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Synthesis And Evaluation Of Cereblon-Recruiting Haloprotacs, Britton Ody

Chemistry Dissertations

The development of proteolysis-targeting chimeras (PROTACs) is a useful approach to develop new therapeutics. PROTACs work by tagging proteins for degradation by the ubiquitin-proteasome system (UPS) in the cell. Especially, in this work, we use Halotag, a genetically modified bacterial dehalogenase that forms covalent bonds with ligands functionalized with terminal chlorohexyl moieties. Reported here the development of bifunctional HaloPROTAC ligands that recruit the cereblon (CRBN) E3 ubiquitin ligase to degrade target proteins fused with a HaloTag in the cell. The dissertation will start with a review of the ubiquitin-proteasome system (UPS) system, PROTACs, and induced protein degradation. Next, I will …

Carbon Monoxide Donors: From Synthetic Applications To Reassessment Of Commercially Available Compounds, Nicola Bauer

Chemistry Dissertations

Carbon monoxide (CO) is known for various reasons. The general public sees CO as the “silent killer”, while organic chemists see CO as a reagent for carbonylation, and medicinal chemists and biologists see CO as an endogenous signaling molecule with therapeutic potential. Along these lines, we have developed and assessed CO donors for wide ranging applications.

In terms of synthetic organic chemistry, our lab has developed a light-activated CO donor for Pd-catalyzed and light-mediated carbonylation. This low-molecular-weight, solid CO surrogate that only requires a low-power LED for activation to release 2 equivalents of CO can be universally implemented in various …

Raspberry Polyphenols Target Molecular Pathways Of Heart Failure, Rami Najjar

Chemistry Dissertations

Approximately 650,000 new cases of heart failure (HF) are diagnosed annually with a 50% five-year mortality rate. HF is characterized by reduced left ventricular ejection fraction (EF) and hypertrophy of the left ventricular wall. The pathophysiological remodeling of the heart is mediated by increased oxidative stress and inflammation. Reactive oxygen species (ROS) derived from NADPH-oxidases (NOX), xanthine oxidase (XO), and mitochondria are the primary drivers of oxidative stress. In addition, damage associate molecular patterns (DAMPs) released from apoptotic cells can trigger toll-like receptor (TLR)4 activation, leading to an inflammatory response. Raspberries are rich in polyphenols which may favorably impact enzymes …

Quantum Chemical And Qm/Mm Calculations On Flavin And Flavin-Binding Fluorescent Proteins, Mohammad Pabel Kabir

Chemistry Dissertations

Flavin is the cofactor for the large and diverse family of proteins called flavoproteins. Upon absorption of blue light, the flavin cofactor may undergo one of several potential photophysical processes, depending on the surrounding environment. Possible photophysical processes include fluorescence from the first singlet excited state (S₁), intersystem crossing to a long-lived triplet state (T₁), or photoreduction to form a radical species (D₁). Each of those processes has been exploited for applications such as biosensing, bioimaging, FRET process, optogenetics, and singlet oxygen generation. However, the dependence of flavin’s photophysics (mechanism and kinetics) on its …

Blackberry Consumption Protects Against E-Cigarette-Induced Vascular Oxidative Stress, Maureen Meister

Chemistry Dissertations

There has been a significant rise in the use of electronic cigarettes (e-cigarettes), devices which deliver vaporized nicotine to the lungs. Due to their recent arrival on the market, evidence of their health effects is scant. Acute e-cigarette exposure induces pulmonary and systemic oxidative stress in e-cigarette users and contributes to vascular endothelial dysfunction through reduction in nitric oxide (NO) levels. Polyphenols, such as cyanidin-3-glucoside and ellagic acid, which are abundant in blackberries (BL), mitigate lung and cardiovascular damage. The goal of this study was to determine the effects of e-cigarette exposure on pulmonary, cardiac, and vascular pathologies and to …

Near Infrared Electrogenerated Chemiluminescence And Related Electrochemical Properties Of Gold Nanoclusters, Hedi Ma

Chemistry Dissertations

This dissertation studies the fundamental principles and analysis applications of electrochemiluminescence (ECL) from atomically precise nanoclusters. The nanoclusters under study are composed of a metal core stabilized by a monolayer of covalently attached ligands. Chapter one describes the basics of ECL and an overview of gold nanoclusters including both fundamentals and applications. In chapter two, the kinetics of ECL generation are investigated where nanoclusters are either immobilized on the surface or free diffusing in solution. The ECL intensity-time profiles suggest that bimolecular or pseudo first order reactions limit the ECL generation immediately following the establishment of the applied potentials, while …

