Medicine and Health Sciences Commons^™

Structural Insights Into The Cl-Par-4 Protein: Ionic Requirements, Conformational Transitions, And Interaction With Cisplatin, Krishna Kumar Raut

Chemistry & Biochemistry Theses & Dissertations

Cancer continues to be the leading global cause of death, with challenges in early diagnosis, drug resistance, non-specific drug targeting, and cancer recurrence and metastasis posing formidable obstacles in cancer therapy. In this context, Prostate Apoptosis Response-4 (Par-4), a pro-apoptotic tumor suppressor protein, emerged as a promising therapeutic target due to its ability to selectively induce apoptosis in cancer cells, thereby minimizing the drug-associated adverse effects. However, a comprehensive understanding of the structural features of Par-4, specifically the caspase-cleaved fragment (cl-Par-4), is crucial for therapeutic advancements.

This dissertation investigated the effects of various ions, both monovalent and divalent, on the …

Biophysical Characterization Of The Par-4 Tumor Suppressor: Evidence Of Structure Outside The Coiled Coil Domain And Interactions With Platinum Chemotherapeutics, Andrea Megan Clark

Chemistry & Biochemistry Theses & Dissertations

Prostate apoptosis response-4 (Par-4) is an apoptosis-inducing tumor suppressor protein. Full-length Par-4 has previously been shown to be a predominantly intrinsically disordered protein (IDP) under neutral conditions, with significant regular secondary structure evident only within the C-terminal coiled coil domain. However, IDPs can gain ordered structure through the process of induced folding, which often occurs under non-neutral conditions. Previous work has shown that the Par-4 leucine zipper, which is a subset of the C-terminal coiled coil domain, is disordered under neutral conditions, but forms a dimeric coiled coil at acidic pH. Increase in ionic strength was also shown to increase …

Network Approaches To Elucidate The Determinants Of Protein Topology And Stability, Zeinab Haratipour

Chemistry & Biochemistry Theses & Dissertations

Predicting three-dimensional structures of proteins from sequence information alone, remains one of the most profoundly challenging and intensely studied problems in basic science. It has uniquely garnered the interdisciplinary efforts of biologists, biochemists, computer scientists, mathematicians and physicists. The advancement of computational methods to study fundamental features of proteins also enables insights that are either difficult to explore experimentally or complimentary to further interpret experimental data. In the present research and through the combined development and application of molecular dynamics and network science approaches we aimed to elucidate the role of geographically important amino acids and evolutionarily conserved long-range interactions …

Characterizing The Activity Of Antimicrobial Peptides Against The Pathogenic Bacterium Clostridium Difficile In An Anaerobic Environment, Adenrele Mojeed Oludiran

Chemistry & Biochemistry Theses & Dissertations

Clostridium difficile is an anaerobic Gram-positive pathogen with high treatment costs and mortality and very high antibiotic tolerance. Antimicrobial host-defense peptides (HDPs) produced naturally by animal immune systems are promising candidates to develop novel therapies for bacterial infection because they cause oxidative stress that damages multiple targets in bacterial cells, so it is difficult for bacteria to evolve resistance to these attacks.

Piscidins, fish-derived HDPs that can also form complexes with copper (Cu) to enhance their activities, are very active against multiple bacterial species in an aerobic environment. We examined their activity against C. difficile and other species in an …

Structure Of The Picornavirus Replication Platform: A Potential Drug Target For Inhibiting Virus Replication, Meghan Suzanne Warden

Chemistry & Biochemistry Theses & Dissertations

Picornaviruses are small, positive-stranded RNA viruses, divided into twelve different genera. Members of the Picornaviridae family cause a wide range of human and animal diseases including the common cold, poliomyelitis, foot and mouth disease, and dilated cardiomyopathy. The picornavirus genome is replicated via a highly conserved mechanism involving a presumed cloverleaf structure located at the 5’ noncoding region of the virus genome. The 5’ cloverleaf consists of three stem loops (B, C, and D) and one stem (A), which interact with a variety of virus and host cell proteins during replication. In this dissertation, human rhinovirus serotype 14 (HRV-14) SLB …

Design Of Drug Nano-Carriers For Study Of Multidrug Resistance In Single Live Cells, Pavan Kumar Cherukuri

Chemistry & Biochemistry Theses & Dissertations

Multidrug resistance (MDR) exists in both prokaryotic and eukaryotic cells. MDR is responsible for ineffective treatment of a wide range of diseases, such as infections and cancer. The ATP-binding cassette (ABC) membrane transporters (efflux pumps) are one of the largest and most diverse super-families of membrane proteins found in all living organisms, ranging from bacteria to humans. All ABC transporters share a common structure of four core domains; two transmembrane domains (TMD) with variable sequence and topology and two nucleotide-binding domains (NBD) with conserved sequences. Conventional methods for the study of the efflux functions are radioactively labeled substrates and fluorescent …

