Biochemistry, Biophysics, and Structural Biology Commons^™

Development Of Ultrafast Protein Digestion And Standards Free Quantitation Methods For Mass Spectrometric Analysis, Praneeth Ivan Joel Fnu

Dissertations

A wide variety of biologically important molecules, such as enzymes, antibodies, hormones, transporters and receptors are proteins by composition and they play key roles in biological functions such as cellular regulation, communication, metabolism and physiological function. Protein dysfunctions and abnormalities are associated with numerous diseases, making proteins critical targets for understanding disease mechanisms and developing therapeutic interventions. Protein-based therapeutics such as monoclonal antibodies, hormones and vaccines gained popularity due to their high specificity, efficacy, and ability to treat complex diseases that are often difficult to address with small-molecule drugs.

Given the important role of proteins in a variety of biological …

Elucidating The Mechanisms Of Complex Flavoprotein Dehydrogenases, Tyler Alt

Dissertations

Flavins are ubiquitous redox active cofactors that can catalyze electron transfer between substrates. They offer the advantage of being able to stabilize a range of oxidation and protonation states, as well being amenable to covalent modification, which can further alter their activity. As such, the scope of reactions catalyzed by flavoproteins is broad and flavin has implications in a range of biological processes. The two predominant forms of flavin that serve as redox active cofactors are flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are functionally homologous. A major complication of studying flavoprotein dehydrogenases, and one that has …

Establishment Of A Hek293t Cell Model System To Investigate Direct Intracellular Interactions Between Amyloid-Β (Aβ) And The Nlrp3 Inflammasome, Cristina Sinobas Pereira

Dissertations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia worldwide. While Amyloid-β (Aβ) aggregation is a defining pathological feature, increasing evidence highlights chronic neuroinflammation as a critical contributor to disease progression. Soluble Aβ assemblies can act as damage associated molecular patterns (DAMPs) including activation of the NLRP3 inflammasome, which links amyloid pathology to sustained inflammatory responses in the brain (Heneka et al., 2015; Nakanishi et al., 2018). However, the molecular mechanisms by which specific Aβ conformations influence inflammasome assembly remain incompletely understood.This study aimed to investigate whether distinct aggregation states of amyloid β42 (Aβ42) are …

Entropic Forces Near Fluctuating Surfaces, Rubayet Hassan

Dissertations

Nanoscale structures are inherently dynamic due to persistent thermal fluctuations. Even in solid materials, these random deformations, driven by ambient thermal energy, can become significant when their characteristic scale approaches that of the structure itself and strongly influence their overall mechanical behavior. Examples of such structures include crystalline membranes, appearing in diverse forms such as nanotubes, nanoribbons, and kirigami/origami structures. Similarly, biological nanostructures, including lipid membranes, microtubules, actin filaments, and DNA, exhibit extreme flexibility and responsiveness due to their low bending rigidity. Numerous physiological processes are intrinsically linked to these thermal fluctuations, including exocytosis and endocytosis, membrane fusion, pore formation, …

Analysis Of Antibody Affinity Maturation And Thermal Stability Of Proteins Using Photophysical Approaches, Claire Baxter

Dissertations

Engineered antibodies (Abs) have found applications in disparate fields such as enzyme catalysis and medicinal therapeutics because of their exquisite binding affinity and specificity. To engineer antibodies for these purposes, a firm understanding of the underlying mechanisms that control Ab affinity and specificity is needed, so these properties can be tightly controlled and adapted to the application at hand. My research characterized the binding site of the anti-fluorescein Ab 4-4-20 using an array of biophysical techniques and gives insight into the binding site interactions that give rise to both affinity and specificity. We utilized Fab fragments of the anti-fluorescein Ab …

Development And Implementation Of Novel Mass Spectrometric Methods For Metabolism Studies In Drug Discoveries, Huifang Yao

Dissertations

The field of drug discovery has evolved significantly over the past few decades, with a growing emphasis on understanding the metabolic pathways and biotransformation processes that influence the pharmacokinetics and pharmacodynamics of drug candidates. Novel mass spectrometric methods have emerged as powerful tools in this domain, facilitating the comprehensive analysis of metabolic profiles and enabling the identification of metabolites with implications for safety, efficacy, and overall drug development. However, challenges remain in quantifying metabolic flux—an essential aspect of understanding drug action—particularly due to the limitations of traditional isotope kinetic assays, which often involve lengthy sample preparation and the use of …