Probing The Regulatory Role Of Allostery Via Protein Dynamics Of Biological Systems, Michael G. Souffrant

Chemistry Dissertations

Allostery is a universal process in cellular interaction and function. Allosteric regulation occurs when local changes from a distant site of the biomolecular structure alter the changes at the binding site. Allosteric regulation is an intrinsic property, which is assumed to be a propagation of conformational or dynamical changes through a network of interactions from the regulatory site to the binding site cavity. Despite many approaches, the concept of allostery is vaguely understood, as this phenomenon occurs at different cellular levels for regulation. In this dissertation, we provide more insight on allosteric regulation, using three different protein systems as models …

Defining And Describing The Complex Nature Of Undergraduates’ Chemistry-Specific Mindset Beliefs, Deborah L. Santos

Chemistry Dissertations

In challenging learning contexts like general and organic chemistry, students’ determining factors for persistence or helplessness become more salient in their likelihood of success in the course. Incremental beliefs (or growth mindset) are theorized to result in a series of behaviors that positively influence outcomes. Because of the difficult nature of chemistry as an academic subject, we investigate the role of chemistry mindset as an influencing factor on student outcomes. We propose that chemistry-specific mindset represents a separate construct from intelligence beliefs students hold about other STEM domains for undergraduates and thus should be measured independently. We show that the …

Chemical Biology Tools For Probing Protein Glycosylation And Ubiquitination Pathways, Xuan Fu

Chemistry Dissertations

Protein post-translational modifications (PTMs) increase the functional diversity of the proteome and over 400 different types of PTMs have been identified so far. In this thesis, we focus on glycosylation and ubiquitination, and developed new methods and tools to study these modifications.

Glycosylation is one of the most common PTMs. Accessing large numbers of structural-defined glycoconjugates, and their derivatives is essential for their structural and functional studies to understand glycan-involved biological processes. In Chapter 2, we showed general tolerance of galactosyltransferases toward uncommon donor uridine-diphosphate-galactosamine (UDP-GalN). Such a property of galactosyltransferase was harnessed to develop a two-step chemoenzymatic strategy for …

Water Use Patterns Of A Riparian Forest In A Humid Subtropical Catchment In The Southeastern United States, Jeffrey Riley

Chemistry Dissertations

The role of groundwater in sustaining plant transpiration has been studied for nearly a century. However, the body of literature investigating plant uptake of groundwater has largely been focused on arid and semiarid climates, with few examples from more humid locations. In this dissertation, I attempt to contribute a rigorous evaluation of groundwater transpiration (T_G) from a humid riparian forest to fill in this knowledge gap. In chapter two, I explored the groundwater use by a riparian forest using techniques that exploit diurnal water table fluctuations from groundwater wells. Specifically, I investigated the spaciotemporal variability of T_{G …}

Revealing Plasmonic Coupling Of Gold Nanoparticles On A Mirror Using Spectromicroscopy, Seth Filbrun

Chemistry Dissertations

A spectro-microscopic system was developed that combines reflected dark field microscopy with total internal reflection scattering microscopy. This instrument also includes a high-resolution spectrometer for Raman Spectroscopy and photoluminescence spectroscopy measurements. This instrument was used to analyze single gold nanorods coupled to a gold film system to understand the light-matter interactions. It was found with total internal reflection scattering the single-particle spectroscopy is uniquely different than the scattering from the same particle illuminated with reflected dark field microscopy. This difference is due to the underlying coupling mechanisms between a gold nanorod and the thin gold film. Interestingly, by tuning the …

The Importance Of Fitting In: Investigating The Role Of Protein Dynamics In Dna Sequence Specificity, Kenneth Huang

Chemistry Dissertations

Specificity for protein/DNA complexes is well-studied in context of DNA sequence requirements, but the precise mechanisms for how segregation of affinity in consensus-bearing DNA has not been well characterized. Nonspecific sites are dictated by the lack of a consensus site, but high/low affinity binding rise from discrepancies in flanking sequences. Low-affinity DNA has been recently implicated as a general mechanism used by cells to regulate gene expression. Ets-1, the progenitor member of the ETS transcription family, offers a useful model to understand how cognate site discrimination occurs. We then performed molecular dynamics simulations of Ets-1 unbound and bound to nonspecific, …

Profiling Substrates Of Hect And Rbr Type E3 Ligases By Orthogonal Ubiquitin Transfer Cascade, Ruochuan Liu

