Biochemistry Commons^™

Novel Role Of The Nociceptin System As A Regulator Of Glutamate Transporter Expression In Developing Astrocytes, Logan Meyer

Theses and Dissertations

Our previous results showed that oligodendrocyte development is regulated by both nociceptin and its G-protein coupled receptor, the nociceptin/orphanin FQ receptor (NOPR). The present in vitro and in vivo findings show that nociceptin plays a crucial conserved role in both human and rodent brain astrocytes, regulating the levels of the glutamate/aspartate transporter GLAST/EAAT1. This nociceptin-mediated response takes place during a critical developmental window that coincides with astrocyte maturation and synapse formation. GLAST/EAAT1 upregulation by nociceptin is mediated by NOPR and the downstream participation of a complex signaling cascade that involves the interaction of several kinase systems, including PI-3K/AKT, mTOR and …

Study Of Molecular Interactions Of Glycosaminoglycans And Glycosaminoglycan Mimetics With Their Protein Targets, Daniel K. Afosah

Theses and Dissertations

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE).

Anticoagulants are very important for the treatment of thrombotic diseases. The …

Optimization Of In Vitro Transcription/Translation Conditions For In Vitro Compartmentalization Studies And Synthesis Of 4-Fluorohistidine, Christine Ring

Theses and Dissertations

Genetic code expansion allows the incorporation of non-canonical amino acids with a variety of new functional groups: fluorescent amino acids,^1-3 azides,^4-6 alkynes,^5-10 and photocrosslinkers.^4,11,12 This incorporation requires the evolution of new tRNA/aminoacyl tRNA sythetase pairs. Traditionally screenings of novel tRNA/aminoacyl tRNA synthetase pairs have been done in vivo. While these in vivo screenings have proven robust, they are limited in multiple ways: non-canonical amino acids (ncAAs) must be nontoxic and bioavailable. Furthermore, library size is limited by transformation efficiency. Lastly, in vivo screenings require substantial amounts of the target ncAA, which is often not available …

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 …

Cannabinoid Receptor Interacting Protein Suppresses Agonist-Driven Cb1 Receptor Internalization And Regulates Receptor Replenishment In An Agonist-Biased Manner, Lawrence C. Blumes, Sandra Leone-Kabler, Deborah J. Luessen, Glenn S. Marrs, Erica Lyons, Caroline E. Bass, Rong Chen, Dana E. Selley, Allyn C. Howlett

Neurology Publications

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminus-associated protein that modulates CB1R signaling via G proteins, and CB1R down-regulation but not desensitization (Blume et al. [2015] Cell Signal., 27, 716-726; Smith et al. [2015] Mol. Pharmacol., 87, 747-765). In this study, we determined the involvement of CRIP1a in CB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB(1)Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1R and CRIP1a, and exhibits a well-characterized endocannabinoid signaling system. We developed stable CRIP1a-over-expressing and CRIP1a-siRNA-silenced knockdown …

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 …

Reduction Of Oxidative Stress And Storage Lesions (Rcsl) In Red Blood Cells - Analysis Of Ascorbic Acid (Aa), N-Acetylcysteine Amide (Ad4), And Serotonin (5-Ht), Shanmuka Gadiraju

Undergraduate Research Posters

Oxidative stress is a common occurrence in red blood cell (RBC) storage in blood banks throughout the world. Typically RBC units stored under routine standard protocol (stored in SAGM-CPD additive solution) can only be kept up to 42 days for transfusion usage before being discarded. I am studying the effects of Ascorbic Acid (AA), N-acetylcysteine amide (AD4), and Serotonin (5-HT) as additives in blood bank storage because I want to find out if these additives can reduce storage-induced oxidative stress on red blood cells (RBCs), in order to help my reader understand how potential blood storage additives can affect the …

A Pipeline For Creation Of Genome-Scale Metabolic Reconstructions, Shaun W. Norris

