Biochemistry, Biophysics, and Structural Biology Commons^™

Development And Biological Evaluation Of Selective Small-Molecule Inhibitors Of The Human Cytochrome P450 1b1, Austin Hachey

Theses and Dissertations--Chemistry

The human cytochrome P450 1B1 (CYP1B1) is an emerging target for small- molecule therapeutics. Several solid tumors overexpress CYP1B1 to the degree that it has been referred to as a universal tumor antigen. Conversely, its expression is low in healthy tissues. CYP1B1 may drive tumorigenesis through promoting the formation of reactive toxins from environmental pollutants or from endogenous hormone substrates. Additionally, the expression of CYP1B1 in tumors is associated with resistance to several common chemotherapies and with poor prognoses in cancer patients. However, inhibiting CYP1B1 with small molecules has been demonstrated in cellular and murine model systems to reverse this …

Synthesis Of 6,6- And 7,7-Difluoro-1-Acetamidopyrrolizidines And Their Oxidation Catalyzed By The Nonheme Fe Oxygenase Lolo, Nabin Panth

Theses and Dissertations--Chemistry

One of the remarkable steps in loline alkaloid biosynthesis is the installation of an ether bridge between two unactivated C atoms in 1-exo-acetamidopyrrolizidine (AcAP). LolO, a 2-oxoglutarate-dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of AcAP and the resulting alcohol's cycloetherification to give N-acetylnornoline (NANL). The mechanism of hydroxylation is well understood, but the mechanism of the oxacyclization is not. I synthesized difluorinated analogs of AcAP in an attempt to further understand the mechanism of the unusual cycloetherification step.

I prepared 6,6-F₂-AcAP in eight steps from N,O-protected 4-oxoproline. The key step was a Dieckmann …

The Role Of Charge On Dna Packaging And Integrity Within Reconstituted Peptide-Dna Assemblies, Ehigbai Oikeh

Theses and Dissertations--Chemistry

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20^th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, …

Development Of Fluorescence Based Approaches To Understand Astrocyte Biology In The Context Of Nicotine And Nicotinic Receptor Activity, Surya P. Aryal

Theses and Dissertations--Chemistry

Smoking and tobacco use (STU) is a major global health problem and worldwide more than six million people die due to tobacco related diseases each year. Although majority of smokers try to quit smoking several times in their life, traditional therapeutic approaches, which focus only on neuronal cells, have a very low success rate. Understanding the effect of nicotine on glial cells, synaptic communication and blood vasculature in the brain can provide further insights on the neurobiology of substance abuse and can potentially help to design better therapeutic approaches. Glial cells are non-excitable cells in the brain which do not …

Flavin Modification And Redox Tuning In The Bifurcating Electron Transfer Flavoprotein From Rhodopseudomonas Palustris: Two Arginines With Different Roles, Nishya Mohamed-Raseek

Theses and Dissertations--Chemistry

Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Developing Synthetic Strategies For Multifaceted Applications Of Stable Gold-Based Complexes, Randall Tyler Mertens

Theses and Dissertations--Chemistry

Development of stable gold-based complexes has been a rapidly advancing field due to the popularity of gold complexes, particularly for use in biomedical research and catalytic transformations. Given that auranofin, a gold(I) complex with FDA approval for the treatment of rheumatoid arthritis is used in the clinic, the development of stable gold-based molecules of clinical relevance is urgently needed. Herein are reported, synthetic strategies used for the development of new classes of gold(I) and gold(III) complexes for advancement in mitochondrial modulation for use as chemotherapeutics as well as application to gold catalysis due to the unique geometry of complexes presented …

Computational Insights On Medicinal Chemistry Targeting Cyp450s, Alexander D. Fenton

Theses and Dissertations--Chemistry

Modern-day medicinal chemistry has provided researchers with a wide variety of tools to not only gather greater insight from their data, but also to generate data in new ways. One such tool is the construction of computational protein models from crystallographic datasets, and their subsequent use to understand the structure-activity relationships of protein-ligand complexes. These models can be utilized for their predictive power to inform the synthesis of, and improvement of, lead compounds. It is the goal of this work to employ such models to the CYP450 enzyme system such that potent and selective inhibitors can be designed, evaluated biologically, …

A Biophysical Investigation Of Stability, Ligand Binding, And Iron State Of Cyp102a1, Catherine A. Denning-Jannace

Theses and Dissertations--Chemistry

Cytochrome P450s (CYPs) are cysteine ligated Fe-heme monooxygenases that are found in all domains of life. In mammals, they have a role in xenobiotic metabolism and steroid synthesis, making them a fundamental requirement for survival. In addition, their ability to perform a variety of chemical reactions on an array of substrates makes CYPs highly sought for biotechnical applications such as wastewater remediation, production of potential drug candidates, and creation of drug metabolites. By mutating specific amino acids, these enzymes can be engineered to change their substrate binding profiles and achieve stereo- and regio-specific chemistry. While these mutations are essential to …

