Molecular Biology Commons^™

Preconditioning Of The Tumor Microenvironment By Means Of Low Dose Chemotherapies For An Effective Immunotherapy Of Breast Cancer, Hussein F. Aqbi

Theses and Dissertations

Breast cancer mortality is mainly due to distant recurrence of the disease arising from dormant tumor cells established by cancer therapies. Patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting …

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 …

An Investigation On The Role Of Β-Arrestin 2, Protein Kinase C And Sex On The Mechanism Of Morphine Tolerance In The Mouse Ileum, Karan Hitesh Muchhala

Theses and Dissertations

Opioids such as morphine are frequently used in the clinic to treat pain. However, the perennial bane of chronic opioid use is the rapid development of tolerance to the analgesic effects but delayed development of tolerance to the respiratory depressant and constipating effects. As constipation is one of the most common opioid-related adverse effects in humans, it is important to delineate mechanisms that drive opioid tolerance in the ileum and lack of it in the colon. The overarching goal of this thesis was to investigate mechanisms of morphine tolerance in the ileum by comparing the mechanism of morphine tolerance in …

Mathematical Modeling Of Nutrient Signaling And Growth In Saccharomyces Cerevisiae, Amogh P. Jalihal, Pavel Kraikivski, T.M. Murali, John J. Tyson

Biology and Medicine Through Mathematics Conference

No abstract provided.

Zebrafish Model Of Mll-Rearranged Acute Myeloid Leukemia, Alex J. Belt

Theses and Dissertations

Acute myeloid leukemia (AML) is the second most common type of leukemia and accounts for 80% of adult acute leukemia cases and is characterized by the accumulation of poorly or undifferentiated myeloid blast cells. Standard treatment includes chemotherapy, which if unsuccessful, is followed by more rigorous chemotherapy as well as stem cell transplantation. Considering most patients are over the age of 45, these more rigorous therapies are not always possible, and as such, new therapies must be developed. Furthermore, AML patients harboring a chromosomal rearrangement involving Multiple Lineage Leukemia (MLL) that results in the expression of an MLL fusion protein …

In Vitro Genetic Code Expansion And Selected Applications, Emil S. Iqbal

Theses and Dissertations

The ability of incorporation non-canonical amino acids (ncAAs) using translation offers researchers the ability of extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the three projects where the addition of ncAAs to in vitro translation systems creates useful chemical biology techniques. In the first, a fluorinated histidine derivative is used to create a novel affinity tag that allows for the selective purification of peptides from a complex mixture of proteins. In the second, the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P) …

The Interaction Between Ceramide-1-Phosphate And Group Iva Cytosolic Phospholipase A2 And Its Role In Wound Healing, Patrick Macknight

Theses and Dissertations

The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A₂ (cPLA₂a) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA₂a with an ablated C1P interaction site (KI) to examine the cPLA₂a/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was dramatically enhanced by loss of the C1P/cPLA₂α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii …

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 …

Chronic Clozapine Treatment Impairs Functional Activation Of Metabotropic Glutamate Receptor 2 Via An Hdac2-Depedent Mechanism, Travis M. Cuddy

Theses and Dissertations

Schizophrenia is a chronic mental disorder affecting millions worldwide. It has no known cure. Current pharmaceutical treatments have shown efficacy in only one of the three symptom clusters of schizophrenia, providing little or no benefit in the other two. Furthermore, the current standard-of-care drugs, known as atypical antipsychotics, carry risks of severe side effects affecting multiple body systems. Most patients opt to discontinue drug therapy within two years of initiation due to lack of efficacy and/or preponderance of adverse effects. Previous findings have shown that chronic usage of atypical antipsychotics causes a 5-HT_2A-dependent upregulation of histone deacetylase 2 …

Survival Model Of Intrahepatic Cholangiocarcinoma; Sex As A Biological Variable, Mary A. Phillippi, Justin L. Mott, Cody J. Wehrkamp, Ying Xie, David Oupicky, Ashley M. Mohr, Bailey A. Stringham

Hepatobiliary Cancers: Pathobiology and Translational Advances

No abstract provided.

Oncogenic Nelfe Enhances Myc-Induced Hepatocellular Carcinogenesis, Hien Dang, Atsushi Takai, Marshonna Forgues, Yosawat Pomyen, Haiwei Mou, Wen Xue, Debashish Ray, Kevn Ha, Quiad Morris, Timothy Hughes, Xin Wei Wang

Hepatobiliary Cancers: Pathobiology and Translational Advances

No abstract provided.

Glypican-3 Targeting Immunotoxins For The Treatment Of Liver Cancer, Bryan D. Fleming, Daniel Urban, Lu Chen, Matthew Hall, Ira Pastan, Mitchell Ho

Hepatobiliary Cancers: Pathobiology and Translational Advances

No abstract provided.