Theses and Dissertations

The decreasing costs of next generation sequencing technologies and the increasing speeds at which they work have lead to an abundance of 'omic datasets. The need for tools and methods to analyze, annotate, and model these datasets to better understand biological systems is growing. Here we present a novel software pipeline to reconstruct the metabolic model of an organism in silico starting from its genome sequence and a novel compilation of biological databases to better serve the generation of metabolic models. We validate these methods using five Gardnerella vaginalis strains and compare the gene annotation results to NCBI and the …

Probing Allosteric, Partial Inhibition Of Thrombin Using Novel Anticoagulants, Stephen S. Verespy Iii

Theses and Dissertations

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an …

From Seed To Sky: Impacts Of Explosive Compounds On Vegetation Across Spatial And Developmental Scales, Stephen M. Via

Theses and Dissertations

Explosive compounds are broadly distributed across the globe as a result of nearly two centuries of munitions use in warfare and military activities. Two explosive compounds have seen disproportionate use; RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and TNT (2-methyl- 1,3,5-trinitrobenzene), being the most commonly found explosives in the environment. The effects of explosives on biota have been studied in great detail; however, there is a general lack of understanding with regard to broader ecological impacts of these contaminants. My dissertation objective was to follow the impacts of explosive compounds on vegetation across scales. Impacts on vegetation at the species scale alter community composition via …

Celecoxib Enhances Sorafenib/Sildenafil Lethality In Cancer Cells And Reverts Platinum Chemotherapy Resistance, Timothy A. Webb

Theses and Dissertations

The present studies sought to determine whether the lethality of the drug combination [sorafenib + sildenafil] could be enhanced by the anti-inflammatory agent celecoxib, using ovarian cancer and other tumor cell lines as models. Also, in a dose dependent fashion celecoxib enhanced [sorafenib + sildenafil] lethality in multiple ovarian cancer cell lines. In a dose dependent fashion celecoxib enhanced the ability of [sorafenib + sildenafil] to reduce expression of multiple chaperone proteins in parallel with lower levels of the drug efflux pumps ABCB1 and ABCG2. Over-expression of GRP78 and HSP27 maintained pump expression in the presence of drugs. Cell killing …

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 …

The Study Of The Regulon Of Oxyr In Escherichia Coli And Porphyromonas Gingivalis, Christopher K. Pham

Theses and Dissertations

The facultative anaerobe, Escherichia coli and the obligate anaerobe, Porphyromonas gingivalis are two bacteria that reside in our body. Although they reside in separate environments, they are both subject to hydrogen peroxide stress and have mechanisms to regulate the stress. OxyR is the primary transcriptional regulator/sensor of oxidative stress response caused by hydrogen peroxide. OxyR in P. gingivalis is not well-characterized compared to OxyR in E. coli. We sought to characterize and compare the two forms of OxyR in order to gain a better understanding of the protein. We determined the oligomeric state of both proteins: primarily a tetramer for …

Bacterial Forensics: Revolutionizing Biochemical Analysis, Caitlin Cain

AUCTUS: The Journal of Undergraduate Research and Creative Scholarship

Eva Childrey is a junior forensic science and chemistry double major working in Dr. Eh- rhardt’s research laboratory at VCU. The main goal of the research conducted in this laboratory is to explore the lipid profiles of different bacterial species.

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 …

Interaction Between Atm Kinase And P53 In Determining Glioma Radiosensitivity, Syed F. Ahmad

Theses and Dissertations

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Studies have shown that targeting the DNA damage response can sensitize cancer cells to DNA damaging agents. Ataxia telangiectasia mutated (ATM) is involved in signaling DNA double strand breaks. Our group has previously shown that ATM inhibitors (ATMi) sensitize GBM cells and tumors to ionizing radiation. This effect is greater when the tumor suppressor p53 is mutated.