Identification Of Persistent Long Range Interactions In GA95 And GB95 Through Thermal Unfolding Simulations, Milen Redai Tesfamariam

Chemistry & Biochemistry Theses & Dissertations

For over five decades, different experiments have been performed to research how proteins attain their native three dimensional structures. However, the folding problem continues to be a puzzle in modern science. The design of two proteins that have maximal sequence identity but different folds and functions is one method that is being used to study the relationship between protein structure and amino acid sequence. In particular, mutant proteins of Streptococcus protein G, G_A and G_B, have 95% sequence identity and a 3a helix fold and β4/a fold, respectively. Molecular dynamics simulations of G_A95 …

Natural And Synthetic Viniferins Associated With The Grapevine Disease Young Vine Decline, David Michael Mcginnis

Chemistry & Biochemistry Theses & Dissertations

Grapevine disease has been thc subject of intense research amongst viticulturists over the last few decades, especially during the 1990's. There has been discoveries that suggest grapevine disease is commonly caused by fungal pathogens. One of the most common fungi that the vine may become infected by is known as Botrytis cinerea. B. cinerea is capable of attacking the grapevine which in turn will lead to bunch rot in the grape clusters. This disease has been researched in great detail during the past several years and is one of only few microorganisms that have actually been identified. Phaeoacremrmium chnlmydospnrum …

Production Of A Monoclonal Antibody Against Benzo[Α]Pyrene Diol Epoxide Dna Adducts, Brian Peden Austin

Chemistry & Biochemistry Theses & Dissertations

Benzo[α]pyrene is a ubiquitous pollutant produced from the incomplete combustion of organic material such as fossil fuels. It is found in the workplace, urban air, drinking water, and the food supply. Recently, it has been proposed that benzo[α]pyrene may be the causative agent in the formation of lung adenocarcinomas among some Taiwanese women exposed to cooking oil fumes without adequate ventilation. In this study, calf thymus DNA was modified in vitro with benzo[α]pyrene-diol epoxide (BPDE) to a level consistent with that found in biological samples. This DNA of low modification was used as an immunogen in the production of a …

Positive Regulation Of Pka On Human Gonadotropin-Releasing Hormone (Hgnrh) Gene Expression In Human Placental Jeg-3 Cells, Zhaoyang Wen

Chemistry & Biochemistry Theses & Dissertations

Using the human placental choriocarcinoma JEG-3 cell line as an in vitro human placental model, we studied the mechanisms of the PKA positive regulation of the hGnRH gene expression in the human placenta. Studies in JEG3 cells showed that through the PKA catalytic subunit a, human GnRH upstream promoter activity was stimulated by PKA signaling pathway in a cAMP dependent mechanism. The sequence between —202 (Afl II) and —554 (BamH I) base pair in the human GnRH upstream promoter region appeared to be responsible for the PKA positive regulation of the gene expression. Furthermore, Western blot analysis demonstrated the involvement …

Induction Of Apoptosis In Human Prostate Cancer Cells By Resveratrol, Gary Zulfikar Morris

Chemistry & Biochemistry Theses & Dissertations

Recently attention has been brought to trans-resveratrol's {TR) anticancer activity, as determined through a number of cultured cancer cell models. This activity was attributed to TR behaving as an estrogen, and the orientation of TR' s hydroxyl groups. Based on this work it was of interest to determine whether TR would also be toxic in prostate cancer cells; if toxic, did TR induce necrosis or apoptosis in the cells; was it toxic through hormone mediated pathways; and were TR's hydroxyl groups responsible for its biological activity. To this end, cellular viability was assessed in two different prostate cancer cell …

A Study On The Effects Of The N-Terminal Amino Acid Sequence On The Activation Of Human T-Cell Leukemia Virus Type 1 Protease, Hidayah Muhammad Kendall

Chemistry & Biochemistry Theses & Dissertations

Human T-cell leukemia virus type 1 (HTL V-1) is dependent upon the enzymatic activity of its protease for maturation. Maturation of the protease is facilitated by cleavage of specific amino acid residues, followed by dimerization. The effects of the amino acid sequence located N-terminally to the cleavage site on the ability of the protease to become active were the focus of the current study. These amino acid sequences were contributed by the plasmid vector into which the protease gene was inserted.