Hepatocyte-Specific Deletion Of Tristetraprolin Family Of Rna Binding Proteins Result In The Development Of Hepatocellular Carcinomas, Sonika Patial

Hepatobiliary Cancers: Pathobiology and Translational Advances

No abstract provided.

Nitrosative Stress Sensing In Porphyromonas Gingivalis: Structure And Function Of The Heme Binding Transcriptional Regulator Hcpr, Benjamin R. Belvin

Theses and Dissertations

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of …

Characterization Of A Putative Hemolysin Expressed By Sneathia Amnii, A Preterm Birth-Associated Pathogen, Lizette Carrasco, Kimberly Jefferson

Undergraduate Research Posters

The gram-negative bacteria Sneathia amnii is a poorly-characterized commensal of the female urogenital tract frequently associated with adverse clinical outcomes such as bacterial vaginosis (BV), amnionitis, and preterm labor. To investigate its potential role in virulence, we sought to identify and characterize virulence determinants produced by S. amnii in an effort to better understand the pathogenesis of infectious preterm birth. Through sequencing of the Sn35 genome (type strain of S. amnii), we identified two genes with amino acid sequence similarity and structural similarity to the filamentous hemagglutinin (FHA) protein of Bordetella pertussis and its Type Vb transporter. Because S. amnii …

Characterization Of Staphylococcal Nuclease And Tudor Domain Containing Protein 1 (Snd1) As A Molecular Target In Hepatocellular Carcinoma And Non-Alcoholic Steatohepatitis, Nidhi H. Jariwala

Theses and Dissertations

CHARACTERIZATION OF STAPHYLOCOCCAL NUCLEASE AND TUDOR DOMAIN CONTAINING PROTEIN 1 (SND1) AS A MOLECULAR TARGET IN HEPATOCELLULAR CARCINOMA AND NON-ALCOHOLIC STEATOHEPATITIS

Nidhi Jariwala, PhD

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Life Sciences

Virginia Commonwealth University, 2017

Devanand Sarkar, M.B.B.S., PhD.

Associate Professor, Department of Human and Molecular Genetics

Virginia Commonwealth University

Richmond, Virginia

SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). …

Separation Of Blood Mixtures Using Fluorescently Labeled Antibodies, Dani Jabado

Undergraduate Research Posters

Identifying and analyzing biological mixture samples at a crime scene are of paramount concern for forensic scientists, especially if that type of evidence contains only one cell type. The presence of multiple contributors in a biological evidence sample reduces the probative value of DNA evidence and can sometimes lead to its eventual loss of value. As such, this study was performed in an attempt to examine and evaluate flow cytometry analysis as a means to separate blood mixture samples labeled with fluorescent antibodies. Fluorescein Isothiocyanate (FITC) antibodies were specifically targeted and bound to HLA (Human Leukocyte Antigens) markers present on …

Evolution Of Protein Complexes In Bacterial Species, Shwetha Hara Sridhar, Wedad Albalawi, Peter Uetz

Undergraduate Research Posters

Protein complexes are composed of two or more associated polypeptide chains that may have different functions. Protein complexes play a critical role for all processes in life and are considered as highly conserved in evolution. In previous studies, protein complexes from E. coli or Mycoplasma pneumoniae have been characterized experimentally, revealing that a typical bacterial cell has on the order of 500 protein complexes. Using gene homology (orthology), these experimentally-observed complexes can be used to predict protein complexes across many species of bacteria. Surprisingly, the majority of protein complexes is not conserved, demonstrating an unexpected evolutionary flexibility.

The current research …

Sh3 And Multiple Ankyrin Repeat Domain 3 (Shank3) Affects The Expression Of Hyperpolarization-Activated Cyclic Nucleotide-Gated (Hcn) Channels In Mouse Models Of Autism, Nikhil N. Shah

Theses and Dissertations

SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human …

Epigenetic Editing To Validate Findings From Methylome-Wide Association Studies Of Neuropsychiatric Disorders, Robin F. Chan

Theses and Dissertations

DNA methylation is necessary for learning, memory consolidation and has been implicated in a number of neuropsychiatric disorders. Obtaining high quality and comprehensive data for the three common forms of methylation in brain is challenging for methylome-wide association studies (MWAS). To address this we optimized a panel of enrichment methods for screening the brain methylome. Results show that these enrichment techniques approach the coverage and fidelity of the current gold standard bisulfite based techniques. Our MBD-based method can also be used with low amounts of genomic material from limited human biomaterials. Psychiatric disorders have high prevalence and are often chronic …

