Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

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 …

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 …

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 …

Synthesis, Self-Assembly And Biological Properties Of Self-Folding Cu(Ii) Coordination Polymers, Alejandro Arzola-Rubio

Open Access Theses & Dissertations

The synthesis of materials via molecular self-assembly is a powerful bottom-up approach for fabricating matter in the nano- and micro-meter scales. This methodology involves the spontaneous and reversible organization of small molecules that interact with one another to create larger structures driven by non-covalent interactions such as hydrogen bonding, hydrophobic forces and metal-ligand coordination interactions. In this thesis, the design, synthesis and characterization of a new set of molecules that spontaneously self-organize in water to form three-dimensional nanoscale toroidal structures was investigated. These supramolecular structures were designed with an intrinsic affinity to bind and condense DNA strands with the intent …

Changes In Apparent Molar Water Volume And Dkp Solubility Yield Insights On The Hofmeister Effect, Daryl K. Eggers, A. Y. Payumo, R. M. Huijon, D. D. Mansfield, L. M. Belk, A. K. Bui, A. E. Knight

Faculty Publications, Chemistry

This study examines the properties of a 4 × 2 matrix of aqueous cations and anions at concentrations up to 8.0 M. The apparent molar water volume, as calculated by subtracting the mass and volume of the ions from the corresponding solution density, was found to exceed the molar volume of ice in many concentrated electrolyte solutions, underscoring the nonideal behavior of these systems. The solvent properties of water were also analyzed by measuring the solubility of diketopiperazine (DKP) in 2.000 M salt solutions prepared from the same ion combinations. Solution rankings for DKP solubility were found to parallel the …

The Specific Role Of The Mll Cxxc Domain In Mll Fusion Protein Function, Laurie Ellen Risner

Dissertations

The MLL gene was first identified because it is involved in chromosome translocations which produce novel fusion proteins that cause leukemia. The CXXC domain of MLL is a cysteine rich DNA binding domain with specificity for binding unmethylated CpG-containing DNA. The CXXC domain is retained in oncogenic MLL fusions, and is absolutely required for the fusions to cause leukemia. This project explored the role of the CXXC domain by introducing structure-informed point mutations within the MLL CXXC domain that disrupt DNA binding, and by performing domain swap experiments in which different CXXC domains from other proteins, including DNMT1, CGBP and …

Role Of Non-Coding Rna Nc4 In Mll And Cyp33 Mediated Regulation Of Hoxc8, Jessica Arvindbhai Solanki

Dissertations

MLL or Mixed Lineage Leukemia gene is clinically known for its involvement in genetic translocation with more than 70 different partners identified each giving rise to a highly leukemogenic fusion protein. With a poor prognosis of MLL related leukemia, an investigation into its role during hematopoiesis has been a very active field of research. In order to design strategies to combat the MLL leukemia, it becomes essential to delineate the molecular mechanisms behind the function of MLL wild type protein. Wild type MLL is a transcriptional maintenance protein from the Trithorax Group (TrxG) that resides in complex with other chromatin …

Wee1 Is A Biological Target Of The Mir-17-92 Cluster In Leukemia, Sonia Susan Olikara

Master's Theses

MicroRNAs are noncoding RNAs that bind to the 3' untranslated region of their mRNA targets, which causes downregulation of target gene expression. Previous studies have shown that the miR-17-92 cluster, which encodes six miRNAs, is overexpressed in leukemias arising from chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene. In the present study, prediction algorithms identified WEE1, a kinase that inhibits cell cycle progression, as a possible target of five of the six miRNAs. Through luciferase reporter assays, we found that miR-17, miR-20a, and miR-18a specifically target nucleotides 465 to 487 of the 3' UTR of WEE1, while miR-19a and …

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 …

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 …

A Study On The Biochemical And Cellular Effects Of Enviormental Tobacco Smoke On Adult And Developing Rat Brain Biochemistry, Brian Fuller

Theses and Dissertations

Exposure to environmental tobacco smoke (ETS) has been recognized as a significant health risk for adults and children. In adults, ETS exposure has been linked to increased incidences of cardiovascular disease and dementia. In children, exposure has been linked to behavioral and cognitive deficits. Studies on the effects of ETS in the brain have been largely epidemiological, and have lacked a defined explanation of the molecular/biochemical effects of ETS. The present dissertation aims to test whether ETS exposure leads to altered biochemistry in the adult and developing mammalian brain. A rat ETS exposure model was employed to investigate changes at …