Surface probability analyses (SPAs) of the vectors, as well as for native sequences which produce the mature protease and …

Generation Of Human Chorionic Gonadotropin Beta Subunit Specific Monoclonal Antibody Using A Synthetic Peptide, Zhe Huang

Chemistry & Biochemistry Theses & Dissertations

Human chorionic gonadotropin (hCG) is well known for early immunological and bioassay tests for early detection of pregnancy. Since the hormone specific beta subunit confers unique biological and immunological specificity to hCG, immunochemical measurement of hCG has achieved greater specificity and sensitivity in recent years by using either the beta subunit as an immunogen or a partially reduced beta subunit polypeptide chain. However, antiserum raised against the entire hCG beta subunit usually cross-reacts to some extent with human luteinizing hormone (hLH). hCG has been found to be present in the extract of tumors and minute quantities of hCG are synthesized …

Determination Of The N-Terminal Amino Acid Residues On Polypeptides In Secondary Wastewaters, Edward L. Creecy

Chemistry & Biochemistry Theses & Dissertations

The N-terminal amino acid residues on polypeptides and proteins were determined in wastewaters prior to chlorination. The terminal amino groups'ere first derivatized with the well known derivatizing agent dansyl chloride, and then the resulting dansyl amino acid hydrolyzed from the peptide chain by a propionic acid/ hydrochloric acid mixture. The resulting dansyl amino acids were then separated and detected using reversed phase high performance liquid chromatography with fluorescence detection. The majority of N-terminal residues detected were the more polar amino acids. Concentrations ranged from 1 x 10^-10 to 3 x 10^-7 moles/liter. It is suggested that the shorter …

Development Of Fluorescent Techniques In The Kinetic Characterization Of Reverse Transcriptase, Kevin Kevin Howerton

Chemistry & Biochemistry Theses & Dissertations

Reverse transcriptase (RT) is an RNA-directed DNA polymerase isolated from human immunodeficiency virus (HIV) and other retroviruses. It has been the primary target for anti-HIV research since the discovery of its vital role in the retroviral life-cycle. The conventional method for carrying out kinetic characterization and inhibition studies of reverse transcriptase involves the use of radioactive materials. Although this method is effective, it is both time consuming, due to a necessary repeated washing step, and hazardous to the worker and environment. Furthermore, strict laws limit the use of radioactive materials to licensed workers and laboratories. In response to these concerns, …

Developmental And Hormonal Regulation Of The Expression Of Cytosolic Phospholipase A2 Protein Levels In Baboon Placenta, Kathleen Elizabeth Dunstan

Chemistry & Biochemistry Theses & Dissertations

Cytosolic phospholipase A₂ ( cPLA₂) specifically catalyzes the release of arachidonate from membrane phospholipids, thus initiating synthesis of prostaglandins, thromboxanes, and other eicosanoids. The human and nonhuman primate placenta actively synthesize high levels of eicosanoids which play both an autocrine and paracrine role in placental function. Previous studies in Dr. Miriam D. Rosenthal's laboratory have shown that cPLA₂ mRNA levels are developmentally regulated. The present study was designed to determine whether placental cPLA₂ protein levels increased during gestation. Since the marked increases in estrogen levels during normal pregnancy have been shown to enhance morphological and …

The Effect Of Growth Hormone Release On Plasma And Erythrocyte Selenium And Zinc Levels In Children, Maria Estela Legal De Reid

Chemistry & Biochemistry Theses & Dissertations

Research studies have suggested that selenium may be important in the release of growth hormone, GH. A selenium-containing amino-acid residue, selenocysteine, has been identified as part of the active site of the three isoenzymes (Type I, II and III iodothyronine 5' -deiodinase) responsible for the synthesis of active thyroid hormone, T_3, which is reported to stimulate GH secretion. The importance of zinc in growth is well- documented because of its involvement in transcription and expression of GH and its intermediary, insulin growth factor I or IGF- I. Thus, selenium and zinc levels in plasma may be altered in response …

An Enzyme-Linked Immunosorbent Assay For The Detection Of Cardiac Myoglobin Using Monoclonal Antibodies, Deborah F. Kelly

Chemistry & Biochemistry Theses & Dissertations

The most prevalent life-threatening disease worldwide, secondary to coronary artery thrombosis is Acute Myocardial Infarction (AMI). Increases in three clinical markers have been explored as an early means of AMI detection: Cardiac myoglobin, and the two specific cardiac isoforms of Creatine Kinase, CK-MM and CK-MB. However, myoglobin levels have shown to give a more accurate correlation (R = 0.89) than does Creatine Kinase evaluation. Therefore, we have developed a two-site Enzyme-Linked Immunosorbent Assay (ELISA) using monoclonal antibodies against myoglobin isolated from human heart tissue. This assay is based on specific anti-genantibody interactions and is sensitive at the nanogram level. We …

