Biochemistry, Biophysics, and Structural Biology Commons^™

Lysophosphatidic Acid Production And Signaling In Platelets, Zachary Bennett Fulkerson

Theses and Dissertations--Physiology

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease.

The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the catalytic …

Preventing Mother-To-Child Transmission Of Human Immunodeficiency Virus-1 (Hiv-1): Effects Of Intrapartum And Neonatal Single-Dose Nevirapine Prophylaxis And Subsequent Hiv-1 Drug Resistance At Antiretroviral Treatment Initiation, Amanda L. Harmon

CMC Senior Theses

The prevention of mother-to-child transmission is one of the most powerful tools in human immunodeficiency virus type 1 (HIV-1) prevention and has huge potential to improve both maternal and child health. In the absence of any preventative measures, infants born to and breastfed by their HIV-positive mothers have roughly a one-in-three chance of acquiring the infection themselves. HIV can be passed on from mother-to-child during pregnancy, during labor and delivery, and even after during breastfeeding.

Intrapartum and neonatal single-dose nevirapine (sd-NVP) is the foundation of preventing mother-to-child transmission in lower resource settings where it has been used alone or as …

Carbon Monoxide And Nitric Oxide Induced-Heme-Based Modification Of Alpha-2-Antiplasmin And Plasmin Activity, Matthew R. Arkebauer

PCOM Biomedical Studies Student Scholarship

Objectives: Carbon monoxide (CO) derived from cigarette smoke or released from carbon monoxide releasing-molecule 2 (CORM-2), diminishes fibrinolysis. The primary purpose of this study was to determine if CO diminished fibrinolysis by means of enhancing α2-antiplasmin via an alleged heme group. Methods: Plasma, isolated α2-antiplasmin and isolated plasmin were exposed to CO released from CORM-2 (tricarbonyldichlororuthenium (II) dimer) and nitric oxide (NO) via a NO donor to induce carboxyheme and metheme states. Exposed, isolated enzymes were placed in either α2-antiplasmin deficient or normal plasma. Effects of CO and NO on tissue-type plasminogen activator initiated fibrinolysis were determined by thrombelastography. Liquid …

Androgen Receptor-Dependent Effects Of Resveratrol On Tnsin Mrna Levels In Prostate Cancer Cells, Courtney Pisano

PCOM Biomedical Studies Student Scholarship

The chemopreventive effects of resveratrol (RSV) on cancers, including prostate cancer, have been well documented; but the mechanisms are not well known. It has been reported recently that tensin, a matrix-adhesion protein, which is greatly down-regulated in prostate cancer, has been induced by RSV in several cancer cell lines. In order to know if RSV up-regulates tensin in prostate cells, we first treated LNCaP cells with RSV and demonstrated that tensin mRNA levels were upregulated by RSV in a time and dose dependent manner. Since LNCaP cells are androgen receptor (AR) positive and previous findings have shown that RSV down-regulates …

The Role Of Endothelial Nitric Oxide Synthase (Enos) Uncoupling On Leukocyte-Endothelial Interactions In Rat Mesenteric Postcapillary Venules, Maria Kern

PCOM Biomedical Studies Student Scholarship

Endothelial derived nitric oxide (NO) is essential in the regulation of blood pressure and attenuates leukocyte-endothelial interactions associated with vascular injury. However, when endothelial-derived NO is decreased, endothelial dysfunction results and promotes inflammation characterized by increased leukocyte-endothelial interactions. Under normal conditions, eNOS produces NO in the presence of an essential cofactor, tetrahydrobiopetrin (BH4) by facilitating the reduction of molecular oxygen to L-arginine oxidation and generation of L-citrulline. Whereas uncoupled eNOS refers to the electron transfer that becomes uncoupled to L-arginine oxidation and therefore superoxide (SO) is generated when the dihydrobiopetrin (BH2) to BH4 ratio is increased. SO is subsequently converted …

The Effects Of Protein Kinase C Inhibitors On Blood Nitric Oxide And Hydrogen Peroxide Release In Ischemia And Reperfusion Injury, Kyle D. Bartol

