Biochemistry, Biophysics, and Structural Biology Commons^™

A Messenger Molecule Governs Interdependency In An Evolved Pseudomonas Aeruginosa Biofilm Community, Thomas Macelliott Johnson

Honors Theses and Capstones

Biofilm populations are known to harbor great diversity, but the importance of this diversity is not fully understood. A likely contributor to this variation is the second messenger molecule cyclic-di-GMP: low levels associate with a planktonic lifestyle while high levels favor biofilm formation. In ongoing studies of an evolving biofilm population of Pseudomonas aeruginosa (PA), we observed extensive ecological diversification that may relate to this lifestyle switch. In PA, the gene bifA encodes a phosphodiesterase that is known to degrade cyclic-di-GMP and reduce biofilm. This gene was cloned onto a plasmid under control of an inducible promoter and the plasmid …

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 …

Identification And Characterization Of Equine Rotaviruses In Southern Ireland, Crystal Cronin

Theses

Group A Rotaviruses(GARVs) are enteric pathogens which induce acute gastroenteritis in infants, neonates and the young of many animal species. During a 4 year study (2005-2009), 64 fecal samples from symptomatic foals, from several counties, in the south of Ireland, were molecularly characterized to determine the VP& (G type), VP4 (P type), and NSP4 antigenic specificities. Globally, G3 [P12] and G14[P12] are recognized as the predominant equine GARV strains. Twenty-two samples (34.38%) were found to contain G3 viruses, while 21 samples (32.81%) contained G14 viruses. In this study, there was a distinct increase in the incidence of mixed infections circulating …

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 …

Cooperative And Antagonistic Roles For Heterochromatin Proteins In Transcriptional Regulation Of The Drosophila Sex Determination Masterswitch Gene, Hui Li

University of Kentucky Doctoral Dissertations

HOAP was originally identified as a component of an ORC-containing multi-protein complex of Heterochromatin Protein 1 (HP1) from early Drosophila embryos. HOAP immunostaining showed prominent association of it with telomeres, and mutants for HOAP (cav¹) showed it functions along with HP1 in forming a telomere capping complex that prevents telomeric fusions.

Weaker HOAP immunostaining is also observed in regions of pericentric heterochromatin and euchromatin. To examine the role of HOAP at these non-telomeric sites, we applied Affymetric Drosophila Genome Arrays to undertake a microarray expression profiling study of genes that are mis-expressed in cav¹ mutant larvae. The …

Fabrication And Characterization Of Mesoscale Protein Patterns Using Atomic Force Microscopy (Afm), Pei Gao

University of Kentucky Doctoral Dissertations

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing.

As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It …

Characterization Of G10h Promoter And Isolation Of Wrky Transcription Factors Involved In Catharanthus Terpenoid Indole Alkaloid Biosynthesis Pathway, Nitima Suttpanta

University of Kentucky Doctoral Dissertations

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis.

Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a …

Rate-Limiting Step Of Cone Phototransduction Recovery And Oguchi Disease Mechanisms, Frank Chen

Theses and Dissertations

ABSTRACT RATE-LIMITING STEP OF CONE PHOTOTRANSDUCTION RECOVERY AND OGUCHI DISEASE MECHANISMS By Frank Sungping Chen Advisor: Ching-Kang Jason Chen, Ph.D. Retinal photoreceptors provide the first gateway in which light information from the environment is transformed into neuronal signals. The cone and rod photoreceptors are responsible for day and night vision, respectively. Understanding rod and cone phototransduction is to figure out how these cells differ in their temporal and spatial sensitivities to allow perception of a broad dynamic range of stimuli. Phototransduction is mediated through a Gprotein signaling cascade. Light absorption by visual pigment triggers the isomerization of 11- cis-retinal covalently …

Hydrogen Peroxide Probes Directed To Different Cellular Compartments, Mikalai Malinouski, You Zhou, Vsevolod V. Belousov, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Background: Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings: Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed …

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 …

Circadian And Homeostatic Regulation Of Sleep In Cast/Eij And C57bl/6j Mice, Peng Jiang

University of Kentucky Doctoral Dissertations

Sleep is essential for mammals and possibly for all animals. Advancing our knowledge of sleep regulation is crucial for the development of interventions in sleep-related health and social problems. With this aim, this study utilizes laboratory mice to explore sleep regulatory mechanisms at behavioral, molecular, and genetic levels.