Relb Acts As A Molecular Switch To Drive Chronic Inflammation In Glioblastoma Multiforme (Gbm)., Michael R. Waters

Theses and Dissertations

Inflammation is a homeostatic response to tissue injury or infection, which is normally short- lived and quickly resolves to limit tissue damage. In contrast, chronic inflammation has been linked to a variety of human diseases, including cancers such as glioblastoma multiforme (GBM). GBMs are very aggressive tumors with very low patient survival rates, which have not improved in several decades. GBM tumors are characterized by necrosis and profound inflammation; with cytokines secreted by both GBM cells and the tumor microenvironment. The mechanisms by which chronic inflammation develops and persists in GBM regardless of multiple anti-inflammatory feedback loops remain elusive. This …

Biochemical Analysis Of Putative Single-Stranded Nucleic Acid Binding Proteins In Porphyromonas Gingivalis, Steve H. Kokorelis

Theses and Dissertations

Proteins that bind to both DNA and RNA embody the ability to perform multiple functions by a single gene product. These nucleic acid binding proteins in prokaryotes can play a vital role in many cellular processes, including replication, transcription, gene expression, recombination, and repair, to name a few. Nucleic acid binding proteins have unique functional characteristics that stem from their structural attributes that have evolved in a widely-conserved manner. In Escherichia coli (E. coli), the highly-conserved histone-like protein, HU, which predominates as a heterodimer of HUα and HUβ, has been found to bind to both dsDNA and ssDNA. …

Investigation Of Anaplasma Phagocytophilum And Anaplasma Marginale Adhesin-Host Cell Interactions, Kathryn S. Hebert

Theses and Dissertations

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and …

Investigating The Mechanism Of The Compartmentalized Cbp (Creb-Binding Protein) Ubiquitin Ligase Activities, Oluwatoyin E. Akande

Theses and Dissertations

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis.

CBP and its paralogue p300, play double-edged roles in the regulation of p53, a …

Conditional Cardiac-Specific Akap13 Knockout Induces Sex Dependent Biventricular Dilated Cardiomyopathy With Sarcomeric And Mitochondrial Defects, Kimberlyn M. Baig-Ward

Theses and Dissertations

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone …

Role Of Vav2 In Podocyte Inflammasome Activation And Glomerular Injury During Hyperhomocysteinemia, Sabena Conley

Theses and Dissertations

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The …

Atm Phosphorylates Subunit A Of Pp2a Resulting In Its Nuclear Export And Spatiotemporal Regulation Of The Dna Damage Response, Amrita D. Sule

Theses and Dissertations

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and major regulator of the DNA damage response (DDR). One critical ATM target is protein phosphatase 2A (PP2A) known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylation of many proteins during the recovery from the DDR while returning the cell to normalcy. Interestingly, ATM and PP2A are known to form an auto-regulatory yin-yang kinase-phosphatase relationship. Herein, we show that the phosphorylation of the PP2A-Aα structural subunit at S401 by ATM results in nuclear export, which regulates the DDR at multiple levels and affects genomic …

Elucidation Of A Novel Pathway In Staphylococcus Aureus: The Essential Site-Specific Processing Of Ribosomal Protein L27, Erin A. Wall

Theses and Dissertations

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are …

Mysteries Of The Trypanosomatid Maxicircles: Characterization Of The Maxicircle Genomes And The Evolution Of Rna Editing In The Order Kinetoplastida, Preethi Ranganathan Iyengar

Theses and Dissertations

The trypanosomatid protists belonging to Order Kinetoplastida are some of the most successful parasites ever known to mankind. Their extreme physiological diversity and adaptability to different environmental conditions and host systems make them some of the most widespread parasites, causing deadly diseases in humans and other vertebrates.

This project focuses on their unique mitochondrion, called the kinetoplast, and more specifically involves the characterization of a part of their mitochondrial DNA (also called kinetoplast DNA or kDNA), the maxicircles, which are functional homologs of eukaryotic mitochondrial DNA in the kinetoplastid protists. We have sequenced and characterized the maxicircle genomes of 20 …

Characterization Of A Novel Protease In Staphylococcus Aureus, Adam L. Johnson

Theses and Dissertations

A newly discovered cysteine protease, Prp, has been shown to perform an essential, site-specific cleavage of ribosomal protein L27 in Staphylococcus aureus. In Firmicutes and related bacteria, ribosomal protein L27 is encoded with a conserved N-terminal extension that must be removed to expose residues critical for ribosome function. Uncleavable and pre-cleaved variants were unable to complement an L27 deletion in S. aureus, indicating that this N-terminal processing event is essential and likely plays an important regulatory role. The gene encoding the responsible protease (prp) has been shown to be essential, and is found in all organisms …