Chemistry Dissertations

SMAD specific E3 ubiquitin protein ligase 2 (Smurf2), a member of the HECT domain family of E3 ubiquitin (UB) ligases, participates in many crucial biological and physiological processes in the cell. Through the E1-E2-E3 ubiquitin transfer cascade, Smurf2 regulates its substrates' stability by ubiquitination-mediated degradation. E6-associated protein (E6AP), another HECT domain E3 ligase, pairs with a papillomavirus oncoprotein E6 as a complex to recruit substrates that result in cervical cancers. Ring finger protein 216 (RNF216) is an RBR type E3 ligase and is involved in neurodegenerative and neurodevelopmental diseases by transferring UB to its substrates. Due to the importance of …

Profiling Substrate Proteins Of Ring And Rbr Type E3 Ligases By Orthogonal Ubiquitin Transfer And The Development Of A Peptide Activator Targeting Hect-Type E3 Ligase, Li Zhou

Chemistry Dissertations

RING finger protein 38 (RNF38) is a member of the RING family of E3 ubiquitin (UB) ligases while HHARI is one of RBR family members: both regulate key processes in the cell. Through the E1-E2-E3 cascades, RNF38 and HHARI transfer UB to cellular proteins and regulate their stability, subcellular localization, and interaction with other proteins. Identifying the ubiquitination targets of RNF38 and HHARI holds the key to deciphering their roles in cell regulation. In this study, we used phage display to engineer the RING domains of the two E3 ligases for their incorporation into an orthogonal UB transfer (OUT) cascade …

Improving Potency Of Existing Drugs: From Developing Bacterial Sensitizers For Existing Antimicrobials Towards Multi-Drug Resistant Strains To Targeting Cancer Drugs To The Mitochondria, Manjusha Roy Choudhury

Chemistry Dissertations

Multi-drug resistant (MDR) bacteria is one of the major health threats to public health. The rate of development of antibiotics is unable to keep pace with the rate of emergence of MDR bacteria. Thus, there is an urgent need to find a way to make existing antibiotics effective again against these MDR strains. Along this line, we have developed compounds that sensitize Gram-negative strains towards various existing antibiotics, both broad- and narrow-spectrum. These compounds work by disrupting the outer membrane of Gram-negative bacteria. These sensitizers exhibit a much lower tendency to induce resistance in bacteria than commonly used antibiotics.

Mitochondria, …

Utilizing Molecular Probes To Examine Protein Structure-Function Relationships, James Terrell

Chemistry Dissertations

Within the cell, the majority of functional roles are carried out by polypeptide macromolecules commonly referred to as proteins. Over the past 100 years, various methodologies to examine the high-resolution structural characteristics of proteins have been developed and utilized, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryogenic electron microscopy. The structural information obtained has yielded much insight into the mechanisms by which many of these proteins function; however, limitations to the nature of the structural data have provided challenges when attempting to elucidate protein structure-function relationships and dynamics.

Owing to its nature, x-ray crystallography provides what can be described …

Methods For Dynamical Analysis Of Biomolecular Mechanisms In Molecular Dynamics Simulations, Ka Chun Ho

Chemistry Dissertations

A deep understanding of the role of motions in the functional mechanisms of biomolecules can potentially speed up molecular dynamics (MD) simulations of the interaction of the same drug target with numerous drug candidates by reusing the simulation of the drug target. This deep understanding can also enable using dynamics as an additional tool to steer a drug toward its intended target and avoid side effects. To gain this understanding, we have modeled the internal motions, the structural hierarchy, and the dynamical hierarchy of biomolecules by analyzing MD simulations. We find that the diffusion coefficients of the internal motions of …

Structural And Mechanistic Investigation Of D-Arginine Dehydrogenase And L-Arginine Dehydrogenase From Pseudomonas Aeruginosa, Archana Iyer

Chemistry Dissertations

Pseudomonas aeruginosa has a two-enzyme D-arginine conversion system that enables it to utilize D-arginine as a nutrient. The coupled-enzyme system consists of D-arginine dehydrogenase (PaDADH) and L-arginine dehydrogenase (PaLADH). PaDADH has been characterized structurally and mechanistically and established as an FAD-dependent enzyme that catalyzes the oxidation of D-arginine to iminoarginine, which can be non-enzymatically hydrolyzed to ketoarginine and ammonia.

A tyrosine 249 to phenylalanine variant of PaDADH (PaDADH-Y249F) was expressed and purified as a mixture of two cofactors, one protein population with regular FAD and one with a green-colored modified FAD. The …

Glycopeptide Preparation And Application For Alzheimer?S Diagnostics And Influenza Immunization, Christopher Gibbons

Chemistry Dissertations

Glycoproteins and glycopeptides are ubiquitous in nature but present challenges in clinical application. From a synthetic standpoint, they combine the difficulties of glycochemistry, namely regio- and stereo-specificity limitations, with the challenges inherent in peptide production. Yet their biological significance makes them impossible to ignore.

