Digital Commons Network^™

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 …

The Role Of Mdm2 In Estrogen-Mediated Breast Cancer Cell Proliferation, Angelika Brekman

Dissertations, Theses, and Capstone Projects

Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the …

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 …

Development Of Molecular And Cellular Imaging Tools To Evaluate Gene And Cell Based Therapeutic Strategies In Vivo, Jixiang Xia

Electronic Theses and Dissertations

Molecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic (“gene gun”) gene targeting to tissues with non-invasive bioluminescence imaging (BLI) methods. Plasmids carrying the firefly luciferase reporter gene were transfected into mouse skin and liver using biolistics, and BLI was measured at various time points after transfer. With optimized DNA …

Mechanisms Of Decreased Cholesterol Absorption Mediated By Phytosterols In The Intestinal Lumen, Andrew W. Brown

Department of Nutrition and Health Sciences: Dissertations, Theses, and Student Research

Phytosterols and their fatty acyl esters have been known for decades to lower LDL cholesterol, making them powerful nutraceuticals in lowering cardiovascular disease risk. The mechanisms by which phytosterols lower cholesterol, though, have been incompletely characterized. Three studies were executed to examine three aspects of cholesterol and phytosterol interactions in the intestinal lumen. In the first study, the ability of pancreatic cholesterol esterase to hydrolyze phytosterol esters was examined. Pancreatic cholesterol esterase hydrolyzed phytosterol esters, but the rate of hydrolysis proved sensitive to the structures of both the sterol and ester components. In the second study, cholesterol micellarization was challenged …

Inhibition Of Calcium Oxalate Calculi By Ndga - A Spectroscopic Study, Preethi Dacha

Open Access Theses & Dissertations

Formation of calculi in urinary tract leads to kidney stones. This process is known as Urolithiasis. Some of the causes of urolithiasis are metabolic disorders, genetic factors, and anatomic abnormalities in the urinary system. Nowadays, urolithiasis is very common disease in population of all over the world. The present study strives to understand calcium oxalate calculi formation as well as its inhibition by using Nordihydroguaiaretic acid (NDGA), which is a chemical extract of the Larrea Tridentata bush, a plant that is traditionally employed in the El paso region as a natural treatment for kidney stones and bladder diseases. Thus, in …

Phospholipid Analysis In Trypanosoma Cruzi, Melissa Rashonda Harris

Open Access Theses & Dissertations

Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi is one of the most endemic and deadly infectious diseases. T.cruzi is distributed throughout most of South and Central America, where it infects 12 to 19 million people, with an annual incidence of 561,000. Another 35 million are exposed to infection, 2 to 3 million people have clinical symptoms of chronic Chagas disease, and about 45,000 of these may die every year due to cardiac failure. In addition to the growing number of incidences in other countries the disease is an emerging infectious disease in the U.S due to the migration …

Free Radical Stress-Induced Parkinsonian Lewy-Like Aggregation Prevented Through Polyphenolic Phytochemical Analog Intervention: Implications For Subcellular Trafficking And Neurodegenerative Disorders, Rituraj Pal

Open Access Theses & Dissertations

Protein disulfide isomerase (PDI), the chief endoplasmic reticulum (ER)-resident oxidoreductase chaperone, is known to catalyze the maturation of disulfide-bond-containing proteins primarily through oxidation-reduction and isomerization functions. The rate-determining step in the oxidative regeneration path of disulfide-bond-containing proteins generally couples chemical thiol-disulfide-exchange reactions to a physical conformational folding reaction. I have determined the impact of PDI and its subdomains on the rate-determining step in ribonuclease A folding and on the physical structure-forming step of select ER-processed proteins including RNase A. This was facilitated through application of a novel chemical tool to exclusively populate native-disulfide-containing intermediates in unstructured forms. The described biochemical …

Molecular Mechanisms Of Poly [Adp-Ribose] Polymerase-1 In Hiv-1 Infection, Daniel Reyes

Open Access Theses & Dissertations

Poly (ADP-ribose) polymerase-1 (PARP-1) is a cellular enzyme involved in genome stability and transcriptional regulation. The role of this protein in HIV-1 infection is largely controversial. Some reports indicated a fundamental role of PARP-1 in HIV-1 DNA integration and results from other laboratories do not support these conclusions. An important characteristic in all these experiments is that the HIV-1 target cells that were used express, in addition to PARP-1, the functional homologue PARP-2. We evaluated the role of PARP-1 in the chicken B lymphoblastoid cell line DT40. These cells naturally lack PARP-2 and support the early steps of HIV infection. …