The goals of this work include validation of a new ATM inhibitor, AZ32, and elucidation of how ATMi and p53 status interact to promote cell death after radiation. We propose that ATMi and …

Regulation Of Tie2 Extracellular Complex Formation In Angiogenesis, Annamarie Dalton

Theses and Dissertations

Pathological angiogenesis is an essential component of tumor growth, development, and metastasis for which few effective therapeutic options exist. Though many cancer therapies target the function of cell surface receptors, mechanisms regulating membrane receptor crosstalk remain unclear. Two important families of receptors in angiogenesis, the Ties and Integrins, respond to the extracellular environment via outside-in and, in the case of Integrins, also inside- out signaling. Recent reports showed that the endothelial specific tyrosine kinase receptor, Tie2, forms complexes with two of the endothelial Integrin heterodimers, α₅β₁ and α_Vβ₃, providing a convenient mechanism for …

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 …

The Role Of Srsf3 In Control Of Alternative Splicing Of Cpeb2 In Triple Negative Breast Cancer, Brian P. Griffin

Theses and Dissertations

In the presented study, we identified that SRSF3 controls the alternative splicing of CPEB2 and consequently promotes a metastatic phenotype in triple negative breast cancer (TNBC). TNBC causes thousands of deaths annually, frequently due to a lack of effective treatments and a high rate of metastasis in patients. Alternative splicing has been found to be dysregulated in numerous cancers, while splicing factors such as SRSF3 are variably expressed. In this study we performed a siRNA panel to screen potential splicing factors, then used specific siRNA to study the effect of its knockdown on cellular function. These results showed that SRSF3 …

Dysregulation Of Micrornas In Blood As Biomarkers For Diagnosing Prostate Cancer, Rhonda W. Daniel

Theses and Dissertations

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers.

Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such …

Pemetrexed, A Modulator Of Amp-Activated Kinase Signaling And An Inhibitor Of Wild Type And Mutant P53, Stuti Agarwal

Theses and Dissertations

New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human …

Nitric Oxide Synthase Activity And Its Modulation In The Treatment Of Colorectal Cancer, Asim Alam

Theses and Dissertations

The American Cancer Society estimates more than 141,000 new cases of and about 50,000 deaths from colorectal cancer every year. Treatment options include surgery, radiation therapy and targeted therapies such as anti-angiogenics. However, no therapies address the key driving factor of colorectal cancer: inflammation. It is well known that chronic inflammatory conditions such as Crohn’s Disease, ulcerative colitis, diabetes, obesity and cigarette smoking all elevate the risk of developing colorectal cancer. One of the hallmarks of chronic inflammation is the elevated levels of reactive oxygen/nitrogen species (ROS/RNS). A primary source of these ROS/RNS is uncoupled Nitric Oxide Synthase (NOS). Under …

Evolution And Divergence Of The Structural And Physical Properties Of Dna Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, Jason Cramer

Theses and Dissertations

The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of …

Atm, Brca1, And Aurora A: Mechanisms Of G2/M Checkpoint Control In Human Embryonic Stem Cells, Jason Beckta

Theses and Dissertations

When cultured in vitro, human embryonic stem cells (hESCs) acquire genetic abnormalities that have slowed their therapeutic use. As hESCs have a “leaky” G1/S boundary, the pressure of ensuring genetic integrity falls on the G2/M checkpoint, which can be activated by failed chromosomal decatenation (among other stimuli). It is hypothesized that hESCs have a deficient decatenation checkpoint, but little data supports this. Evidence suggests that the ataxia telangiectasia mutated (ATM) kinase controls the G2/M decatenation and DNA damage checkpoints, though previous reports are conflicting on this point. My work demonstrates that inhibition of decatenation activates ATM and arrests hESCs in …

Folate Conjugated Dendrimers For Targeted Anticancer Therapy, Shannon Andrews

Theses and Dissertations

Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of …

Group Via Calcium-Independent Phospholipase A2 Regulates Bcl-Xl Protein Levels In Mice Lung, Sang-Jin Nam