The pathology of Alzheimer’s Disease (AD) is characterized by the extracellular deposition of amyloid β peptides (Aβ) and intracellular accumulation of hyperphosphorylated tau protein (p-tau). The hyperphosphorylation of tau is known to occur in concert with the reduction of O-GlcNAc loading. Indeed, it is known that artificially forcing an increase in O-GlcNAc results in reduced phosphorylation, …

Kinetic Studies And Investigation Of Active Site Loop Dynamics In A Flavin-Dependent Enzyme, D-Arginine Dehydrogenase, Daniel Ouedraogo

Chemistry Dissertations

Enzymes are highly flexible biocatalysts in biological organisms. X-ray crystallography, nuclear magnetic resonance studies, and computational simulations data indicate that enzymes are dynamic networks of amino acid residues and undergo multiple conformational changes during catalysis. Understanding the interconnection between enzyme dynamics and catalysis or catalysis-linked dynamics is a broad subject of interest for modern scientists. However, the role of enzyme dynamics in catalysis is still not fully understood. This dissertation used D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) as a model enzyme to investigate enzyme dynamics and catalysis.

PaDADH is part of a two-enzyme system that catalyzes the …

Characterization Of The Contributions Of Hydration In Protein-Dna Specificity Via Structurally Conserved Ets Family Transcription Factors Pu.1 And Ets-1, Amanda V. Albrecht

Chemistry Dissertations

Protein-DNA interactions involve a redistribution of conformational dynamics and hydration properties. As a model DNA-binding protein, structural and thermodynamic studies have shown that ETS transcription factor PU.1 DNA selection is dependent on solvent reorganization. PU.1 high-affinity DNA binding is osmotically sensitive, whereas low-affinity binding is not, which provides a model for examining the role of hydration and DNA dynamics in protein-DNA specificity. Characteristic of ETS members, PU.1 recognizes DNA sequences harboring a central 5’-G₀G₁A₂A₃-3’ consensus, where an Arg sidechain contacts O⁶ of G₁ in the major groove. This conserved interaction …

The Cd98 Is A Potential Target For Non-Alcoholic Fatty Liver Disease Treatments, Heliang Song

Chemistry Dissertations

CD98 is a glycoprotein with many important biological functions including amino acid transportation, endocytosis regulation, and activation of the integrin β signal pathway. Studies have shown the expression levels of CD98 are increased in the livers from fatty degeneration, although the role of CD98 in non-alcoholic fatty liver disease (NAFLD) is not clear. The inflammatory stress inside of the liver is considered one of the main driving forces for NAFLD development. It has been well-studied that the inhibition of CD98 protects mice from colitis (inflammation). To understand the role of CD98 in NAFLD, CD98 siRNA was loaded into the poly …

A Reporter Platform For Characterizing Transcription Factor-Dna Interactions In Live Cells, Hye Mi Kim

Chemistry Dissertations

Reporters are genetic constructs that place the biosynthesis of an exogenous probe, such as an indicator enzyme or fluorescent protein, under the control of a cloned promoter. Benefiting from advances in molecular cloning, both the regulatory and indictor elements can be highly customizable to meet specific experimental needs. Reporters are therefore flexible tools for investigating mechanisms of transcriptional regulation in live cells, where detailed characterization of protein/DNA complexes remains challenging. In addition, reporters are attractive cell-based indicators in library screening and downstream pre-clinical characterization of hit compounds. To obtain a better understanding of the mechanism of action of PU.1, a …

Chemistry Of A Dehydrogenase And Di-Heme Enzyme Related To Tryptophan Oxidation, Christopher Ian Davis

Chemistry Dissertations

Tryptophan is an essential amino acid that is used as a building block to construct proteins, the biosynthetic precursor for several essential molecules, and is modified to serve as a cofactor in some enzymes. This dissertation focuses on two enzymes involved in tryptophan oxidation, AMSDH and MauG. AMSDH is a dehydrogenase in the kynurenine pathway, which is the main metabolic route for tryptophan catabolism. In addition to breaking down tryptophan, the kynurenine pathway is also involved in regulating the innate immune response, NAD biosynthesis, and some neurodegenerative. As such, enzymes of the kynurenine pathway are of fundamental interest for study. …

Development And Optimization Of A Human Collagen-Targeted Protein-Based Mri Contrast Agent For Early Detection Of Chronic Human Diseases, Oluwatosin Ibhagui

