Biochemistry 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 …

Structural Basis Of Bacterial Flagellin For Naip5 Binding And Nlrc4 Inflammasome Activation And The Mechanism Of Flagellin Induced Release Of Cytokines In Vivo, Jian Cui

Theses and Dissertations--Chemistry

The bacterial flagellum is a whip-like structure that protrudes from the cell membrane and is one of the most complex and dynamic biological molecular machines that propels bacteria to swim toward beneficial environments and the sites of infection. It is composed of a basal body, a hook, and a long filament. The flagellar filament contains thousands of copies of the protein flagellin (FliC) monomer arranged helically and ending with a filament cap composed of oligomer protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases …

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 …

Antibiotic Permeation In Gram-Negative Bacteria And Contribution Of Inflammasome Activation And Pyroptosis In Pathogenesis Of Salmonella Systemic Infection, Ankit Pandeya

Theses and Dissertations--Chemistry

Antibiotic resistance is one of the major global issues in the field of public health and medicine. Good antibiotic candidates need to be selectively toxic, inhibit cellular target, and effectively penetrate and accumulate in bacterial cells. The last factor is a formidable barrier in the development of antimicrobials effective in Gram-negative bacteria, due to the presence of two layers of cell envelope. The first half of my thesis focuses on understanding the permeation of small molecules through this formidable cell envelope, distribution inside the cell of Gram-negative bacteria, and design of novel methods to make small molecules effectively cross the …

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 …

Clpxp Degradation System In Escherichia Coli, A Study Of Its Energy Sources And Its Applications In Managing The Expression Levels Of Targeted Membrane And Soluble Proteins, Thilini Abeywansha

Theses and Dissertations--Chemistry

ClpXP is an Escherichia coli protease that carries out energy-dependent intracellular proteolysis. In recent years, this system has been widely studied due to its importance as protein regulatory machinery and a virulence factor. Protein substrates of ClpXP contain degrons with a specific protein sequence. SsrA tag is one of the five degrons known to designate proteins for ClpXP degradation. SsrA is an 11 amino acid peptide added to the C-terminus of nascent polypeptide chains translated from aberrant messenger RNAs lacking stop codons via a process called trans-translation.

ClpXP was known to targets only cytosolic proteins with degrons until recently, …

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 …

Deconvolution Tools For Extracting Insight From Challenging Two-Flavin Systems, Dallas Michael Bell

Theses and Dissertations--Chemistry

Flavoproteins have long been explored for their ubiquity among a number of metabolic and energetic reactions. The flavin cofactor has the inherent benefit of distinct spectral changes associated with redox transitions; however, the double-edged sword is incurred as these distinct signatures overlap and take up much of the UV-vis spectral range. Therefore, it is crucial to create a method to demarcate the expressed redox transitions for studying these systems. The first portion of these studies discusses the creation of a program that deduces spectra for redox transitions in a single-flavin containing model protein: flavodoxin. The latter portions discuss the application …

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 …

Studies Of Oxidative Damage, Brain Proteome, And Neurochemical Metabolites In Cognitive And Neurodegenerative Disorders: (1) Chemotherapy-Induced Cognitive Impairment; (2) Parkinson Disease Rat Model, Xiaojia Ren

Theses and Dissertations--Chemistry

The rate of cancer patients is increasing as the development of science and technology. Twenty million cancer survivors are estimated living in the United States by 2025. However, many cancer survivors show cognitive dysfunction, negatively affecting the quality of life. These cognitive impairments are recognized as chemotherapy-induced cognitive impairment (CICI), also called "chemo brain" by cancer survivors, including the diminished ability of memory and learning, hard to concentrate and focus, as well as diminution of executive function and processing speed. The etiologies and pathologies of CICI are complicated, especially in most cases the anti-cancer drug cannot cross the blood-brain barrier …

Illuminating Dna Packaging In Sperm Chromatin: How Polycation Lengths, Underprotamination And Disulfide Linkages Alters Dna Condensation And Stability, Daniel Kirchhoff

Theses and Dissertations--Chemistry

During spermiogenesis, somatic chromatin is remodeled and a vast majority (> 90%) of DNA histones are replaced by short arginine-rich peptides called protamines. This compaction is immense, with protamine-DNA self-assembly in sperm chromatin resulting in a final volume roughly 1/6th of a somatic nucleus. This near crystalline organization of the DNA in sperm is thought crucial both for the transport of the paternal genes as well as for the protection of genetic information as sperm chromatin is transcriptionally inactive and all DNA repair mechanisms are shut down.

