Medicine and Health Sciences Commons^™

Characterization Of The Mechanism Of Pparγ-Mediated Neointima Formation In Rodents, Ryoko Tsukahara

Theses and Dissertations (ETD)

Lysophosphatidic acid (LPA) and its ether analog alkyl glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for …

Compound 49b: A Novel Beta-Adrenergic Receptor Agonist In The Treatment Of Diabetic Retinopathy, Kimberly Williams-Guy

Theses and Dissertations (ETD)

Diabetic retinopathy is the leading cause of blindness in working Americans. While there are therapeutic regimens for the disease, more effective methods are needed. We have previously shown that a non-specific beta-adrenergic receptor agonist, isoproterenol, was effective in preventing functional and morphological changes associated with diabetic retinopathy in the rat. Isoproterenol also produced left ventricle remodeling suggesting it entered the systemic circulation. We therefore synthesized various novel beta-adrenergic receptor compounds and screened these compounds in vitro for their ability to reduce markers of inflammation and apoptosis. Of the various compounds tested, Compound 49b was able to reduce both inflammation and …

Novel Insights Into Ubiquitin-Like Protein E1-E2 Interactions, Asad Taherbhoy

Theses and Dissertations (ETD)

Posttranslational modification of macromolecules by ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo and NEDD8 regulate a vast array of processes in the cell. Transfer of UBLs to their target generally occurs by a series of molecular handoffs down an E1‑E2‑E3 cascade. We are interested in understanding how E1‑E2 pairs interact and mediate UBL transfer. To this effect, we studied two E1‑E2 pairs: the Sumo pathway (Sumo utilizes a canonical E1 and E2) and the Atg8 pathway (Atg8 is a UBL involved in autophagy that utilizes a non-canonical E1‑E2 pair).

Sumo conjugation is initiated by the heterodimeric Aos1‑Uba2 E1 enzyme (in …

Inflammatory Proteins, Genetic Variation, And Environmental Influences On Health Care Associated Infection Development In Sepsis, Reba Antoinette Umberger

Theses and Dissertations (ETD)

The purpose of this study was to determine the impact of baseline systemic inflammation (pro‑inflammatory cytokine, anti‑inflammatory cytokine, and their ratio), genetic variability, and environment on the development of health care associated infections (HAI) among sepsis patients during their ICU stay (up to 28 days).

Methods: A prospective observation study was conducted at the Veterans Affairs Medical Center in the Medical Intensive Care Unit over an 18 month period. A total of 78 patients were enrolled within 72 hours of presenting to the ICU with sepsis. Patient were excluded if they were receiving immunosuppressants (chemotherapy or greater than one mg/kg …

The Design And Synthesis Of Autotaxin Inhibitors And Analogs Of Lysophosphatidic Acid, Renuka Niranjan Gupte

Theses and Dissertations (ETD)

Lysophosphatidic acid (LPA) is a naturally occurring bioactive phospholipid. It has a wide array of biological effects like cell proliferation, survival, migration, apoptosis invasion, wound healing and angiogenesis. Autotaxin (ATX) was identified as an autocrine tumor cell motility factor from A2058 melanoma conditioned medium. ATX has lysophospholipase D enzyme activity and is responsible for the cleavage of lysophophatidylcholine (LPC) leading to the generation of LPA. Antagonists of Autotaxin would have a potential therapeutic application in cancer research. Chapter 1 is an introduction of LPA and autotaxin. It provides the background and significance of the research. Chapter 2 explores the synthesis …

Discovery Of Quinic Acid Derivatives As Oral Anti-Inflammatory Agents, Kui Zeng

Theses and Dissertations (ETD)

Quinic acid (QA) esters found in hot water extracts of Uncaria tomentosa (a.k.a. Cat’s claw) exert anti-inflammatory activity through mechanisms involving inhibition of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB). Herein, we described the synthesis and biological testing of novel QA derivatives. Inhibition of NF-κB was assessed using A549 (Type II alveolar epithelial-like) cells that stably express a secreted alkaline phosphatase (SEAP) reporter driven by an NF-κB response element. A549- NF-κB cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of QA derivative for 18 hours followed by measurement of SEAP activity. Amide substitution at …

The Role Of Interferon Gamma In The Regulation Of Il-18 Binding Protein And The Development Of Autoimmune Arthritis In A Genetically Non-Susceptible Mouse Strain, Timothy Daniel Kayes

Theses and Dissertations (ETD)