Structural And Functional Investigation Of Φ-El-Chaperonin Mediated Protein Folding, Sudheer Kumar Molugu

Open Access Theses & Dissertations

Chaperonins are ubiquitous, sequence related protein complexes that aid in the folding of nascent and misfolded polypeptides in an ATP driven pathway. Recently a GroEL-like, 860 kilo Dalton chaperonin protein complex was identified and isolated from the bacteriophage EL, a virus that infects the Gram-negative bacterium Pseudomonas aeruginosa. The bacteriophage EL contains 201 predicted open reading frames and is the only known phage that encodes for its own chaperonin known as Φ-EL-chaperonin.

To understand the importance of Φ-EL-chaperonin in phage EL life cycle, the recombinant Φ-EL-chaperonin protein was expressed in Escherichia coli (E. coli) purified to homogeneity and the structure …

Identification And Characterization Of Fkbp52-Specific Inhibitors For The Treatment Of Prostate Cancer, Johanny Tonos De Leon

Open Access Theses & Dissertations

Steroid hormone receptors require the ordered assembly of various chaperone and cochaperone proteins in order to reach a functional state. The final stage in the receptor maturation process requires the formation of a mutimeric complex consisting of Hsp90 dimer, p23, and one of several large immunophilins. Studies conducted previously demonstrated that the large immunophilin FKBP52 acts to potentiate glucocorticoid, androgen, and progesterone receptor signaling pathways. The aim of these studies was to identify and characterize FKBP52-specific inhibitors that would not only serve as tools for the pharmacological analysis of FKBP52-receptor interactions, but may also lead to novel drugs with significant …

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 …

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 …

Human Trophoblast Survival And Invasion In The Developing Placenta: Autocrine Regulation By Hbegf, Philip Jessmon

Wayne State University Dissertations

HBEGF is a multifunctional protein in early pregnancy that induces cytotrophoblast (CTB) cell differentiation to an invasive phenotype, protects against apoptosis, and is involved in an autocrine signaling mechanism that leads to its own protein synthesis. CTBs exist in a low O₂ environment during the first 10 weeks of implantation, during which they invade the decidualized uterine stroma. Inhibitors of intracellular signaling pathways demonstrated that at 20% O₂ HBEGF induces an increase in cell migration through the ERK, MAPK14, JNK, or PIK3 pathways downstream of signaling through its ERBB receptors. Also downstream of these four pathways, HBEGF induces …

Destruction Of Biological Tetrapyrrole Macrocycles By Hypochlorous Acid And Its Scavenging By Lycopene, Dhiman Maitra

Wayne State University Dissertations

Hypochlorous acid (HOCl) is a potent oxidant generated by the hemoprotein myeloperoxidase. Although HOCl plays an important role in the innate immune response,sustained high levels of HOCl has been implicated to play a harmful role. In several pathological conditions such as atherosclerosis, endometriosis and sickle cell disease where HOCl is elevated there are reports of significant free iron accumulation. Free iron is toxic since it can lead to the generation of other secondary free radicals such as hydroxyl radical by Fenton reaction. The exact source and mechanism by which the free iron is generated is not clearly understood. This work …

Mechanical Forces And Tumor Cells: Insight Into The Biophysical Aspects Of Cancer Progression, Indrajyoti Indra

Wayne State University Dissertations

Mechanical forces play an important role in the regulation of cellular behavior and physiological processes including adhesion, migration, proliferation, tissue repair, embryogenesis and development. In addition, a number of diseases including cancer, have been linked to changes in cellular and extracellular mechanical properties. However, whether a correlation exists between the progression of cancer towards metastasis and mechanical factors has not been clearly defined. Additionally, how a cell responds to changes in extracellular mechanical cues as it gains metastatic abilities is poorly understood. To address these questions, we have utilized a panel of murine breast cancer cell lines with progressive metastatic. …

Rox1 Function In Dosage Compensation: Structural / Functional Analysis Of A Non-Coding Rna, Ying Kong

Wayne State University Dissertations

roX1 is a long non-coding RNA involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation in Drosophila melanogaster occurs by a global two-fold increase of transcription from the single male X chromosome. This essential process compensates for X chromosome monosomy. The male-specific lethal (MSL) complex, containing five proteins, localizes to the male X chromosome and alters chromatin to modify gene expression. roX1 and roX2 RNAs are redundant components of MSL complex that are required for its exclusive X-localization. Recent studies in our lab have revealed a second role of roX RNAs in heterochromatic gene …