Biochemistry 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 …

Clostridioides Difficile Biofilm And Spore Production In Response To Antibiotics And Immune Stress, Adenrele M. Oludiran

Chemistry & Biochemistry Theses & Dissertations

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. C. difficile, an anaerobic spore-forming Gram-positive pathogenic bacterium, is a major cause of hospital-acquired infections. C. difficile persists in the environment and spreads the infection to new hosts in the form of dormant spores and can persist within hosts as surface-attached biofilms. These studies investigate bacterial vegetative cell survival, biofilm formation, and sporulation in response to stress. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating …

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 …

Genetically Engineered Thermosynechococcus Elongatus Bp1: Assessment Of Potential Biorisks And Biofuel Production, Thu Ho Anh Nguyen-Jones

Chemistry & Biochemistry Theses & Dissertations

According to the International Energy Outlook 2019, released by the U.S. Energy Information Administration, it is projected that the energy consumption will increase up to 50% between 2018 and 2050 worldwide. As fossil fuel being a finite source of energy with the risk of depletion, many countries are now facing an energy security crisis. Therefore, it is important to develop other renewable and sustainable energy sources that will allow countries to shift away from depending on fossil fuels. Among several types of renewable energy, biofuel production using genetically engineered cyanobacteria is capturing much interest due to its many advantages. Different …

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 …

Computational Analysis Of Type 3 Iodothyronine Deiodinase: Potential Inhibitors, Substrate Binding, And Dimer Structure, Eric Scott Marsan

Chemistry & Biochemistry Theses & Dissertations

Thyroid hormones (THs) in mammalian tissues are crucial for development and maintaining metabolic homeostasis. Iodothyronine deiodinases (Dios) remove iodines from THs by a selenocysteine (Sec) residue, which either activates or inactivates them. Halogen bonding (XB) has been proposed to describe the interaction between the Se and I atoms of the T4-Dio complex. Disruption of TH homeostasis by xenobiotics, such as polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) can cause deleterious effects on the endocrine system. Experimental studies have indicated that PBDEs and PCBs could disrupt TH homeostasis by inhibiting Dio through XB formation. However, no current quantitative study exists …

Improvement Of Biochar Through Ozonization And Biosafety Of Genetically Engineered Cyanobacteria, Oumar Sacko

Chemistry & Biochemistry Theses & Dissertations

Through the innovative technique of biochar post-production surface oxygenation by ozonization, we were able to improve certain properties of biochar. In project one, the incubation of an insoluble phosphate rock material (hydroxyapatite) with the wet ozonized pine 400 biochar and its filtrate resulted in a solubilization of 80 times more phosphate from hydroxyapatite (569.0 mg/L ± 6.4) compared to the pure water-hydroxyapatite control (7.2 mg/L ± 0.3). The ozonized biochar may provide a new possible way to unlock the phosphorus from insoluble phosphate mineral phases. The cation exchange capacity (CEC) is a key property of biochar when used as a …

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 …

Evaluating The Role Of The Stringent Response Mechanism In Clostridioides Difficile Survival And Pathogenesis, Astha Pokhrel

Chemistry & Biochemistry Theses & Dissertations

The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventive and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stresses, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile. This dissertation explores the ability of C. difficile to mount the stringent response. The bacteria encode a full-length, canonical bifunctional Rel/Spo Homolog or RSH enzyme. C. difficile RSH is incapable of utilizing GTP as a substrate but readily synthesizes putative 5’-pGpp-3’ alarmones. …

Computational And Experimental Investigation Into The Determinants Of Protein Structure, Folding, And Stability In The Β-Grasp Superfamily, John T. Bedford Ii

Chemistry & Biochemistry Theses & Dissertations

Elucidating the mechanisms of protein folding and unfolding is one of the greatest scientific challenges in basic science. The overarching goal is to predict three-dimensional structures from their amino acid sequences. Understanding the determinants of protein folding and stability can be facilitated through the study of evolutionarily related but diverse proteins. Insights can also be gained through the study of proteins from extremophiles that may more closely resemble the primordial proteins. In this doctoral research, three aims were accomplished to characterize the structure, folding and unfolding behavior within the β-grasp superfamily. We propose that the determinants of structure, stability, and …