Flavodoxin, The Hydrogen Atom Of Flavoproteins: A 19f Nmr Study Of Dynamics And Conformational Changes Utilizing Flavodoxin From Rhodopseudomonas Palustris, Taylor Varner

Theses and Dissertations--Chemistry

Flavodoxin is a small, highly stable protein that contain a single FMN cofactor used to transfer single electrons at low potentials. The organism Rhodopseudomonas palustris contains a long-chain flavodoxin. Long-chain flavodoxins are characterized by a 20 amino acid loop that is proposed to allow interactions with partner proteins. We plan to utilize this protein as a model to build our repertoire with protein fluorination and fluorine NMR in flavoproteins. This tool kit will then be applied to the study of a partner protein of interest that is capable of performing electron bifurcation. We have incorporated m-fluoro tyrosine into flavodoxin and …

Investigation Of Amyloid Β Oligomer Dissociation Mechanisms By Single Molecule Fluorescence Techniques, Hope Cook Abdalla

Theses and Dissertations--Chemistry

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the …

Protein Suppression Of Flavin Semiquinone As A Mechanistically Important Control Of Reactivity: A Study Comparing Flavoenzymes Which Differ In Redox Properties, Substrates, And Ability To Bifurcate Electrons, John Patrick Hoben

Theses and Dissertations--Chemistry

A growing number of flavoprotein systems have been observed to bifurcate pairs of electrons. Flavin-based electron bifurcation (FBEB) results in products with greater reducing power than that of the reactants with less reducing power. Highly reducing electrons at low reduction midpoint potential are required for life processes of both aerobic and anaerobic metabolic processes. For electron bifurcation to function, the semiquinone (SQ) redox intermediate needs to be destabilized in the protein to suppress its ability to trap electrons. This dissertation examines SQ suppression across a number of flavin systems for the purpose of better understanding the nature of SQ suppression …

Applications Of Cell-Derived Vesicles: From Single Molecule Studies To Drug Delivery, Faruk H. Moonschi

Theses and Dissertations--Chemistry

Single molecule studies can provide information of biological molecules which otherwise is lost in ensemble studies. A wide variety of fluorescence-based techniques are utilized for single molecule studies. While these tools have been widely applied for imaging soluble proteins, single molecule studies of transmembrane proteins are much more complicated. A primary reason for this is that, unlike membrane proteins, soluble proteins can be easily isolated from the cellular environment. One approach to isolate membrane proteins into single molecule level involves a very low label expression of the protein in cells. However, cells generate background fluorescence leading to a very low …

Synthesis And Development Of Zwitterionic Pei (Zpei) For Optimized Delivery Of Nucleic Acids, Joseph Raleigh Duke Iii

Theses and Dissertations--Chemistry

Gene therapy holds promise for the treatment a wide range of diseases ranging from cystic fibrosis to cardiovascular disease to cancer. The need for safe and efficient gene delivery methods remains the primary barrier to human gene therapy. Non-viral vector materials, including polymers, can be designed to be biocompatible and non-immunogenic, but lack the efficiency to be clinically relevant. Gene therapy awaits the development of new materials that are both safe and efficient. Here, we have synthesized a series of modified zwitterionic polymers based on the common transfecting agent polyethylenimine (PEI). Using a variety of biochemical and biophysical methods we …

The Development Of Colorimetric Assays To Determine The Identity And Frequency Of Specific Nucleobases In Dna Oligomers, Elizabeth Marie Thomas

Theses and Dissertations--Chemistry

Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this dissertation, …

Investigations Into Modulation Of Brain Oxidative Stress By Various Interventions, Jessica Lynn Harris

Theses and Dissertations--Chemistry

In this thesis study we examined glycogen synthase kinase-3β (GSK-3β) and its effects over Nrf2 and Pin 1 as it relates to Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized by a prolonged high oxidative environment. Transcription factor Nrf2 is vital in the brain’s defense against oxidative insults through its up-regulation of over 100 antioxidants. Depletion of the brain’s antioxidant defense system results in intolerance to an oxidative environment, contributing to the progression of AD. The regulatory Pin 1 protein promotes cellular homeostasis, and when down-regulated results in increased deposits of neurofibrillary tangles (NFTs) and amyloid-β (Aβ) plaques, the …

Bifunctional Bisphosphonates For Delivering Biomolecules To Bone, Jivan N. Yewle

Theses and Dissertations--Chemistry

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted …