theses and Dissertations

The fabrication of implant surgical guides through stereolithographic 3D printing has become a staple in dental implant guided surgery over the last couple decades. These surgical guides have typically utilized metal sleeves to assist in guidance of the drills during osteotome preparation. The metal sleeves can be costly and potentially cause deviations if improperly placed during post-processing of the guide. This research explored a novel method for the utilization of sleeve-free surgical guides by comparing the dimensional and angulational deviations between the implant guides with and without a metal sleeve. To achieve this goal, two separate aims were pursued. Our …

Computational Analysis And Prediction Of Intrinsic Disorder And Intrinsic Disorder Functions In Proteins, Akila I. Katuwawala

Theses and Dissertations

COMPUTATIONAL ANALYSIS AND PREDICTION OF INTRINSIC DISORDER AND INTRINSIC DISORDER FUNCTIONS IN PROTEINS

By Akila Imesha Katuwawala

A dissertation submitted in partial fulfillment of the requirements for the degree of Engineering, Doctor of Philosophy with a concentration in Computer Science at Virginia Commonwealth University.

Virginia Commonwealth University, 2021

Director: Lukasz Kurgan, Professor, Department of Computer Science

Proteins, as a fundamental class of biomolecules, have been studied from various perspectives over the past two centuries. The traditional notion is that proteins require fixed and stable three-dimensional structures to carry out biological functions. However, there is mounting evidence regarding a “special” class …

Muscarinic Excitation Of Dopamine Neurons In The Ventral Tegmental Area Via Activation Of A Trpc-Like Cation Conductance, Yu Tzu Chen

Theses and Dissertations

Dopaminergic (DA) neurons in the ventral tegmental area (VTA) play a crucial role in reward and motivational behaviors, including the development of drug addictions. VTA DA neurons receive excitatory cholinergic inputs from the mesopontine tegmentum. Blockage of the M5 muscarinic receptor in DA neurons has been shown to attenuate drug-induced DA release and abuse-related behaviors, but the molecular mechanism is unknown. In this study, experiments were designed to identify the electrophysiological effects of muscarinic agonism in the modulation of action potential kinetics and firing patterns in VTA DA neurons of mice. Pharmacology of the muscarinic receptor-evoked current was also characterized. …

Interclass Gpcr Heteromerization Affects Localization And Trafficking, Rudy Toneatti

Theses and Dissertations

Class A serotonin (5-hydroxytryptamine) 2A (5-HT_2AR) and class C metabotropic glutamate 2 receptors (mGluR2) are seven transmembrane receptors (7TMRs or G protein-coupled receptors – GPCRs) involved in multiple neuropsychiatric disorders including schizophrenia. Previous findings from our laboratory reported that 5-HT_2AR and mGluR2 are dysregulated in the prefrontal cortex of patients suffering from this psychiatric condition, although 5-HT_2AR’s expression was recovered in antipsychotic-medicated patients. Genome-wide association studies on schizophrenia reported that endosomal trafficking that regulates cell surface abundance of another 7TMR implicated in this disease (dopamine D2 receptor) can be altered. Ligand-activated receptors, including the …

Single Molecule Investigations Of Holliday Junction Binding Protein Ruva, Dalton Reed Gibbs

Theses and Dissertations

DNA breaks are inevitable as they mainly occur due to cells’ own reactive oxygen species (ROS). While DNA breaks can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired, it can lead to genetic instability and serious health issues including cancers. One way dsDNA breaks can be repaired is via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA itself and DNA …

A Mechanism Behind The Mechanotransduction Of Surface Characteristics In Osteoblasts, Otto J. Juhl Iv

Theses and Dissertations

Biomaterials for use in bone regeneration and healing range from metal and metal alloy implants to hydrogel-based solutions. These materials can be optimized to increase bone healing and integration by improving the mechanical and biological properties. Regardless of the material itself, the cell-substrate interaction is key to the success of the biomaterial once implanted. Substrate surface characteristics such as roughness, wettability, and particle density are well-known contributors to a substrate’s overall osteogenic potential, and therefore the substrate's overall success. Unfortunately, it is still unknown how these substrate surface characteristics are transduced into intracellular signals by cells, preventing specific tailoring of …

A Novel Review Of Heat Shock Protein 110 Kda: A Basis For Research And Continued Experimentation Through Biochemical Analysis, Crist W. Cuffee

Theses and Dissertations

Heat shock protein 110 kDa, Hsp110, is a distinct cellular protector, different in form and function from Hsp70, a close relative of Hsp110. Functioning primarily as a holdase or in tandem with other molecular chaperones, a review of current accomplishments elucidates the uniqueness of this protein and the continued mysteries that surrounds it. Found only in eukaryotes, Hsp110 has been linked to many diseases, ranging from parasitic infection to neurodegenerative disorders. While still lacking, studies of this protein have provided much in the realm of speculation on the mechanisms behind Hsp110s’ contribution to different pathologies. This review will serve as …