PCOM Biomedical Studies Student Scholarship

Vascular endothelial dysfunction is a key component initiating oxidative stress in ischemia/reperfusion (I/R). Endothelial dysfunction is characterized by an increase in hydrogen peroxide (H2O2) and a decrease in the bioavailability of nitric oxide (NO). Previous studies using protein kinase C (PKC) inhibitor Gö 6983 or PKC Beta (β) II inhibitor improved cardiac function in myocardial I/R, decreased leukocyte-endothelial interactions and leukocyte superoxide (SO) release and increased endothelial-derived NO release in vitro. This study examined the effects of Gö 6983 or PKC β II inhibitor on realtime H2O2 and NO release in femoral vein I/R in vivo. NO or H2O2 microsensors …

Molecular, Cellular And Systemic Effects Of Atrazine On The Xenopus Laevis Tadpole, Renee Maryanne Zaya

Dissertations

Atrazine is one of the two most commonly used herbicides in the US and it is the most studies are among the first to link physiological effects of atrazine to changes in tissue and gene expression in Xenopus laevis tadpoles. These studies provide the groundwork for future studies into the mechanisms behind responses to chemical stressors generating data resulting in a better understanding of how these chemicals affect us all. commonly found herbicide in ground water. As a result, a great deal of attention has been placed on its use and safety. It has also been implicated to play a …

Repression Of Protein Kinase C Delta In Human Squamous Cell Carcinomas By Ras, Fyn And Nf-Kappa B Signaling, Vipin Yadav

Dissertations

The delta isoform of Protein Kinase C (PKC-delta) is widely expressed in many normal tissues, including epidermal keratinocytes, and has a critical role in UV-induced apoptosis. However, PKC-delta is frequently lost in chemically or UV-induced mouse skin tumors, as well as in human cutaneous squamous cell carcinomas (SCC). Furthermore, re-expression of PKC-delta in human SCC lines is sufficient to induce apoptosis and suppress tumorigenicity, making PKC-delta a potential tumor suppressor gene for SCCs. The objective of this dissertation is to investigate the mechanism of PKC-delta loss in human SCCs.

To determine the mechanism of PKC-delta loss in human SCCs, we …

Phosphodiesterase-5 Inhibition: A Novel Strategy To Improve Stem Cell Therapy In The Heart, Nicholas Hoke

Theses and Dissertations

Several studies have shown cellular replacement therapy as a treatment strategy of myocardial infarction but results have been limited. Therefore, enhancing the therapeutic potential of stem cells injected into ischemic microenvironments by novel preconditioning (PC) techniques is critical for improving cellular therapy. Recent studies have shown that inhibition of phosphodiesterase-5 (PDE-5) is a powerful strategy to precondition the heart and cardiomyocytes against ischemia/reperfusion injury. We therefore tested the hypothesis that inhibition of PDE-5 with sildenafil (Viagra®) or selective knockdown with a silencing vector in adipose derived stem cells (ASCs) would improve their survival after ischemia/reoxygenation in vitro and enhance cardiac …

Synthesis, Evaluation And Structural Studies Of Antiproliferative Tubulin-Targeting Azetidin-2-Ones, Niamh O'Boyle, Lisa M. Greene, Orla Bergin, Jean-Baptiste Fichet, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

Articles

A series of azetidin-2-ones substituted at positions 2, 3 and 4 of the azetidinone ring scaffold were synthesised and evaluated for antiproliferative, cytotoxic and tubulin binding activity. In these compounds, the cis double bond of the vascular targeting agent combretastatin A-4 is replaced with the azetidinone ring in order to enhance the antiproliferative effects displayed by combretastatin A-4 and prevent the cis/trans isomerization that is associated with inactivation of combretastatin A-4. The series of azetidinones was synthetically accessible via the Staudinger and Reformatsky reactions. Of a diverse range of heterocyclic derivatives, 3-(2-thienyl) analogue 28 and 3-(3-thienyl) analogue 29 displayed the …

Direct Cloning Of Double-Stranded Rnas From Rnase Protection Analysis Reveals Processing Patterns Of C/D Box Snornas And Provides Evidence For Widespread Antisense Transcript Expression, Manli Shen, Eduardo Eyras, Jie Wu, Amit Khanna, Serene Josiah, Mathieu Rederstorff, Michael Q. Zhang, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern …

Glucosamine-Induced Insulin Resistance In Primary Rat Hepatocytes And The Role Of Selenium As An Insulin Mimetic, Sandhya N. Adiyodi Veetil