Chapter 1 will include an overview of the topics discussed in this document, …

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 …

Multi-Scale Computational Studies Of Calcium (Ca2+) Signaling, Bin Sun

Theses and Dissertations--Chemistry

Ca²⁺ is an important messenger that affects almost all cellular processes. Ca²⁺ signaling involves events that happen at various time-scales such as Ca²⁺ diffusion, trans-membrane Ca²⁺ transport and Ca²⁺-mediated protein-protein interactions. In this work, we utilized multi-scale computational methods to quantitatively characterize Ca²⁺ diffusion efficiency, Ca²⁺ binding thermodynamics and molecular bases of Ca²⁺-dependent protein-protein interaction. Specifically, we studied 1) the electrokinetic transport of Ca²⁺ in confined sub-µm geometry with complicated surfacial properties. We characterized the effective diffusion constant of Ca²⁺ in a cell-like environment, which helps to understand …

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 …

Protein Engineering In The Study Of Protein Labeling And Degradation, Xinyi Zhang

Theses and Dissertations--Chemistry

Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, …

Surface And Structural Modification Of Carbon Electrodes For Electroanalysis And Electrochemical Conversion, Yan Zhang

Theses and Dissertations--Chemistry

Electrocatalysis is key to both sensitive electrochemical sensing and efficient electrochemical energy conversion. Despite high catalytic activity, traditional metal catalysts have poor stability, low selectivity, and high cost. Metal-free, carbon-based materials are emerging as alternatives to metal-based catalysts because of their attractive features including natural abundance, environmental friendliness, high electrical conductivity, and large surface area. Altering surface functionalities and heteroatom doping are effective ways to promote catalytic performance of carbon-based catalysts. The first chapter of this dissertation focuses on developing electrode modification methods for electrochemical sensing of biomolecules. After electrochemical pretreatment, glassy carbon demonstrates impressive figures-of-merit in detecting small, redox-active …

Assembly And Trafficking Of The Cystic Fibrosis Transmembrane Conductance Regulator And Associated Proteins, Zhihui Zhang

Theses and Dissertations--Chemistry

Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which …

Study Of The Mechanism Of Action For Ru(Ii) Polypyridyl Complexes As Potential Anticancer Agents, Yang Sun

Theses and Dissertations--Chemistry

Application of chemotherapeutic agents in current cancer treatment has been limited by adverse effects as poor selectivity results in systemic toxicity; most chemotherapy approaches also experience inherited or acquired drug resistance which lead to reduced treatment outcome. Research efforts have focused on the discovery of novel chemotherapies that overcome the limitations mentioned above. Ru(II) polypyridyl complexes with anti-cancer properties have been extensively studied as traditional cytotoxic agents and photodynamic therapy agents due to their photophysical and photochemical characteristics.

Most research has focused on the design of Ru(II) polypyridyl complexes that have affinities to nucleic acids as inspired by the classic …

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 …

Illuminate The Pathway Of Membrane Protein Association And Degradation, Zhaoshuai Wang

Theses and Dissertations--Chemistry

Escherichia coli transporter protein AcrB and its homologues are the inner membrane components of the Resistance-Nodulation-Division (RND) family efflux pumps in Gram-negative bacteria. It is well accepted that soluble proteins are only marginally stable, but such insight is missing for membrane proteins. The lack of stability data, including thermodynamic stability and oligomer association affinity is a result of intrinsic difficulties in working with membrane proteins. In addition, the degradation of soluble proteins in E. coli has been extensively studied whereas the degradation process of membrane proteins remains unclear. A focus of my thesis is the validation and development of methods …