The Impact Of Aging And Mechanical Injury On Alveolar Epithelial And Macrophage Responses In Acute Lung Injury And Inflammation, Michael S. Valentine

Theses and Dissertations

Patients with severe lung pathologies, such as Acute Respiratory Distress Syndrome (ARDS), often require mechanical ventilation as a clinical intervention; however, this procedure frequently exacerbates the original pulmonary issue and produces an exaggerated inflammatory response that potentially leads to sepsis, multisystem organ failure, and mortality. This acute lung injury (ALI) condition has been termed Ventilator-Induced Lung Injury (VILI). Alveolar overdistension, cyclic atelectasis, and biotrauma are the primary injury mechanisms in VILI that lead to the loss of alveolar barrier integrity and pulmonary inflammation. Stress and strains during mechanical ventilation are believed to initiate alveolar epithelial mechanotransduction signaling mechanisms that contribute …

Characterization Of The Dyrk1a Protein-Protein Interaction Network, Varsha Ananthapadmanabhan

Theses and Dissertations

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is a protein kinase encoded by a dosage-dependent gene. An extra copy of DYRK1A contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe mental retardation and autism. DYRK1A is involved in phosphorylation of several proteins that regulate cell cycle control and tumor suppression. However, the function and regulation of this kinase is not well understood and current knowledge does not fully explain dosage-dependent function of this important kinase. Our previous proteomic studies identified several novel DYRK1A interacting proteins including RNF169, FAM117B, TROAP, LZTS1, LZTS2 and DCAF7. In this …

Characterization Of The Tsc/Dyrk1a Interaction, Supriya Joshi

Theses and Dissertations

The Tuberous sclerosis complex (TSC) includes TSC1, TSC2 and the TBC1D7 subunits that together function as a principal inhibitor of the mTOR protein kinase complex 1 (mTORC1). mTORC1 is a master regulator of cell growth and proliferation that responds to signaling cues such as growth factors and nutrient availability. Proteomic studies in our lab revealed an interaction between the TSC subunits and DYRK1A, a ubiquitous protein kinase encoded by a gene located in the Down syndrome (DS) region on human chr21. In this study, we sought to validate the interaction of the TSC components with DYRK1A and to determine the …

The Role Of Manganese In Streptococcus Sanguinis, Tanya M. Puccio

Theses and Dissertations

Streptococcus sanguinis is primarily associated with oral health as a commensal bacterium. As an opportunistic pathogen, S. sanguinis is capable of colonizing heart valve vegetations, leading to the disease infective endocarditis. Previous studies from our lab have identified the high-affinity manganese transporter SsaACB as important for endocarditis virulence. The impact that manganese depletion has on S. sanguinis had never been evaluated and a secondary manganese transporter has not been identified. Thus, we employed the use of a fermentor to control large-scale growth over time and depleted manganese in an ΔssaACB mutant using a metal chelator, EDTA. The changes in …

Investigation Of The Interactions Between The Dream Complex And Hpv16, Kevin Ko

Theses and Dissertations

According to the American Cancer Society, it has been estimated that in 2019 alone, there will be approximately 53,000 new cases of oropharyngeal cancers. Oropharyngeal cancers are the largest subset of head and neck squamous cell carcinomas (HNSCCs), which are the sixth most common cancer across worldwide populations. They, along with other HNSCCs, fall under a category of cancers known as Human papillomavirus (HPV)-associated cancers, and it has been found that upwards of 70% of these cancers can be attributed to high-risk HPV infections.

Specifically, the high-risk HPV gene, E7, plays a key role in relieving cell cycle repression by …

Electrostatic Networks And Mechanisms Of Δph-Dependent Gating In The Human Voltage-Gated Proton Channel Hv1, Ashley L. Bennett

Theses and Dissertations

The structure of the voltage-gated proton (H⁺) channel Hv1 is homologous to the voltage sensor domain (VSD) of tetrameric voltage-gated Na⁺, K⁺ and Ca²⁺ channels (VGCs), but lacks a pore domain and instead forms a homodimer. Similar to other VSD proteins, Hv1 is gated by changes in membrane potential (V), but unlike VGCs, voltage-dependent gating in Hv1 is modulated by changes in the transmembrane pH gradient (DpH = pH_o - pH_i). In Hv1, pH_o or pH_i changes shift the open probability (P_OPEN)-V relation by ~40 mV per …