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 …

Study Of Abc Membrane Transporters In Single Live Cells, Preeyaporn Songkiatisak

Chemistry & Biochemistry Theses & Dissertations

The multidrug ATP-binding cassette (ABC) membrane transporters (efflux pumps) are found in both prokaryotes and eukaryotes and they can extrude diverse structurally unrelated substrates, such as antibiotics and chemotherapeutic agents out of the cells. The efflux pumps are responsible for multidrug resistance (MDR) and the failure of numerous treatments in infections and cancers. All ABC membrane transporters share a common modular topology containing two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). The underlying molecular mechanisms regarding how the similar structural ABC membrane transporters could selectively extrude a wide variety of substrates and cause MDR, are not yet fully …

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 …

Bioenergetics: Experimental Demonstration Of Excess Protons And Related Features, Haitham A. Saeed

Chemistry & Biochemistry Theses & Dissertations

Over the last 50 years, ever since the Nobel-prize work of Peter Mitchell’s Chemiosmotic theory, the question whether bioenergetics energy transduction occurs through localized or delocalized protons has been a controversial issue among scientists. Recently, a proton-electrostatics localization hypothesis was formulated which may provide a new and clear understanding of localized and delocalized proton-coupling energy transduction in many biological systems. The aim of this dissertation was to test this new hypothesis.

To demonstrate the fundamental behavior of localized protons in a pure water-membrane-water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated …

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 …

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 …

Exploring The Effect Of Climate Change On Biological Systems, Nardos Sori

Chemistry & Biochemistry Theses & Dissertations

The present and potential future effect of global warming on the ecosystem has brought climate change to the forefront of scientific inquiry and discussion. For our investigation, we selected two organisms, one from cyanobacteria and one from a cereal plant to determine how climate change may impact these biological systems. The study involved understanding the physiological and adaptive responses at both the genetic and protein function levels to counteract environmental stresses. An increase in atmospheric carbon dioxide is a key factor in global climate change and can lead to alterations in ocean chemistry. Cyanobacteria are important, ancient and ubiquitous organisms …

Density Functional Theory Modeling Of It-Stacking And Electrophilic Donor-Acceptor Interactions With Application To Therapeutic Targeting Of Zinc-Finger Proteins, Patricia Beall Lutz

Chemistry & Biochemistry Theses & Dissertations

Several viruses, including viruses that cause cancer, contain conserved zinc finger (ZF) proteins that are essential for viral reproduction, making them attractive drug targets for cancer and viral treatment. ZFs are small protein domains that have Zn²⁺ tetrahedrally coordinated to at least 2 Cys and His. They form three classes of ZFs depending on the amino acid ligands, CCHH, CCCH, and CCCC. Zn²⁺ is stable towards redox reactions; however, the Cys thiolates are redox active. Oxidation of the Cys thiolates release Zn²⁺, and the ZF loses its tertiary structure and can no longer bind DNA or …

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 …

Computational Investigation Of The Bioactive Selenium Compounds Ebselen And Selenious Acid, Sonia Antony

Chemistry & Biochemistry Theses & Dissertations

Selenium, a toxic element, is required in trace quantities for the proper functioning of biological systems. The experimental mechanistic study of the reactions of ebselen and selenious acid is difficult due to complexity of the reaction mixtures and the presence of short-lived intermediates. Computational modeling of the reactivity of these species can give us an insight into their mechanisms, but the process is complicated by proton exchanges associated with the mechanistic steps. In gas phase modeling, this may be corrected to a certain level using the solvent assisted proton exchange (SAPE) method. SAPE is a modeling technique that mimics solvent …

Bioinformatics, Thermodynamics And Kinetics Analysis Of An All Alpha Helical Protein With A Gree-Key Topology, Hai Li

Chemistry & Biochemistry Theses & Dissertations

Computational and experimental studies focusing on the role of conserved residues for folding and stability is an active and promising area of research. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all β-sheet immunoglobulin and mixed α/β-plait superfamilies. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native …

Conformational Change And Topological Stability Of Proteins, Jeffrey Andrew Tibbitt

Chemistry & Biochemistry Theses & Dissertations

The conformation and topology of a protein changes when stabilizing forces are absent, but the mechanisms by which these changes occur remains elusive. This dissertation aims to broaden the understandings. On the conformational level, the M20 loop conformers of E. coli dihydrofolate reductase are interrogated to identify factors responsible for their stability as well as to determine how one conformer might change into another. Molecular dynamics is used to simulate the open, closed and occluded conformers (observed in X-ray crystal structures) under a series of different single ligand conditions. Analysis shows that all open conformers move to a similar new …