Theses and Dissertations

With previous indication of the Group VIA phospholipase A2 (iPLA2β) enzyme regulating ER-stress induced apoptosis in β-cells by regulating the anti-apoptotic protein Bcl-xL via alternative splicing, our lab postulated iPLA2β to be utilizing a similar mechanism to regulate apoptosis in mice lung. Our previous lab work has shown implications of lung function compromise in iPLA2β-/- mice, and we speculated the cause to be due altered lung architecture stemming from the attenuation of apoptosis. The western blot analysis in this study suggested that iPLA2β is involved in the regulation of Bcl-xL, but the mRNA ratios of the splice variants suggested that …

The Cytotoxic Effect Of The Bcl-2 Family Of Proteins In Breast Cancer Cells, Yamileth Chin

Theses and Dissertations

Breast cancer is the second leading cause of death amongst women ages 20 to 59. Despite advancements in cancer therapies, more research is necessary to improve the diagnoses and treatment of several types of breast cancer. Paclitaxel (Taxol) is a commonly utilized anti-cancer drug for various types of solid tumors. However, the molecular mechanism utilized by paclitaxel to induce cell death is still elusive. Previous studies in our laboratory have shown that the pro-apoptotic BCL-2 family protein, BAK (BCL-2 homologous antagonist/killer) plays an important role in paclitaxel-induced cell death. In untreated breast cancer cells, BAK is associated with the anti-apoptotic …

Modulation Of Gene Expression By Tumor-Derived Mutant P53. Role Of Transactivation In Gain-Of-Function., Mariano J. Scian

Theses and Dissertations

It was hypothesized that the C-terminal sequences for mutant p53 would be required for oligomerization. and oligomerization may be critical for gain-of-function. An N-terminal deletion mutant of p53 that deletes amino acids 1-293 was used as a tool to perform hetero-oligomerization studies. This mutant retains the entire oligomerization domain but dispenses off the transactivation domain and a large portion of the sequence- specific DNA-binding domain. Co-transfection experiments show that p53 del. 1-293 forms hetero-oligomeric complexes with p53-D281G. Also. co-expression of p53 del. 1- 293 with p53-D281G inhibited p53-D28lG-mediated transactivation of the EGFR and MDRl promoters suggesting that hetero-oligomerization inactivates transcriptional …

Chemical Synthesis, Bacterial Expression, And Characterization Of Pro-Gnrh/Gap, A Precursor Protein Of Two Biologically Active Peptide Hormones, Louis E. Mcadory

Theses and Dissertations

Two biologically active peptides, gonadotropin releasing hormone (GnRH) and GnRH associated peptide (GAP) are both derived from a common prohormone precursor protein, pro-GnRH/GAP. Both peptides are cosecreted from hypothalamic neurosecretory granules and are involved in the regulation of mammalian reproduction. A calcium dependent, neutral pH serine protease discovered in this laboratory, GAP-releasing enzyme, is the most likely processing enzyme of pro-GnRH/GAP. GAP-releasing enzyme is immunologically related to PC1/3, a member of the prohormone convertase (PC) class of processing endoproteinases.

GAP-releasing enzyme recognizes the eight residue processing site within pro-GnRH/GAP, G⁶LRPGGKR¹³, and correctly cleaves the R^{13 …}

Molecular Cloning And Analysis Of The Promoter For Rat Hepatic Neutral Cholesterol Ester Hydrolase, Ramesh Natarajan

Theses and Dissertations

Neutral cholesterol ester hydrolase (CEH) is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5'-untranslated region as a primer, 1.3 kb of sequence upstream of the ATG initiation codon was amplified and cloned. Primer extension analysis with total RNA from rat primary hepatocytes identified a transcription initiation site, 60 bases upstream from the initiation codon. No typical TATA-box sequences were found upstream from the transcription start site. However, a consensus GC-box, which can bind the positive transcription factor SP1, was found 35 bases upstream from the transcription start site. In addition the …