Pyruvate Dehydrogenase Kinase 4 Mediates Sex Specific Outcomes In Sepsis Induced Cardiomyopathy, John Yap

Dissertations

During sepsis, males often experience worse cardiac outcomes than females. Here, we identified pyruvate dehydrogenase kinase 4 (PDK4) as a key contributor to this disparity. PDK4 is a mitochondrial protein that regulates glucose utilization by inhibiting pyruvate dehydrogenase (PDH). Using a mouse model of endotoxemia, we induced acute inflammation with a sublethal dose of lipopolysaccharide (LPS), which caused cardiac dysfunction and significantly increased myocardial PDK4 expression in males but not females. Cardiac-specific PDK4 overexpression promoted LPS-induced cardiac dysfunction in both sexes, whereas cardiac-specific PDK4 knockout provided protection. In wild-type males, LPS disrupted cardiac metabolism by decreasing PDH activity and fatty …

Regulatory, Structural And Functional Studies Of The Adp-Glucose Pyrophosphorylase From Photosynthetic And Heterotrophic Organisms, Gabriela Martinez-Ramirez

Dissertations

The production of biofuels like ethanol has increased with the transition to renewable energy. Starch can be converted into ethanol through chemical modification. Increasing the availability of starch is crucial to fight back climate change thus, researchers have focused on the biotechnological modification of plants. This modification is known as transgenics, where a plant is modified by the insertion of genes, to make it as productive as possible. The genes inserted code for enzymes that control pathways responsible for the synthesis of certain biomolecules, in this case starch. The synthesis of starch in plants (as glycogen in both photosynthetic and …

Enzymological Revisionism: Employing Transient State Approaches To Define The Catalytic Sequences Of Poorly Understood Systems, Madison M. Smith

Dissertations

Flavin is a molecule composed of a tricyclic isoalloxazine ring linked to a ribityl. Flavins are utilized as a cofactor by many enzymes to facilitate a myriad of chemistries. Flavoproteins were first discovered in the late 1800 s and have since been an interest to researchers, largely due to the unique spectrophotometric characteristics that make it a valuable reporter molecule. Flavin cofactors (typically either flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN)) are chemically versatile as the isoalloxazine ring can form one and two-electron reduced states. The reduced states are prone to react with molecular oxygen and numerous observations have …

Design, Synthesis, And Optimization Of Allosteric Inhibitors Of Hiv-1 Integrase, Krunal H. Patel

Dissertations

The human immunodeficiency virus type 1 (HIV-1) infection remains a global health crisis, necessitating the development of innovative antiviral strategies. During the integration step, HIV-1 integrase (IN) interacts with viral DNA and the cellular cofactor LEDGF/p75 to effectively integrate the reverse transcript into the host chromatin. Recently, a novel class of antiretroviral agents called Allosteric Inhibitors of HIV-1 Integrase (ALLINI) compounds has emerged as a promising avenue in the fight against HIV-1. While originally designed to inhibit IN-LEDGF/p75 interactions, these compounds have been shown to also impact late-stage viral maturation severely through IN multimerization. Induction of IN multimerization interferes with …

Novel Photobase Generators For Photoinduced Polymerization And Ph Regulation, Shupei Yu

Dissertations

Photochemistry encompasses the investigation of chemical processes instigated by light absorption. As important branches of photochemistry, photosensitive and optical materials have attracted extensive research interests in both academia and industry. Photosensitive and optical materials are composed of polymers / small molecules with photo-responsive properties. These materials not only can absorb light in the desired energy spectrum, but also exhibit chemical / physical reactions, which can be applied to different fields such as photoredox, photo-heat, phototherapy, solar cells, diodes, etc. Among them, photobase generators (PBGs) are a series of photosensitive compounds, which absorb the incident light, then release the basic species …

Heterotypic Interaction Of Α-Synuclein And Tdp-43 In Neurodegenerative Diseases, Shailendra Dhakal

Dissertations

Amyloid aggregates of specific proteins form important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show co-morbidities and exhibit clinical and pathological overlaps between multiple neurodegenerative diseases including α-synucleinopathies and TDP-43 proteinopathies. These overlaps are often accompanied by co-localization of α-synuclein (αS) and TDP-43 enhancing the possibility of direct interactions to generate heterotypic amyloids with distinct cellular consequences. To understand this, we investigate the interaction of α-synuclein (αS) and prion-like domain of TDP-43 (TDP-43 PrLD) in both homogenous and inhomogeneous phases. Firstly, we show that monomeric TDP-43 PrLD and αS interact …