The Regulation Of Endothelial And Neuronal Nitric Oxide Synthase Expression In The Preglomerular Vessels By The Increase In Intracellular Calcium Concentration, Stephanie Thomas Montgomery

Chemistry & Biochemistry Theses & Dissertations

This purpose of this study was to examine the regulation of endothelial and neuronal nitric oxide synthase expression in the preglomerular vessels of mature and immature porcine kidneys by the increase in intracellular calcium concentration. Angiotensin II can increase intracellular calcium concentrations in two ways; either through the mobilization of intracellular calcium stores or through the influx of calcium from the extracellular fluid through calcium channels. The aim of this study was to determine the effects of a calcium control, a calcium-free control, L- and T-type voltage-gated calcium channel blockers, an internal calcium store release agent, KCI, and EDTA on …

Biochemical Profiling Of Gout Patients, Tamera Fayre Schlitt

Chemistry & Biochemistry Theses & Dissertations

The purpose of the research is to investigate the biochemical cause of gout in 82 patients. The gouty patients had been diagnosed with gout according to the criterion of the American Rheumatism Association. This gout patient population consisted of 58 men and 24 women with gout whose ages ranged from 11 to 84 years of age with a mean age of 44 years. Key purine metabolism enzyme activity levels and purine metabolite concentrations in the patients' plasma and urine were compared to the mean values of a healthy control group. The control group consisted of 33 males and 25 female …

Development Of Single Nanoparticle Optical Assays For Imaging Single Living Cells, William John Brownlow

Chemistry & Biochemistry Theses & Dissertations

Multi-drug resistance (MDR) has been reported in both prokaryotes and eukaryotes; the pathogenic gram-negative bacteria Pseudomonas aeruginosa can extrude a variety of structurally and functionally diverse substrates via a number of membrane transport systems leading to MDR. We have developed a novel nanoparticle assay to characterize both the membrane transport system composed of the MexAB-OprM efflux pump and the membrane permeability induced by antibiotics. Gold (Au) and silver (Ag) nanoparticles were investigated for use as probes to explore membrane transport in P. aeruginosa.

The surface plasmon absorption (color) of Au nanoparticle solutions was found to change in the presence …

Bacterial Degradation Of Disinfection By-Products In Drinking Water, Laura Inga Fauntleroy

Chemistry & Biochemistry Theses & Dissertations

Chlorine became a major disinfectant for the removal of microbial contaminants in 1914. Current water chlorination procedures yield halogenated disinfection byproducts (DBPs), such as haloalkanes and haloacetic acids (HAAs), due to the reaction of chlorine with naturally occurring organic compounds. Various water utilities have observed decreased HAAs levels in maximum residence time locations (MRTLs), where they were expected to be higher. These MRTLs have low free chlorine residual and high heterotrophic bacteria plate counts. Xanthobacter autotrophicus, GJ-10, is a bacterium that has been shown to contain dehalogenase enzymes and, therefore, can biodegrade HAAs. A number of water-system bacteria were …

Synthesis And Biological Evaluation Of Novel Capacitative Calcium Entry Blockers, Thadigiri Celina Mala

Chemistry & Biochemistry Theses & Dissertations

Studies have shown that 2-aminoethoxydiphenyl borate (2-APB) inhibits thrombin-mediated influx of Ca^{2 +} through store-operated calcium channels in human platelets. Based on this molecule, two sets (boron analogues and the non-boron analogues) were synthesized and characterized by IR, NMR and CHN analysis and evaluated in human platelets to block thrombin-induced [Ca²⁺] elevation.

The synthesis of the non-boron compounds was performed in a simple one step process. According to the literature, some of these compounds were synthesized previously by a complicated two-step process.

All the compounds synthesized were tested for biological activity and the results showed that they …

Real-Time Study Of Multidrug Resistance Mechanism In Pseudomonas Aeruginosa Using Nanoparticle Optics And Single Live Cell Imaging, Sophia Vasou Kyriacou

Chemistry & Biochemistry Theses & Dissertations

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis …

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 …