Dissertations

Type 2 diabetes is mediated by insulin resistance, the inability of insulin to elicit a normal biological response in insulin responsive tissues. Several cellular models have been utilized to determine the mechanism of induction of insulin resistance but questions remain unanswered. One model, implicates the products of the Hexosamine Biosynthetic Pathway (HBP) in the induction of insulin resistance under hyperglycemia. The major end product of HBP, UDP-GlcNAc, is the substrate for O-GlcNAc transferase, an enzyme that catalyzes the O-GlcNAcylation of numerous proteins. This modification may play a role in induction of insulin resistance and thus needs to be evaluated in …

Targeted Deletion Of The Mouse Mitoferrin1 Gene: From Anemia To Protoporphyria, Marie-Berengere Troadec, David Warner, Jared Wallace, Kirk Thomas, Gerald J. Spangrude, John Phillips, Oleh Khalimonchuk, Barry H. Paw, Diane Mcvey Ward, Jerry Kaplan

Department of Biochemistry: Faculty Publications

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show …

Microbial Ecology And Functional Genomics Of Deep-Water Coral-Associated Microbes, Julia Parker Galkiewicz

USF Tampa Graduate Theses and Dissertations

Lophelia pertusa is a cosmopolitan cold-water coral, often found in aphotic waters (>200m). Aggregations of L. pertusa (reefs) provide important habitat to many invertebrate and fish species and act as biodiversity hotspots in the deep sea. The health and diversity of these reefs is of vital importance to deep-sea ecosystems, and the microbial consortia associated with L. pertusa form the most basic ecological level. Deciphering the diversity and function of these microbes provides insight into the roles they play in maintaining reef health. This dissertation takes microbiological techniques that are used in shallow-water coral microbial research and applies them …

Physical Models Of Amyloid Fibril Assembly, Shannon Elizabeth Hill

USF Tampa Graduate Theses and Dissertations

Formation of large fibers and plaques by amyloid proteins is recognized as the molecular hallmark of an increasing number of human disorders, including Parkinson's disease, Alzheimer's disease and even type II diabetes. The broader objective of my research is to unravel the basic mechanisms that initiate and regulate fibril formation by amyloidogenic proteins. This objective is significant because even basic aspects of how fibril formation proceeds from a soluble, monomeric protein to an insoluble amyloid fibril remain much debated. Furthermore, there is increasingly strong evidence suggesting that intermediates of the aggregation process, with properties distinct from those of mature fibrils, …

Role Of Protein Kinase C-Iota In Neuroblastoma And The Effect Of Ica-1, A Novel Protein Kinase C-Iota Inhibitor On The Proliferation And Apoptosis Of Neuroblastoma Cells, Prajit P. Pillai

USF Tampa Graduate Theses and Dissertations

Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate …

Novel Roles For The Transcriptional Repressor Prdm1 In Human Natural Killer Cells And Identification Of An Inhibitor Of Its Interacting Methyltransferase G9a, Matthew Adams Smith

USF Tampa Graduate Theses and Dissertations

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation.

In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at …

Probing Molecules In Confined Space, Carissa Marie Vetromile

USF Tampa Graduate Theses and Dissertations

Despite the plethora of information regarding cellular crowding and its importance on modulating protein function the effects of confinement on biological molecules are often overlooked when investigating their physiological function. Recently however, the encapsulation of biomolecules in solid state matrices (NafionTM, sol-gels, zirconium phosphate,etc.) has increased in importance as a method for examining protein conformation and dynamics in confined space as well as novel applications in biotechnology. Biotechnological applications include, but are not limited to, bioremediation, biosensors, biocatalysts, etc. In order to better utilize solid state materials as substrates for biological molecules an understanding of the effects of encapsulation on …

A Study Of Complex Systems: From Magnetic To Biological, Douglas Carroll Lovelady

USF Tampa Graduate Theses and Dissertations

This work is a study of complex many-body systems with non-trivial interactions. Many such systems can be described with models that are much simpler than the real thing but which can still give good insight into the behavior of realistic systems. We take a look at two such systems. The first part looks at a model that elucidates the variety of magnetic phases observed in rare-earth heterostructures at low temperatures: the six-state clock model. We use an ANNNI-like model Hamiltonian that has a three dimensional parameter space and yields two-dimensional multiphase regions in this space. A low-temperature expansion of the …

Inhibition Of P53 Dna Binding Function By The Mdm2 Acidic Domain, Brittany Lynne Cross

USF Tampa Graduate Theses and Dissertations

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild …

Analysis Of Ahr Expression And Stability In A Recombinant Yeast Model System, Sarah Elizabeth Cuccinello