Identification And Characterization Of The Human Herpesviruses 6a And 6b Genome Integration Into Telomeres Of Human Chromosomes During Latency, Jesse Herbert Arbuckle

USF Tampa Graduate Theses and Dissertations

While the latent genome of most Herpesviruses persists as a nuclear circular episome, previous research has suggested that Human Herpesvirus 6 (HHV-6) may integrate into host cell chromosomes, and be vertically transmitted in the germ-line. Because the HHV-6 genome encodes a perfect TTAGGG telomere repeat array at the right end direct repeat (DR_R) and an imperfect TTAGGG repeat at the end of the left end direct repeat (DR_L), we established a hypothesis that during latency, the HHV-6A and HHV-6B genome integrates into the telomeres of human chromosomes through homologous recombination with the n(TTAGGG) viral repeats, and …

Development Of An Elisa For Eastern Equine Encephalitis Virus That Can Differentiate Infected From Vaccinated Horses, Andrea Bingham

USF Tampa Graduate Theses and Dissertations

Eastern Equine Encephalitis virus (EEEV) causes a fatal mosquito-borne virus that is vaccine preventable for horses. The conventional serological tests measure antibodies to the structural proteins of EEEV which are also found in the vaccine. This makes it difficult to differentiate infected and vaccinated animals (DIVA). Detection of antibodies to non-structural proteins (NSPs) is a theoretical strategy that would allow you to survey natural infections among vaccinated populations. This test would also allow for more accurate representations of the natural infection rate, vaccination rate, and help identify vaccine failures. The potential uses of the NSPs of Eastern Equine Encephalitis virus …

Lead Discovery And Optimization Strategies Towards The Development Of 4(1h)-Quinolones And 1,2,3,4-Tetrahydroacridone Analogs With Antimalarial Activity, Richard Matthew Cross

USF Tampa Graduate Theses and Dissertations

The goal of our research endeavor was to successfully employ modern lead discovery and optimization strategies towards the development and identification of compounds possessing antimalarial activity. Preliminary data from in vitro screening at the Walter Reed Army Institute of Research identified several chemotypes including 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones to have potent antimalarial activities. Multiple synthetic routes were devised and implemented which enabled the rapid preparation and isolation of over 400 structurally diverse 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones.

Our research towards discovering and optimizing antimalarials was inspired from the severe impact malaria has had on our planet especially on impoverished countries. There are over …

Role Of Protein Kinase C-Iota In Glioblastoma, Shraddha R. Desai

USF Tampa Graduate Theses and Dissertations

The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma.

The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of …

Multivariate Anti-Inflammatory Approaches To Rescue Neurogenesis And Cognitive Function In Aged Animals, Sandra Antonieta Acosta

USF Tampa Graduate Theses and Dissertations

Studies have shown that there is a strong correlation between aging and neurodegenerative diseases. Aging is considered the number one risk factor to develop neuropathologies such as memory loss, senile dementia, Alzheimer's disease (AD), and Parkinson's disease. Neurodegenerative diseases tend to start during adulthood, and aggravate over time, making them difficult to prevent and to treat. In the Unites States, demographic studies by U.S. Bureau of the Census have determined that our aging population of >65 years is expected to increase from the present 35 million to 78 million in 2030. This would result, not only to an increase of …

The Role Of Brca1 Domains And Motifs In Tumor Suppression, Aneliya Velkova

USF Tampa Graduate Theses and Dissertations

Individuals that carry deleterious mutations in the breast and ovarian cancer susceptibility gene 1 (BRCA1) have much more elevated risk to develop breast and/or ovarian cancer than the individuals from the general population. The BRCA1 gene product has been implicated in several aspects of the DNA damage response, but its biochemical function in these processes has remained elusive. In order to probe BRCA1 functions we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. In this dissertation, we report the identification of the actin-binding protein Filamin A (FLNA) …

From Dna To Protein: A Study Of Genomic Instability Candidate Genes During Zebrafish Development, Kristine Griffett