Competitive Enzyme Immunoassay Of Catechin In Human Blood Serum, Jay Fedorowicz

Chemistry & Biochemistry Theses & Dissertations

( +)-Catechin is a type of polyphenolic compound, and it is enriched in fruits, plants, and various beverages. Polyphenolic compounds also play an important role in the human diet. Many scientists, especially wine researchers, have proposed that(+)catechin and its polymers could have many positive health effects. Thus, it is essential to learn more about (+)-catechin and other polyphenolics. Presently, high performance liquid chromatography is the only method to detect ( + )-catechin levels in human blood serum, but this method has the high cost of instrumentation and does not provide good sensitivity. The objective of this project is to develop …

The Kinetic Characterization Of Mutant L289p Of Recombinant Hiv-1 Reverse Transcriptase, Tryn Thomas Stimart

Chemistry & Biochemistry Theses & Dissertations

The Human Immunodeficiency Virus, type 1 (HIV-1) is the causative agent of the Acquired immunodeficiency Syndrome (AIDS). Currently, AIDS is the leading cause of death amongst young people in the United States. The large increase of HIV-1 infections over the past decade has spawned considerable research in fighting the spread of this disease. One area of intense HIV-1 research has focused on the enzyme Reverse Transcriptase (RT) which is an essential enzyme involved in the replication and life cycle of HIV-1. HIV-1 RT is composed of p66 and p51 subunits and is only active as a heterodimer in vivo. …

Structural Characterization Of A Novel Inhibitor Of Hiv Reverse Transcriptase (Hiv Rt), Greggory Jon Woitte

Chemistry & Biochemistry Theses & Dissertations

Human immunodeficiency virus (HIV) infections have become a leading cause of death among young people in the United States today. As the number of HIV infections increases, so too does the cost of treatment. Together, these numbers have prompted an increase in the development of pharmaceutical interventions. HIV reverse transcriptase (HIV RT) has become a suitable target for drug therapy because it is the sole enzyme responsible for HIV replication.

Fucoidan, a sulfated polysaccharide isolated from the brown algae Fucus vesiculosus, has been shown to block a variety of cell adhesion related events including metastasis. In addition, fucoidan has also …

Investigation Of Selenium Status In Hypo-, Hyper- And Euthyroid Children, Ping Sun

Chemistry & Biochemistry Theses & Dissertations

Recently, it has been reported that human type I iodothyronine deiodinase, an enzyme important in the conversion of T₄ to T₃ , is a selenoenzyme. Several studies have reported alterations in plasma selenium level in hypo- and hyperthyroid patients.

Using polarized Zeeman-effect atomic absorption spectroscopy, we measured selenium, zinc, copper and manganese concentrations in the plasma and/or red blood cells in children with or without thyroid disease being seen at the outpatient clinic at Children's Hospital of the King's Daughters. Children with thyroid disease were subdivided into untreated, treated and nonresponsive groups. Data were analyzed using Student's t-test …

The Isolation And Identification Of Polyphenolics From Grape Seeds And Their Activity Towards Specific Cell Lines, Joseph A. Reddy

Chemistry & Biochemistry Theses & Dissertations

Grape seed extract is being marketed in France for cardiovascular use and skin care. The active constituents of both these drugs are certain polyphenols called procyanidins. A similar seed extract has now been obtained from Virginia Chardonnay grape seeds. Ten of the major components in this extract have been isolated in their pure form by the semi-preparative HPLC separation on a YMC ODS-AQ (Octadecylsilane-AQ) column. The ODS-Aq column has been shown to have a better resolving capacity compared to the Spherisorb ODS (Octadecylsilane) column previously described in the literature. This has been attributed to the ODSAQ support which has both …

Longitudinal Study Of Plasma And Erythrocyte Selenium Levels In Type I Diabetic Children During Development And Treatment Of Ketoacidosis, Pi-Hsia Fan

Chemistry & Biochemistry Theses & Dissertations

Most studies on the biological activity of selenium have focused on its role at the active site in the ubiquitous enzyme, glutathione peroxidase (GSH-Px) which catalyzes the removal of hydrogen peroxide and organic hydroperoxides (1). A connection between selenium deficiency and a wide range of diseases in animals and humans has been reviewed (2). Development of various forms of human cancer (3), failure of the immune system (4), as well as some cardiovascular diseases (5) have been reported or suspected to correlate with selenium deficiency. However, the physiological function of selenium in these different diseases is not fully understood, and …