Structural Features Of Hiv-1 Integrase In The Late Stage Of Viral Replication, Tolga Catmakas

Dissertations

HIV-1 integrase (IN) is an essential enzyme for the viral life cycle as it integrates viral genomic material into the host cell genome. In addition to its’ essential catalytic function, IN has another important non-catalytic role where it binds the viral RNA to facilitate successful particle maturation during the viral life cycle. A significant number of IN mutants, addressed as class II IN mutants, show various defects, affecting different steps of the viral life cycle. Class II IN mutants exhibit aberrant morphology where viral ribonucleoprotein particles are mislocalized outside of the capsid core. This phenomenon can be observed in the …

Biophysical Factors Affecting Habitat Suitability For Crassostrea Virginica, Jason D. Tilley

Dissertations

Oyster reefs provide a variety of important ecosystem services. However, the mortality rate of eastern oyster, Crassostrea virginica, the dominant species that produces oyster reefs in the northern Gulf of Mexico, is increasing at an alarming rate due to a variety of abiotic and biological factors. I examined how biophysical factors, including the less-studied fatty acid profiles of the suspended particulate matter on which oysters feed, influenced morphometric condition of C. virginica.

I sampled suspended particulate matter (SPM) and oysters in-situ in the western Mississippi Sound, which historically supported the majority of oyster production in Mississippi waters. Sampling …

Binding Interactions Of Biologically Relevant Molecules Studied Using Surface-Modified And Nanostructured Surfaces, Palak Sondhi

Dissertations

This research focuses on the field of surface nanobioscience, wherein different nanosurfaces that will be used as working electrodes in the electrochemical cell are manufactured and surface modified to understand the critical binding interactions between biologically significant molecules like proteins, carbohydrates, small drug molecules, and glycoproteins. This research is essential if we are to determine whether a synthetic molecule can serve as a therapeutic candidate or diagnose a disease in its early stages. In order to fully understand the binding interactions, the study begins with defining some of the fundamental concepts, principles, and analytical tools for biosensing.

Afterwards, we addressed …

Newer Insights Into Mmtv Replication: The Human Retroviral Proteins Hiv-1 Tat And Htlv-1 Tax Can Transactivate The Mmtv Novel 5’ Element, Thanumol Abdul Khader

Dissertations

The mouse mammary tumor virus (MMTV) is a prototypic betaretrovirus that causes breast cancer in mice. MMTV regulates its gene expression both at the transcriptional and post transcriptional levels. It has hormonally-regulated promoters controlled by negative regulatory factors in a tissue-specific manner. Recently, we identified a novel 24-nucleotide region at the 5' end of the MMTV genome crucial for MMTV gene expression. Its deletion resulted in disruption of MMTV transcript elongation and stability, leading to a complete loss of Gag gene expression. This conserved element is present within the bifurcated stem loop 4, responsible for gRNA dimerization and encapsidation, into …

Molecular Mechanisms Of Amyloid-Like Fibril Formation, Sharareh Jalali

Dissertations

Proteins play a critical role in living systems by performing most of the functions inside cells. The latter is determined by the protein's three-dimensional structure when it is folded in its native state. However, under pathological conditions, proteins can misfold and aggregate, accounting for the formation of highly ordered insoluble assemblies known as amyloid fibrils. These assemblies are associated with diseases like Parkinson's and Alzheimer's. Strong evidence suggests that three mechanisms are critical for forming amyloid fibrils. These mechanisms are the nucleation of amyloid fibrils in solution (primary nucleation) as well as on the surface of existing fibrils (secondary nucleation) …

Exploring Topological Phonons In Different Length Scales: Microtubules And Acoustic Metamaterials, Ssu-Ying Chen

Dissertations

The topological concepts of electronic states have been extended to phononic systems, leading to the prediction of topological phonons in a variety of materials. These phonons play a crucial role in determining material properties such as thermal conductivity, thermoelectricity, superconductivity, and specific heat. The objective of this dissertation is to investigate the role of topological phonons at different length scales.

