Molecular Biology Commons^™

Investigating The Biorisk Of Genetically Engineered Thermosynechococcus Elongatus Bp1, Cherrelle Leah Barnes

Chemistry & Biochemistry Theses & Dissertations

Cyanobacteria, also known as blue-green algae, are an ancient group of microorganisms that use simple materials, such as sunlight, carbon dioxide and water, to produce energy while providing oxygen to the atmosphere by performing photosynthesis. Synthetic biology approaches have been employed with cyanobacteria as a platform to produce a range of products, such as biofuels, by inserting a series of genes into the cyanobacterial genome that will allow the conversion of metabolic intermediates to such desired products. Although these methods are promising, it is important to understand any potential bio-risk they pose. This research evaluates the potential bio-risk of genetically …

Mechanisms Of Stress Survival In Gram Positive Bacterial Pathogens, Asia Poudel

Chemistry & Biochemistry Theses & Dissertations

Anaerobic Gram-positive bacteria are not a well-characterized group but include many human pathogens that are resilient against stresses caused by the human immune system or by antibiotic treatment. This dissertation investigated the survival mechanisms of two clinically relevant Gram-positive organisms, Clostridioides difficile and Cutibacterium acnes under extracellular stresses. The response of the opportunistic skin pathogen Cutibacterium acnes to nanosecond electric pulses is characterized and found that growth in a biofilm, which usually protects bacteria from stress, renders this species more killable by this treatment. In addition, the stringent response (SR), a conserved bacterial stress survival mechanism, is studied in 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 …

Synthesis And Mechanistic Investigations Of Transition Metal Complexes And Ligands For Chemotherapeutic Applications, Jimmie Lee Bullock Jr.

Chemistry & Biochemistry Theses & Dissertations

Transition metal complexes have played a critical role in antitumor therapy with many complexes incorporating platinum, ruthenium, and lanthanum having been investigated in preclinical and clinical trials. The best known transition metal therapeutic is cisplatin, which is utilized in nearly 50% of all cancer therapies, despite its significant toxic side effects. The toxic side effects of current FDA approved platinum-based chemotherapeutics are often overlooked due to the “special status” granted to these drugs due to their ability to fight, what is often considered an incurable disease with life expectancies often measured in months. Oncology drug development has therefore now focused …

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 …

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 …

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. …

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 …

Separation Of Transfer Rna Isoacceptors By Reversed Phase W-Porex C-4 High Pressure Liquid Chromatography, Christine D. Witte

Chemistry & Biochemistry Theses & Dissertations

Transfer RNA (tRNA), is a complex class of small molecules which have been proven to play a major role in protein synthesis. Transfer RNA is composed of 20 families of isoacceptors which may recognize only one particular amino acid. This family of isoacceptors may differ from each other by primary sequence or modified nucleosides. Queuine is a modified nucleoside which is found in the anticodon regions of tRNA isoacceptors for asparagine, aspartic acid, tyrosine and histidine. Queuine has gained the interest of many researchers since queuine deficient tRNA was found in many neoplastic and undifferentiated cell-lines. It is often difficult …

Regulation Of Queuine Insertion Into Transfer Rna: Effects Of Tumor Promoters, Bonnie Jean Brooks

Chemistry & Biochemistry Theses & Dissertations

The purpose of this study was to treat normal human fibroblasts with known tumor promoters and observe queuine modification of tRNA. The queuine insertion enzyme, tRNAguanine ribosyltransferase, was studied in vivo and in vitro. Tumor promoter-treated human fibroblast cultures exhibited variable queuine-insertion rates with a transient inhibition that correlated with variable levels of queuine modified tRNA over time in culture from passages 3 — 8. In contrast the in vitro studies showed that phorbol cetera and saccharin actually increased insertion with strong evidence indicating phosphorylation as a positive modulating force of the enzyme activity. It is proposed that chronic stimulation …

Microwell Based Competitive Enzyme Immunoassay For Quantifying Serum Triiodothyronine And Thyroxine Levels, Yiwen Yang

Chemistry & Biochemistry Theses & Dissertations

A microwell based competitive enzyme immunoassay for determination of total thyroxine and triiodothyronine in human serum was developed with use of horseradish peroxidase conjugate. The assay, which can be performed in only 1.5 hours at room temperature, involves simultaneous incubation of T₃- or T₄- HRPO conjugate and serum sample in anti-T₃ or anti-T₄ gamma globulin fraction coated polystyrene microwells.

Isolation of anti-T₃ and anti-T₄ gamma globulin fractions were accomplished from whole antiserum by using DEAE-cellulose chromatograph. A carbodiimide coupling method was described to prepare the T₄- and T₃-HRPO …

Hypoxanthine-Induced Differentiation Of Cultured Human Leukemia Cells, Gayle Jennette Singleton

Chemistry & Biochemistry Theses & Dissertations

Human cultured leukemia cells appear to have a decreased amount of inosine in their tRNA. When cells with inosine deficient tRNA are placed in a hypoxanthine fortified media, they incorporate hypoxanthine into their tRNA by the action of the enzyme tRNA-hypoxanthine ribosyl transferase. This generates the nucleoside inosine in the tRNA. The cultured human leukemia cell lines, CCRF-CEM, HL-60, and HGPRT(-) HL- 60, incorporate hypoxanthine into their tRNA, as determined by tRNA isolation, hydrolysis, and HPLC analysis. Hypoxanthine treatment dramatically inhibited cell growth in conjunction with partial induction of differentiation in the CCRF-CEM, HL-60, and HGPRT ( - ) HL-60 …