Separation And Quantification Of Queuine-Modified And Unmodified Transfer Rna Isoacceptors, Jorge Miguel Pereira De Oliveira Da Silva Santos

Chemistry & Biochemistry Theses & Dissertations

The discovery of a base-exchange modification in the first position of the anticodon of four transfer RNA (tRNA) isoacceptor families has been shown to be correlated with a decrease in the rate of cell division in several tumorigenic tissues. This fact led to the hypothesis that wobbling in the anticodon might be a control point wich mediates the rate of translation. A suitable method for separation of different tRNA isoacceptor families had to be created in order to generate a means for quantitatively measuring degrees of this base modification in tRNA. Radio labelled amino acids were used to charge tRNA …

Kinetic Characterization Of A Recombinant C-Terminal Mutant Of Reverse Transcriptase From The Human Immunodeficiency Virus, Thomas S. Heard

Chemistry & Biochemistry Theses & Dissertations

The human immunodeficiency virus (HIV) reverse transcriptase (RT) (EC 2.7.7.49) is the central replication enzyme for HIV. In general, the kinetic mechanism for this and all other polymerases involves the ordered binding of two substrates: a primer-template (PT) followed by a deoxyribonucleoside triphosphate (dNTP). Previous investigations prompted this research when it was discovered that the substrate dNTP, in absence of PT, could protect a recombinant c-terminal mutant HIV-1 RT from inhibition by pyridoxal-5'-monophosphate (PLP), an active-site dNTP inhibitor. In contrast, the non-mutant recombinant HIV-1 RT required both substrates for protection from PLP inhibition. This investigation sought to determine if this …

Biochemical Investigation Of Gout And Its Familial Incidences, Chin-Ling Hsu

Chemistry & Biochemistry Theses & Dissertations

Gout is a chronic metabolic disorder caused by deposition of uric acid on the joint. It is categorized into two types: primary and secondary. Primary gout is uric acid overproduction, caused by excessive synthesis of the purine precursors. Secondary gout occurs also as the result of overproduction or decreased renal excretion of uric acid resulting from other disease processes or medication.

The two purine bases, hypoxanthine and xanthine, liberated from ribosides by the action of nucleoside phosphorylase, are degraded to uric acid as the final excretory product in the purine degradation pathway. Hypoxanthine and xanthine are the physiological substrates of …

High Performance Liquid Chromatographic Separation Of Different Types Of Collagen, Yongjoo Chung

Chemistry & Biochemistry Theses & Dissertations

The objective of this research was to develop a method for the separation of different types of collagen using a large pore size (100 nm) reversed-phase C-8 column. Three different types of collagen (type I, III, and V) were sufficiently separated on the column using a mobile phase containing water-acetonitrile-trifluoroacetic acid. Collagens found in each HPLC peak were identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assay the column effluents.

Trifluoroacetic acid (TFA) was used as an ion pair reagent. This compound improved the chromatographic profile for these proteins on a reversed-phase column. It was observed that with …

Activation In Vitro Of Transfer Rna-Guanine Ribosyltransferase By Protein Kinase C, Panayota Eriotou

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to isolate and purify the enzymes, transfer RNA-guanine ribosyltrasferase and protein kinase C, and to determine whether the phosphorylating enzyme activates the insertion enzyme in vitro. Transfer RNA-guanine ribosyltransferase lost activity within several days after isolation and total, complete reactivation was accomplished in the presence of protein kinase C. This demonstrated that ribosyltransferase's instability is related to the degree of its phosphorylation. It is proposed that modification of tRNA is controlled by protein kinase C. Deactivation of the insertion enzyme leads to hypomodified tRNA which in turn is associated with neoplasia. Potential use …

Development Of A Solid Phase Enzyme Immunoassay For Total Serum Testosterone, Li Li

Chemistry & Biochemistry Theses & Dissertations

An enzyme-labeled immunoassay of sufficient range and sensitivity was developed for serum testosterone determination. In this method, the testosterone in the serum sample and testosterone-HRPO conjugate compete with each other for the limited antibody binding sites on the anti-testosterone gamma globulin coated microwell. The assay can be performed in only 2.5 hours.

The gamma globulin fraction was isolated from the anti-testosterone whole serum by DEAE-cellulose column chromatography. Testosterone-peroxidase conjugate was prepared by a carbodiimide coupling method. The interference which arises from the binding of testosterone to steroid hormone binding protein was eliminated by the addition of 17 beta-estradiol. All assay …