USF Tampa Graduate Theses and Dissertations

The zebrafish, Danio rerio, is a type of freshwater minnow often used to model human diseases including cancer, anxiety and aging diseases. The overall biology of zebrafish is strikingly similar to that of humans, allowing these fish to be used for drug discovery and toxicology studies for preclinical trials. In this study, zebrafish embryos were used to identify and characterize several candidate genes within two known regions of genomic instability on chromosome 18 and chromosome 4. This fish that were used in this study had been previously classified as genomic instability (gin) mutants due to increased incidence of somatic mutation …

Expression Of Genes Involved In A Radiation-Induced Bystander Effect, Hayley Furlong

Conference Papers

The radiation induced bystander effect is relevant to carcinogenesis, it may have significant implications for risk estimation for radiation exposure. Currently the mechanisms and cellular events are the subject of intense investigation, because little is known. It is thought that the radiation induced bystander response is due to a bystander factor secreted in the medium post irradiation. However the biological nature of this factor is currently unknown, but it is thought to be a protein of some sort that may be involved in the apoptosis cascade. Materials and Methods: HaCaT epithelial cells were used in this study and exposed to …

Lead Identification Of Β-Lactam And Related Imine Inhibitors Of The Molecular Caperone Heat Shock Protein 90, Niamh O'Boyle, Andrew Js Knox, Trevor P. Price, D. Clive Williams, Daniela M. Zisterer, David G. Lloyd, Mary J. Meegan

Articles

Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the identification of inhibitors based on β-lactam and imine templates. β-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC₅₀ values of 5.6 μM, 14.5 μM and 22.1 μM respectively. The binding affinity displayed …

Synthesis, Biochemical And Molecular Modelling Studies Of Antiproliferative Azetidinones Causing Microtubule Disruption And Mitotic Catastrophe, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Niall O. Keely, Andrew Js Knox, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

Articles

The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC₅₀ value of 0.22 μM. The mechanism of action was demonstrated to be by inhibition of tubulin. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for …

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 …

Tca Cycle Inactivation In Staphylococcus Aureus Alters Nitric Oxide Production In Raw 264.7 Cells, Chandirasegaran Massilamany, Arunakumar Gangaplara, Donald J. Gardner, James M. Musser, David J. Steffen, Greg A. Somerville, Jay Reddy

Jay Reddy Publications

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts …

Gender Differences In Cns Autoimmunity Induced By Mimicry Epitope For Plp 139–151 In Sjl Mice, Chandirasegara Massilamany, Sivasubramani Thulasingam, David Steffen, Jay Reddy

Jay Reddy Publications

Development of multiple sclerosis (MS) is more prevalent in females than in males, but the underlying mechanisms are not clear. Microbial infections have been suspected as triggers of MS and it is not known whether gender differences in reactivity to environmental antigens contribute to the disease pathogenesis. We demonstrated that ACA 83–95, a mimicry epitope from Acanthamoeba castellanii for proteolipid protein (PLP) 139–151, induces clinical signs of encephalomyelitis in both male and female SJL mice. Conversely ACA 83–95-induced effector cells from males fail to induce disease in female mice. Although we found no gender differences in the frequencies of antigen-specific …

Rnai Screening Reveals Requirement For Host Cell Secretory Pathway In Infection By Diverse Families Of Negative-Strand Rna Viruses, Debasis Panda, Anshuman Das, Phat X. Dinh, Sakthivel Subramaniam, Debasis Nayak, Nicholas J. Barrows, James L. Pearson, Jesse Thompson, David L. Kelly, Istvan Ladunga, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genomewide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of …

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 …

Y-Like Retinal Ganglion Cells Innervate The Dorsal Raphe Nucleus In The Mongolian Gerbil (Meriones Unguiculatus), Liju Luan, Chaoran Ren, Benson Wui-Man Lau, Jian Yang, Gary E. Pickard, Kwok-Fai So, Mingliang Pu

School of Veterinary and Biomedical Sciences: Faculty Publications

Background: The dorsal raphe nucleus (DRN) of the mesencephalon is a complex multi-functional and multi-transmitter nucleus involved in a wide range of behavioral and physiological processes. The DRN receives a direct input from the retina. However little is known regarding the type of retinal ganglion cell (RGC) that innervates the DRN. We examined morphological characteristics and physiological properties of these DRN projecting ganglion cells.

Methodology/Principal Findings: The Mongolian gerbils are highly visual rodents with a diurnal/crepuscular activity rhythm. It has been widely used as experimental animals of various studies including seasonal affective disorders and depression. Young adult gerbils were used …