Chemistry Dissertations

Chronic diseases are leading cause of death in US and globally. About 60 % of adults in the US have at least one form of chronic disease while 40 % suffer from 2 or more chronic diseases. Noninvasive, early detection, quantitative diagnosis, and staging of these diseases with high-resolution imaging remain a pressing unmet medical need. Type I collagen a major constituent of extracellular matrix is highly expressed in the microenvironment of various chronic and acute human diseases such as hepatocellular carcinoma and liver and lung fibrosis. Its expression level and spatial crosslink pattern are stage-dependent and is an attractive …

Structure And Function Of Viral Rna-Host Protein Interactions Among Flaviviruses, Jessica Siemer

Chemistry Dissertations

All viruses are dependent on interactions with host cellular proteins to perpetuate their lifecycles. The genus Flavivirus, one of the four genera that comprise the family Flaviviridae, contains members that cause significant morbidity and mortality worldwide. Among the mosquito-transmitted flaviviruses, West Nile Virus (WNV), dengue (DENV), and Zika virus (ZIKV) have emerged in the Western hemisphere, which now joins Southeastern Asia and Africa as an endemic region. There are no effective antiviral therapies for treating individuals with flavivirus infections, and only a few flavivirus vaccines are available for humans.

Current knowledge about the complex mechanisms that differentially regulate …

Characterizing Novel Filovirus Proteins And 3-Deazaneplanocin A Derivatives As Antivirals Against Non-Segmented Negative Sense Rna Viruses, Joyce Sweeney Gibbons

Chemistry Dissertations

Filoviruses belong to a family of RNA viruses that includes deadly emerging zoonotic pathogens such as Ebola and Marburg viruses. A concern of public health is whether recently discovered filoviruses have the potential to infect humans and cause disease. Filoviruses encode proteins that suppress innate immune signaling and this is postulated as a contributing determinant of virulence in animals. Měnglà virus (MLAV), a recently discovered bat filovirus, can infect human cells using a vesicular stomatitis virus (VSV)-MLAV GP pseudotype system. In Chapter 2, we characterize MLAV’s VP35, VP40 and VP24 proteins on their ability to regulate both human and bat …

Integration Of Calcium Signaling By Dynamically Tuned Calcium Indicators And Calcium Sensing Receptor, Xiaonan Deng

Chemistry Dissertations

As a first and second messenger, calcium (Ca²⁺) regulates numerous biological processes. Its homeostasis is modulated by intracellular Ca²⁺ stores, membrane channels, pumps, and/or receptors (etc. calcium sensing receptor (CaSR)). To integrate multi-level Ca²⁺ dynamics, which exhibit a wide range of temporal kinetics, there is a pressing need to integrate Ca²⁺ signaling intracellularly and extracellularly, using a novel class of genetically encoded Ca²⁺ indicators (GECIs) with rapid kinetics and fully understanding the molecular mechanism of CaSR, respectively.

In the present study, we first report a green Ca²⁺ indicator called “G-CatchER⁺” that …

Development Of Esterase-Sensitive Persulfide Prodrugs And Studies Of Co-Independent Reactivities From Ruthenium-Based Co-Releasing Molecules, Zhengnan Yuan

Chemistry Dissertations

As endogenously produced small molecules, hydrogen sulfide (H₂S) and carbon monoxide (CO) have been demonstrated in extensive studies to mediate cellular signaling. More importantly, the therapeutic effects of these small molecules have been validated in vitro and in vivo. As such, development of H₂S- and CO-based therapeutic agents have attracted great interests in recent years.

H₂S is known to undergo redox transformations in biological systems to produce various sulfur species such as glutathione persulfide (GSSH) and cysteine persulfide (CysSSH). Several studies have indicated the enhanced ability for persulfides to modify protein through S-persulfidation and …

Computational Investigations Of Biomolecular Mechanisms In Genomic Replication, Repair And Transcription, Thomas Dodd

Chemistry Dissertations

High fidelity maintenance of the genome is imperative to ensuring stability and proliferation of cells. The genetic material (DNA) of a cell faces a constant barrage of metabolic and environmental assaults throughout the its lifetime, ultimately leading to DNA damage. Left unchecked, DNA damage can result in genomic instability, inviting a cascade of mutations that initiate cancer and other aging disorders. Thus, a large area of focus has been dedicated to understanding how DNA is damaged, repaired, expressed and replicated. At the heart of these processes lie complex macromolecular dynamics coupled with intricate protein-DNA interactions. Through advanced computational techniques it …