Targeting Sphingosine Kinase 2 As A Treatment For Cholangiocarcinoma, Anthony D. Stillman

Theses and Dissertations

Cholangiocarcinoma (CCA) has a high mortality rate and its occurrence is rising. This increase prompts the need for improved CCA treatments. Studies have suggested that CCA is highly reliant on the sphingosine-1-phosphate-receptor-2 (S1PR2) and sphingosine kinase 2 (SphK2). Recently, a competitive SphK2 inhibitor, ABC294640, has been approved for clinical trial. ABC294640 has the potential to treat CCA, which is support by a phase I clinical study that was able to temporarily treat a patient suffering from metastasized CCA with ABC294640. To determine the viability of ABC294640 as a treatment for CCA, this study focused on determining the effects of ABC294640 …

Regorafenib Enhances Lethality Of Sildenafil And Curcumin In Colorectal Cancer Cells, Kervin Benjamin Owusu

Theses and Dissertations

In the United States, more than 130,000 people will be diagnosed with colorectal cancer (CRC) each year and an estimated 50,000 people will die from the disease. Standard of care (SOC) therapies for CRC combine multiple cytotoxic chemotherapeutic drugs. These combinations have varying degrees of effectiveness and can often result in significant patient morbidity. For second recurrence patients, the multi-kinase inhibitor, regorafenib, is an approved agent, but is often poorly tolerated at current doses. In the current study, we propose to develop therapeutic regime of combining agents with modest toxicity profiles: curcumin and sildenafil with regorafenib. Using clinically achievable enterohepatic …

Eralpha Isoforms Modulate The Tumorigenicity Of 24r,25(Oh)2d3 In Estrogen-Responsive Cancer, Anjali Verma

Theses and Dissertations

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D₃ levels have better prognoses than patients with low serum vitamin D_3. However, double-blind clinical trials on the efficacy of vitamin D₃ supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D₃ supplements who had ‘estrogen receptor positive’ …

Cellular Mechanisms By Which Alcohol Promotes Hiv Protease Inhibitor-Induced Hepatotoxicity, Michael Hinton

Theses and Dissertations

CELLULAR MECHANISMS BY WHICH ALCOHOL PROMOTES HIV PROTEASE INHIBITOR-INDUCED HEPATOTOXICITY

Michael Hinton, B.S.

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University

Virginia Commonwealth University, 2019

Major Director: Huiping Zhou

Professor, Department of Microbiology and Immunology

The development of highly-active-antiretroviral therapy(HAART) has allowed management of HIV and extended the lives of those infected. Alcohol abuse, which is very common in HIV-1 infected patients, is one of the most important co-morbid risk factors for liver injury and has been associated with the occurrence of serious metabolic syndrome and subsequent discontinuation …

The Role Of Irf1 In The Brain And In Adaptive Responses Of Astrocytes, Andrew Hoskins

Theses and Dissertations

In neurodegenerative diseases, the CNS becomes inflamed through activation of pathways, including the NF-B pathway. Some of the therapies for those diseases target neuroinflammatory pathways. Here, we explore the mechanisms for the upregulation of a subset of genes following a restimulation of the NF-B pathway. We discover that this upregulation occurs independent of IRF1 expression and type 1 interferon signaling. A knockdown of IRF1 using siRNA and an inhibition of JAK proteins using inhibitor AG490 both had no effect on priming. A secreted factor was found to upregulate the expression of both this subset of genes and genes encoding pro-inflammatory …

Processing Of 3′-Blocked Dna Double-Strand Breaks By Tyrosyl-Dna Phosphodiesterase 1, Artemis And Polynucleotide Kinase/ Phosphatase, Ajinkya S. Kawale

Theses and Dissertations

DNA double-strand breaks (DSBs) containing unligatable termini are potent cytotoxic lesions leading to growth arrest or cell death. The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted …

Cellular Inhibitor Of Apoptosis Protein2 – A Critical Regulator Of Neuroinflammation, Debolina Dipankar Biswas

Theses and Dissertations

Inhibitors of apoptosis (IAPs) modulate cell death and play critical role in signal transduction that promotes inflammation. Recently, Smac mimetics, which are IAP antagonists, have attracted attention as novel cancer therapeutics. Cellular Inhibitor of Apoptosis 2 (cIAP2), a member of IAP family, positively affects both NF-κB and MAPK activation in response to many inflammatory stimuli. We observed that the lack of cIAP2 ablates LPS-induced neuroinflammation. Also, cIAP2^-/- macrophages demonstrated diminished antigen presentation potential that could contribute to ablated immunity. In addition to these functions, we have previously reported that cIAP2 also regulates the activation of Interferon Regulatory Factor 1 …