Sleep is regulated by the interaction of circadian and homeostatic processes. The circadian clock facilitates sleep to occur at a favorable time of the day. Normal mice, such as the C57BL/6J (B6) strain, sleep mostly during the day and initiate activities at dark onset. Here, I show mice of the CAST/EiJ (CAST) …

Loss Of Bloom Syndrome Protein Causes Destabilization Of Genomic Architecture And Is Complemented By Ectopic Expression Of Escherichia Coli Recg In Human Cells, Michael Wayne Killen

University of Kentucky Doctoral Dissertations

Genomic instability driven by non-allelic homologous recombination (NAHR) provides a realistic mechanism that could account for the numerous chromosomal abnormalities that are hallmarks of cancer. We recently demonstrated that this type of instability could be assayed by analyzing the copy number variation of the human ribosomal RNA gene clusters (rDNA). Further, we found that gene cluster instability (GCI) was present in greater than 50% of the human cancer samples that were tested. Here, data is presented that confirms this phenomenon in the human GAGE gene cluster of those cancer patients. This adds credence to the hypothesis that NAHR could be …

Computer Simulation Of A Hollow-Fiber Bioreactor: Heparan Regulated Growth Factors-Receptors Binding And Dissociation Analysis, Changjiang Zhang

University of Kentucky Doctoral Dissertations

This thesis demonstrates the use of numerical simulation in predicting the behavior of proteins in a flow environment.

A novel convection-diffusion-reaction computational model is first introduced to simulate fibroblast growth factor (FGF-2) binding to its receptor (FGFR) on cell surfaces and regulated by heparan sulfate proteoglycan (HSPG) under flow in a bioreactor. The model includes three parts: (1) the flow of medium using incompressible Navier-Stokes equations; (2) the mass transport of FGF-2 using convection-diffusion equations; and (3) the cell surface binding using chemical kinetics. The model consists of a set of coupled nonlinear partial differential equations (PDEs) for flow and …

Composition - Structure - Property Relationships In Strontium / Zinc Doped Glass-Ceramics., Mark Looney

Theses

Calcium silicate ceramics are amongst a group of ceramic materials currently utilised as bone graft materials. However, current calcium silicate grafts offer little therapeutic potential and poor mechanical properties. The objective of this project was to examine the composition-structure-property relationships arising from the incorporation of therapeutic ions of strontium (Sr) and zinc (Zn) into calcium silicate glass-ceramics (GC) in order to allow for healthy bone regeneration and antibacterial efficacy. Five compositions were studied with varying mole fractions of Sr and Zn. The GC’s were characterised using differential scanning calorimetry (DSC), x-ray diffraction (XRD) at room and ascending temperatures, x-ray tomography …

Molecular Details Of The Catalytic Activity Of Carboxylesterases, Xiaozhen Yu

Electronic Theses and Dissertations

Carboxylesterases (CEs; EC 3.1.1.1) are ubiquitous enzymes responsible for the detoxification of xenobiotics. CEs hydrolyze carboxyl esters into their corresponding alcohol and carboxylic acid. Because of their biological functions, especially their roles in converting inactive prodrugs, such as the anti-cancer drug CPT-11, to their active metabolites, a good understanding of the mechanism of the hydrolysis reaction will give us a better direction for drug design. In this study, we used a multidisciplinary approach (computational simulation, molecular biology techniques and enzyme kinetic methods) to study the dynamic motions of CEs and the potential role of these motions in the catalytic mechanism …