The etiology of the autoimmune disease rheumatoid arthritis (RA) is unknown, but the role of cytokines, including IFN-g, as effectors of immune cell function has been established by the examination of cytokine production in RA patients and through the use of animal models. C57BL/6 (B6) mice that express MHC class II molecules of the b haplotype (I-A^b) are not typically susceptible to collagen-induced arthritis (CIA), the most widely studied animal model of RA. When the gene encoding IFN-g is removed by genetic deletion, however, susceptibility to CIA is conferred. In addition, T cell responses against the immunogen that …

Contribution Of Organic Cation Transporter 2 (Oct2) To Cisplatin-Induced Nephrotoxicity, Kelly K. Filipski

Theses and Dissertations (ETD)

Cisplatin is the most widely used anticancer agent; however, the cellular pharmacokinetics are poorly understood. Cisplatin is predominantly eliminated through the urine via active secretion and is associated with nephrotoxicity. Currently, prehydration therapy is employed to prevent toxic renal side effects; however it has not been completely ameliorated. The studies described herein aim to determine the mechanism in which cisplatin enters the kidney cell from the blood and how it is subsequently secreted into the urine. Organic cation transporter 2 (OCT2) and ABCC2 are highly expressed in the kidney on the basolateral and apical membrane, respectively. We determined the contribution …

Insights Into Btb-Cul3 Ubiquitin Ligases From The Structures Of Spop-Substrate Complexes, Min Zhuang

Theses and Dissertations (ETD)

Cullin-Ring ubiquitin ligases (CRLs) are E3 complexes that specifically recognize substrates through substrate adaptors. In the largest CRL subfamily, Cul3 binds a BTB domain, and a protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH and BTB domain containing protein, SPOP, which regulates diverse signaling pathways. First, we identified a conserved SPOP Binding Consensus (SBC) motif in the transcriptional regulator Ci, the protein phosphatase Puc, and the chromatin component MacroH2A. The SBC motif specifically binds the MATH domain of SPOP, and is required for Puc ubiquitination in vitro and in …

Abcb6 Is A Porphyrin Transporter With A Novel Trafficking Signal That Is Conserved In Other Abc Transporters, Yu Fukuda

Theses and Dissertations (ETD)

The ATP-binding cassette (ABC) transporters play an important role as a barrier to protect cells from the accumulation of toxic xenobiotics and metabolites due to their ability to translocate a wide array of compounds across lipid bilayers. However, many ABC transporters, especially the ones localized in the intracellular organelles, are involved in critical biological processes such as antigen presentation. The core unit of ABC transporters contains two functional domains: the membrane spanning domain (MSD) and the nucleotide binding domain. The full transporters contain two of these units in tandem in a single polypeptide, whereas the half transporters only contain one …

Manipulation Of The Moloney Murine Leukemia Virus Envelope Protein In An Effort To Develop Directly And Indirectly Targeted Retroviral Vectors For Use In Human Gene Therapy, Geneva M. Vasser

Theses and Dissertations (ETD)

Highly effective, targeted therapies against cancer would revolutionize the way people recover from this devastating illness. Gone would be the lingering side effects of the current non-specific treatments and in their place would be faster recovery times, better quality of life both during and after treatment, and less ambiguity about whether or not treatment was effective. This concept will elude modern medicine until treatments can be tailored to the patient's individual and unique disease. This concept of a transient, targeted, and tailored vehicle aimed at cancer cells lends itself to the use of replication deficient retroviral gene therapy vectors with …

C-Reactive Protein Polymorphism And Serum Levels As An Independent Risk Factor In Sickle Cell Disease, Elizabeth A. Chismark

Theses and Dissertations (ETD)

This study explored the relationship of a dinucleotide repeat polymorphism in the intron of the CRP gene and serum CRP levels as independent risk factors for end-organ dysfunction (mild vs. severe) in adults with sickle cell disease. The pathogenesis of secondary complications of sickle cell disease is complex and poorly understood. Predicting the severity of these complications could assist in therapeutic decision-making.

The study measured serum CRP levels and the number of CA intron repeats located on the CRP gene in 29 adults (31.74 ± 11.54 years) with sickle cell disease The hemoglobin genotypes were distributed as Hgb SS 48.6% …

Regulation Of The Human Parainfluenza Virus (Hpiv3) Fusion Protein, Amanda Ruth Chapman

Theses and Dissertations (ETD)

Paramyxoviruses include a number of important human pathogens, including measles virus, mumps virus, and the human parainfluenza viruses (hPIV) 1-4, as well as several animal pathogens, such as Sendai virus, Nipah virus and Hendra virus. The creation of effective drugs and vaccines against this family of viruses would play an important role in decreasing the prevalence of these viruses and contributing to the health of both humans and animals worldwide. The purpose of this work was to determine how the fusion (F) protein is regulated with a focus on the heptad repeat B (HRB) region of the F protein located …