USF Tampa Graduate Theses and Dissertations

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of …

Microbial Landscapes Of Corals And Ctenophores, Camille Arian Daniels

USF Tampa Graduate Theses and Dissertations

As technology and engineering allow mankind to survey nature at finer scales, the importance of bacteria has been elucidated in their metabolic diversity, ability to transfer genetic information, involvement in biogeochemical cycling, and sheer abundance. With an individual domain of life unto themselves, this diverse group of microorganisms plays an integral role in facilitating life on land and in the oceans, and is second only to viruses in abundance on Earth. They carve niches in a wide range of environments, including those inhospitable to other life forms, and reside in concert or to the detriment of other microbes and/or hosts …

Development Of A Non-Invasive Electrode For Intradermal Electrically Mediated Dna Vaccination, Amy Lynn Donate

USF Tampa Graduate Theses and Dissertations

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our …

Infection With Chlamydia Pneumoniae In Neuronal Cells Alters The Expression Of Genes Involved In Apoptosis And Autophagy Pathways, Annette K. Slutter

PCOM Biomedical Studies Student Scholarship

Dysfunctions in cellular mechanisms such as apoptosis and autophagy have been implicated in the neurodegeneration associated with Alzheimer’s disease (AD). Autophagy in AD pathogenesis has been linked to the endosomal-lysosomal system, which has been shown to play a role in amyloid processing. Studies have suggested that apoptosis may contribute to the neuronal cell loss observed in AD; however, there is no evidence of the apoptotic process leading to terminal completion. Aβ1-42 has been shown to induce apoptosis in neurons and may be an initiating factor in AD. Our previous studies demonstrated that neurons infected with C. pneumoniae are resistant to …

Cardiac Calsequestrin Phosphorylation And Trafficking In The Mammalian Cardiomyocyte, Timothy Mcfarland

Wayne State University Dissertations

Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significant quantities of Ca2+ and altering ryanodine receptor activity at the junctional sarcoplasmic reticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site of biosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multiple antibodies specific to classic rough ER markers, and with the creation of CSQ-DsRed tetramer fusion protein, we were able to establish a juxtanuclear localization of rough ER in cardiomyocytes. Using fluorescence confocal microscopy, the translocon complex …

Crystallographic, Molecular Dynamics, And Enzymatic Studies Of Multi-Drug Resistant Hiv-1 Protease And Implications For Structure Based Drug Design (Project 1); Crystallographic Studies Of Human Myelin Protein Zero (Project 2), Zhigang Liu

Wayne State University Dissertations

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistance, leading to the therapy failure in anti-HIV treatment.Tthe multidrug-resistant 769 (MDR) HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) is selected for the present study to understand drug resistance issue.

Ten additional mutations are introduced to MDR769 HIV-1 protease to study the structural influences brought by these mutations. We get crystal structures of four variants (I10V, A82F, A82S and A82T) of MDR769 HIV-1 protease. All these mutations fail to further open the flaps and expand the active site cavity of MDR769 …

Amphiregulin (Areg) And Epidermal Growth Factor (Egf): Disparate In Egfr Signaling And Trafficking, Andrea Jacqueline Baillo

Wayne State University Dissertations

We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the …

Frazzled And Abelson Interact To Regulate The Actin Cytoskeleton In Drosophila, Bridget Elsa Varughese

Wayne State University Dissertations

Guidance receptors such as Frazzled affect cell shape and motility by directly or indirectly modulating the cytoskeleton. Fra is particularly needed for the formation of the posterior commissures in a developing Drosophila embryo. The cytoplasmic tyrosine kinase, Abelson Kinase (Abl) enhances the loss of commissures observed in fra mutant. Abl physically interacts with Frazzled to help guide commissural axons across the midline. Furthermore, the loss of commissural axons is only seen when the actin dynamics are perturbed. Abl is also known to regulate actin-dependent processes underlying formation of filopodia, microspikes and membrane ruffles. So, we established a Drosophila S2 cell …

Molecular Mechanisms Of Snare Assembly And Expulsion Of Intravesicular Contents In Cell Secretion, Leah Jiyoung Zhang

Wayne State University Dissertations

For nearly half a century, it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the `all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. …

A Novel In Vivo Protein Refolding Technique, Yuefei Huang

Wayne State University Dissertations

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer's disease, Parkinson's disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics.

Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and …