The Role Of Gln146 To The Stability And Activity Of Manganese Superoxide Dismutase, Ting Wang

Theses and Dissertations--Chemistry

Gln146 is a highly conserved outer-sphere amino acid residue at the active site of MnSOD. It serves as a hydrogen bond donor to both the solvent molecules at the active site and Tyr 34, the conserved “gateway” amino acid residue. This dissertation develops our understanding of the effect of amino acid Gln146 at the second shell of the active site of metalloprotein MnSOD in facilitating metal binding, the modulation of redox potential, and the optimization of catalytic activity and structure stability. Different from the wild-type MnSOD, Q146E is always purified as a completely apo-protein with zero active metal ion and …

Assembly And Degradation Of A Trimeric Membrane Protein Acrb, Qian Chai

Theses and Dissertations--Chemistry

Multidrug efflux pumps are membrane proteins that actively transport foreign objects out of cells. The active efflux of these pumps is a critical self-defense mechanism that enables the survival of bacteria under hostile environments. Efflux pump AcrB is a member of the Resistance-Nodulation-Division (RND) super family. In E. coli, it associates with periplasmic protein AcrA and outer membrane channel TolC to extrude a variety of noxious compounds out of cell from both the cytoplasm and the periplasm. My dissertation research focused on two aspects of this multidrug efflux pump: the oligomerization process during the biogenesis of AcrB and its …

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, …

Understanding Dna Condensation By Low Generation (G0/G1) And Zwitterionic G4 Pamam Dendrimers, Min An

Theses and Dissertations--Chemistry

Cationic polymers have shown potential as gene delivery vectors due to their ability to condense DNA and protect it from cellular and restriction nucleases. Dendrimers are hyperbranched macromolecules with precisely defined molecular weights and highly symmetric branches stemming from a central core. The nanosize, tunable surface chemistries and ease of surface functionalization has made dendrimers an attractive alternative to conventional linear polymers for DNA delivery applications. The commercially available, cationic dendrimer poly(amidoamine) or PAMAM is the most widely studied dendrimer for use as a gene delivery vector. The aim of this dissertation is to provide an increased understanding of the …

Nanoparticle Behavior In Biological Gels And Biofluids: The Impact Of Interactions With Charged Biogels And The Formation Of Protein Coronas On Nanoparticles, Xiaolu Zhang

Theses and Dissertations--Chemistry

With the rapid growth of nanotechnology, situations where nanomaterials will interact with biological systems will unquestionably grow. Therefore, it is increasingly understood that interactions between nanomaterials and biological environments will play an essential role in nanomedicine. Biological polymer networks, including mucus and the extracellular matrix, serve as a filter for the exchange of molecules and nanoparticles. Such polymer networks are complex and heterogeneous hydrogel environments that regulate transport processes through finely tuned particle-network interactions. In chapters 3 and 4, we investigate the role of electrostatics on the basic mechanisms governing the diffusion of charged molecules inside model polymer networks by …

Stability Studies Of Membrane Proteins, Cui Ye

Theses and Dissertations--Chemistry

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms.

The first direction of my research is in the area …

Increase Of Basal Oxidative Stress Levels And Impairment Of Heme Oxygenase-1/Biliverdin Reductase Post-Translational Modification By The Defect Of Parkinson-Related Gene Of Pink1, Zhaoshu Zhang

Theses and Dissertations--Chemistry

Parkinson disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. PINK1, PTEN-induced kinase 1, functions as a serine/threonine kinase as well as a protector of mitochondrial function. Mutations in PINK1 gene result in either mitochondria dysfunction or disruption of kinase signaling pathways involved in the pathogenesis of PD.

In this thesis, oxidative stress levels were examined in the brain of PINK1 knockout mice, and also how heme oxygenase-1 and biliverdin reductase are affected in brain of PINK1 knockout mice. In addition, posttranslational modifications are a way to control the behavior of proteins, so posttranslational …