The N-Terminal Domain Of The Y-Box Binding Protein Yb-1 Plays A Major Role In Cell Proliferation And Apoptosis, Payal Khandelwal

Theses and Dissertations (ETD)

Y-box binding protein, YB-1, is a member of the cold shock domain superfamily of proteins. It is involved in a plethora of cellular functions, including cell proliferation. The molecular mechanisms governing the involvement of YB-1 in cell proliferation are still unclear. Earlier studies done in chicken pre-B lymphocyte DT-40 cells in our laboratory have shown that a targeted disruption in one allele of chicken YB-1

(Chk-Yb-1b) gene at its N-terminal domain resulted in multiple abnormalities in the heterozygous mutants, including slower growth rate, abnormal cell morphology, increased cell size, increased genomic DNA content and significant changes in levels of cell …

Cdc45 Function Alters Cell Sensitivity To Dna Topoisomerase I Poisons, Cynthia Sue Lancaster

Theses and Dissertations (ETD)

Eukaryotic DNA topoisomerase I (Top1) is a highly conserved enzyme that functions to manage the torsional strain of DNA during cellular processes such as transcription, replication, chromatid condensation and recombination. The enzyme binds duplex DNA and through a series of strand cleavage and religation reactions removes positive or negative supercoils relieving torsional strain. Top1 is the sole cellular target of the anticancer agent camptothecin, which stabilizes the covalent complex. CPT cytotoxicity is S-phase dependent. It has been suggested that the mechanism of this S-phase toxicity is due to the advancing replication forks either colliding with the stabilized drug-enzyme-DNA intermediate or …

Transcriptional Regulation Of Azole Antifungal Resistance In Candida Albicans, Teresa T. Liu

Theses and Dissertations (ETD)

Candida albicans is a pathogenic fungi found in the mucosa, gastrointestinal, and urogenital tracts of humans. Oropharyngeal candidiasis (OPC), an opportunistic mucosal infection caused by C. albicans, occurs most frequently in patients infected with human immunodeficiency virus (HIV). OPC is usually treated with azole antifungals, a class of antifungals that target ergosterol biosynthesis, at low doses over long periods of time. This course of treatment allows for the development of azole resistance.

Two major mechanisms of azole resistance exist in C. albicans, the up-regulation of genes encoding efflux pumps and the up-regulation of ERG11, a gene encoding the azole drug …

The Structural And Functional Study Of Git1 Paxillin Binding Domain, Ziwei Zhang

Theses and Dissertations (ETD)

The G protein coupled receptor (GPCR)-kinase (GRK) interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of p21-activated kinase (PAK) and PAK-interactive exchange factor (PIX) to focal adhesions, and its activation is regulated by the interaction between its C terminal paxillin-binding domain (PBD) and the LD motifs of paxillin.

In this dissertation, we determined the solution structure of rat GIT1 PBD by nuclear magnetic resonance (NMR) spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting …

Enzyme Architecture And Flexibility Affect Dna Topoisomerase I Function, Mariè Van Der Merwe

Theses and Dissertations (ETD)

DNA topoisomerase I (Top1) is a highly conserved enzyme composed of four domains: a positively charged N-terminus; a DNA binding/core domain that circumscribes duplex DNA; a non-conserved linker domain; and a C-terminal/catalytic domain. Top1 catalyzes changes in DNA topology by transient cleavage of a single DNA strand and the concomitant formation of a phosphotyrosyl linkage between the enzyme and the 3’ DNA end. This covalent Top1-DNA complex is the binding site for camptothecin (CPT), which selectively inhibits religation of the cleaved DNA strand. CPT binding stabilizes the covalent complex, while the collision of replication forks with CPT-Top1-DNA adducts produces DNA …

The Role Of Multi-Drug Resistance Associated Protein 4 And P-Glycoprotein In Resistance Of Neuroblastoma To Topotecan And Irinotecan, Patricia Kellie Turner

Theses and Dissertations (ETD)

High-risk neuroblastoma presents a significant therapeutic challenge because the 5-year survival rate remains less than 30% despite the use of surgery, multi-agent chemotherapy, radiation, and autologous bone marrow transplant. Novel therapeutic modalities are under development. The camptothecin analogs topotecan and irinotecan have been identified as successful cytotoxic agents. For topotecan, pharmacokinetically guided dosing to achieve a systemic exposure associated with preclinical anti-tumor activity in neuroblastoma xenograft models is feasible and has elicited favorable responses in children with high-risk neuroblastoma. However, some children with high-risk disease did not respond to the putatively effective topotecan systemic exposure. These children represent a subset …

Tyrosine Phosphorylation Of Villin: Effects On Actin Dynamics, Cell Morphology And Cell Migration, Alok Tomar

Theses and Dissertations (ETD)