A Method For The Large Scale Purification Of Hcg (Human Chorionic Gonadotropin) From The Urine Of Pregnant Women, Chun-Shyan Kuo

Chemistry & Biochemistry Theses & Dissertations

A large scale preparation of human chorionic gonadotropin (HCG) from the urine of pregnant women was accomplished by using kaolin adsorption, NH₄OH extraction and DEAE-cellulose ion exchange. A recovery about 81% of HCG and a specific activity in the 2,000-3,000 IU/mg range was achieved using this purification method.

Two HCG activity fractions, HCG I & HCG II, were isolated from DEAE-cellulose chromatography by a stepwise sodium chloride gradient elution. HCG I was eluted at 0.01-0.02 M NaCl while HCG II was eluted at 0.12-0.02 M NaCl.

The differences between HCG I and HCG II have been studied. The …

Modulation Of Queuine Uptake In Cultured Human Fibroblasts By Phorbol Esters And Interferons, Debra L. Crane

Chemistry & Biochemistry Theses & Dissertations

Queuosine, a nucleoside found exclusively in the first position of the anticodon of transfer RNA (tRNA), is generated post-transcriptionally in an exchange of guanine for queuine by the modifying enzyme tRNA-guanine ribosyltransferase. Phorbol ester tumor promoters were shown to induce queuine hypomodified tRNA, and that phorbol ester action was due, in part, to inhibition of queuine transport across the cell membrane. An unidentified protein designated conditioned medium factor (CMF) that potentiated a phorbol-induced inhibition of queuine uptake was also documented. We suggest here that phorbol ester-induced inhibition of queuine uptake is not a significant factor in inducing queuine-deficient tRNA although …

Determination Of Erythrocyte Zinc Concentrations Using Zeeman Effect Atomic Absorption Spectroscopy, Tanya Maranda Lewis

Chemistry & Biochemistry Theses & Dissertations

The purpose of this research project was to develop assay procedures for determination of zinc in erythrocytes. Three methodologies were developed. The methods were evaluated on the basis of within-run and between-run precision. The most reproducible method, the 213.856 nm flame methodology, was used to determine red blood cell zinc concentrations in more than 200 samples obtained from the Neonatal Intensive care Unit at the Children's Hospital of the King's Daughters.

This study demonstrated that erythrocyte zinc levels in premature infants were similar to those of healthy adults. A linear relation between plasma and red blood cell zinc concentrations was …

Isomers Of 5.8s Ribosomal Rna, David Smith

Chemistry & Biochemistry Theses & Dissertations

When whole cell RNA was separated by high resolution PAG (i.e., polyacrylamide gel) electrophoresis, 5.8S rRNA migrated as three higher mobility intense bands, and three faint bands of lower mobility. The three intense bands represent conformational isomers which migrated as a single band under denaturing conditions on PAG. The three faint bands represent conformational isomers of a minor form of 5.8S rRNA which is elongated at the 5' terminus of the molecule. This minor form migrates as a single band of lower mobility than the major form when electrophoresed on a denaturing PAG. The minor form was determined to have …

High Resolution Two-Dimensional Electrophoretic Study Of Human Seminal Plasma Proteins, Edward E. Gaunt

Chemistry & Biochemistry Theses & Dissertations

A comprehensive biochemical screening procedure for the evaluation of seminal plasma constituents is needed which, when used in conjunction with semen analysis, can provide the physician with both qualitative and quantitative information regarding the fertility status of an individual.

High resolution two-dimensional electrophoresis is a technique whereby proteins in a complex mixture such as a biological fluid are separated in one dimension by molecular charge and in the second dimension by molecular mass. It has been the purpose of this investigation to adapt and evaluate this technique for the analysis of human seminal plasma.

Our efforts have shown that this …

The Association Of 5.8 S With 28s Ribosomal Rna, Nandita Banerjee

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is noncovalently bonded to the larger ribosomal subunit 28S rRNA; through its 3' end and through its 5' end. This interaction is an integral part of the ribosome, and plays an important role in the ribosome structure and function.

There is a high degree of homology between the 5.8S rRNA primary structures of rat, turtle and chicken. The base sequence of rat 5.8S rRNA differs only in one position from that of turtle and in three positions fr.om that of cl1.icken. Tl1ere is a single purine substitution at the 5' …

Multiple Methods Of Analysis Of 2'-O-Methylation In 5.8s Rrna, Sharon H. Ryan

Chemistry & Biochemistry Theses & Dissertations

5.8S rRNA is a low molecular weight ribosomal RNA which is found hydrogen bonded to 28S rRNA in the eucaryotic cell. There are two nucleotides which have 2'-0-methylations; a guanine residue at position 77 is fully methylated and a uridine residue at position 14 which is partially methylated. This partial 2'-0-methylation of the uridine residue has been found to vary with the tissue source, with the highest level in normal tissue and the lowest level in neoplastic tissue. In order to study the significance of this site-specific methylation, a reliable and convenient method of analysis was needed.

Early studies on …