Cell migration is a key aspect of many normal and abnormal biological processes, including embryonic development, defense against infections, wound healing, and tumor cell metastasis. In this study we demonstrate that an epithelial cell actin-binding protein, villin, plays a crucial role in the process of cell migration. Overexpression of villin in doxycyline-regulated HeLa Tet-off and MDCK Tet-off cells enhanced cell migration. We further demonstrate that tyrosine phosphorylation of villin by c-src is required for villin-induced cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. I further identified six new sites in the carboxylterminal region …

Protein-Protein Interactions And Muscle Cell Signaling Via Syntrophin, Shilpa A. Oak

Theses and Dissertations (ETD)

Absence of dystrophin results in Duchenne muscular dystrophy (DMD), a lethal neuromuscular d isorder that afflicts 1 in 3500 live male births. In the sarcolemma, dystrophin is associated with a complex of proteins and glycoproteins, known as the dystrophin glycoprotein complex. The DGC constituents are dystrophin, a-dys troglycan, b-dystroglycan, syntrophin, a-sarcoglycan, b-sarcoglycan, g-sarcoglycan, d-sarcoglycan, and sarcospan. Not al l of these are single protein species. The syntrophins consists of a group of three homologous proteins composed of acidic (a) and basic (b) components. Syntrophins are known to self-associate to form oligomers.

In this dissertation, syntrophin's oligomerization and its interactions with …

Developmental Expression Of Rat Target Of The Antiproliferative Antibody (Rtapa) Protein In The Brain, Clyde Dale Sullivan

Theses and Dissertations (ETD)

The present study defines the expression pattern of rTAPA (CD81) in the developing rat brain. rTAPA is a member of the tetramembrane spanning family of proteins, and like other members of this family, appears to be associated with the stabilization of cellular contacts. On immunoblots of the brain, rTAPA is present in higher levels than any other tissue examined: muscle, tendon, peripheral nerve, cartilage, liver, kidney, skin, and testicle. Immunohistochemical methods were used to define the distribution of rTAPA in the brain. This protein is expressed by ependyma, choroid plexus, astrocytes, and oligodendrocytes, and rTAPA is dramatically upregulated at the …

Regulation Of Luteinizing Hormone And Catecholamine Release, Benjamin Aaron Adler

Theses and Dissertations (ETD)

These studies tested the interrelated hypotheses that the ovarian hormones produce their positive feedback effects on luteinizing hormone (LH) secretion through activation of noradrenergic and adrenergic systems in specific hypothalamic regions. Furthermore, the ovarian hormones may alter the activity of opioid neuropeptide and Gamma-Aminobutyric Acid (GABA) systems to produce these alterations in catecholamine transmission and gonadotropin secretion. Radioimmunoassays were utilized to determine plasma LH and median eminence LHRH, and hypothalamic catecholamine concentrations were measured by radioenzymatic assay.

The first two studies tested whether epinephrine (EPI) synthesis inhibition blocks the accumulation of median eminence LHRH that precedes the ovarian hormone-induced LH …

Characteristics Of The Recovery Of The Coenzyme A-Synthesizing Protein Complex From Saccharomyces Cerevisiae, Stanley Joseph Tarnowski Jr.

Theses and Dissertations (ETD)

A multienzyme complex contained in Bakers' yeast (Saccharomyces cerevisiae) which synthesizes CoA has been named the coenzyme A-synthesizing protein complex (CoA-SPC). The CoA-SPC has been shown to be insoluble in the crude Bakers' yeast cell lysate formed by exposing the yeast cell to ether and dry ice. Only after solubilization has this multienzyme complex been shown to catalyze the formation of bound dephospho-CoA utilizing the substrates adenosine triphosphate, D-pantothenic acid and L-cysteine. A low molecular weight component or components of the soluble fraction of the yeast cell and chloride ion appears to be responsible for the solubilization of CoA-SPC. This …

The Purification, Properties And Subunit Structure Of Glycerol Dehydrogenase, Michael James Barrett

Theses and Dissertations (ETD)

The inducible, NAD-linked glycerol dehydrogenase (E.C. 1.1.1.6) of a guanine requiring mutant of A. aerogenes has been purified to homogeneity. The molecular weight of the pure enzyme was found to be 3.4 x 10⁵ daltons. The sedimentation coefficient of the enzyme was 10.7 x 10^-13 sec.^-1. The diffusion constant was 3.07 x 10^-7 cm²/sec.

The partial specific volume calculated from the amino acid composition was 0.72 ml/g. The following kinetic parameters were determined; K_m glycerol, 2.4 x 10^-3 m, K_m NAD, 2.8 x 10^-4 M, K_m dihydroxyacetone, 5.1 …