Firstly, the acoustic resonator properties of tubulin proteins, which form microtubules, will be explored The microtubule has been proposed as an analog of a topological phononic insulator due to its unique properties. One key characteristic of topological materials is the …

Reactive Oxygen Species And Weak Magnetic Fields: Unraveling The Mechanisms Of Planarian Regeneration And Tissue Growth, Luke James Kinsey

Dissertations

Reactive oxygen species (ROS) play a critical role in basic cellular behaviors such as apoptosis, proliferation, and differentiation. These molecules once thought to be harmful byproducts of metabolism, are also required for many complex and ubiquitous processes like wound healing, development, and regeneration. This study investigates the influence of ROS on planarian regeneration, with a particular focus on ROS dynamics in stem cell-mediated tissue growth. Data reveal that modulation of ROS levels leads to changes in cellular behaviors and tissue growth outcomes. Inhibition of ROS accumulation blocks stem cell proliferation and tissue growth, preventing planarian regeneration, while partial ROS accumulation …

Molecular Mechanisms Of Natural And Synthetic Compounds Against Breast And Colon Cancer, Aysha Hamad Alneyadi

Dissertations

Cancer is a complex disease with various causes, including genetic mutations, viruses, chemical agents, and lifestyle factors. Although numerous studies have been conducted to develop a cure or novel medications for cancer, no standout treatment has been found. In this project, the use of various natural and synthetic compounds as potential cancer treatments was investigated using triple-negative breast cancer (TNBC) and colon cancer cells. The cancer therapy strategies investigated were based on plant-derived therapuetic approaches including an essential oil (Origanum majorana essential oil), a pure phytochemical (Carnosol), and newly developed synthetic drugs (Chromenes drevatives; C1 and C2). This study …

Continuum Modeling Of Active Nematics Via Data-Driven Equation Discovery, Connor Robertson

Dissertations

Data-driven modeling seeks to extract a parsimonious model for a physical system directly from measurement data. One of the most interpretable of these methods is Sparse Identification of Nonlinear Dynamics (SINDy), which selects a relatively sparse linear combination of model terms from a large set of (possibly nonlinear) candidates via optimization. This technique has shown promise for synthetic data generated by numerical simulations but the application of the techniques to real data is less developed. This dissertation applies SINDy to video data from a bio-inspired system of mictrotubule-motor protein assemblies, an example of nonequilibrium dynamics that has posed a significant …

Effects Of Membrane Components On The Oligomerization Of Amyloid-Β (Aβ) In Alzheimer Disease, Jhinuk Saha

Dissertations

Deposition of extracellular proteinaceous plaques of amyloid-β (Aβ) is one of the major hallmarks of Alzheimer disease (AD). Although, insoluble high molecular fibrils of Aβ are the main constituents of the senile plaques in AD, low molecular weight soluble Aβ oligomers have emerged as the key toxic species involved in memory impairment and neuronal cell death in AD. The process for generation of oligomers can be highly diverse and can involve homotypic interactions of Aβ as well as heterotypic interaction of Aβ with other macromolecules. Previously, the generation of Aβ oligomers with distinct biochemical and biophysical characteristics catalyzed non-esterified fatty …

Characterization And Genomic Analysis Of Two Escherichia Coli O157:H7 Bacteriophages Isolated From Pigeons, Mohamad I. Alolama

Dissertations

Enterohemorrhagic Escherichia coli, also known as EHEC, is a subset of Shiga toxin-producing E. coli (STEC), and it has recently been identified as one of the principal foodborne pathogens. E. coli O157:H7 is the most important serotype of STEC for its role in causing foodborne illnesses. E. coli O157:H7 could cause various gastroenteritis symptoms such as diarrhea, hemolytic uremic syndrome, hemorrhagic colitis, and thrombotic thrombocytopenic purpura and may cause death. Elimination of E. coli O157:H7 during food processing and storage is a possible solution. Bacteriophages have a significant impact on bacterial populations in nature due to their ability to …

Characterization Of Tick And Host-Associated Factors Linked To Induction Of Alpha-Gal Syndrome., Surendra Raj Sharma, Surendra Raj Sharma

Dissertations

Alpha-Gal Syndrome (AGS) is a delayed allergic reaction caused by specific IgE antibodies to the oligosaccharide galactose-α-1,3-galactose (α-gal) present in red meat. AGS is a growing concern globally, with over 34,000 confirmed cases in the United States alone. Studies have linked AGS to tick bites, which sensitizes hosts against α-gal antigens and elevates α-gal specific IgE. This dissertation investigates the role of tick and host-associated factors in AGS development. Microbiome analysis of Am. americanum ticks revealed a comprehensive core microbiome profile and showed that new Fransiella-like endosymbionts dominate and outcompete conventional tick Coxiella endosymbionts. Ticks subjected to tetracycline treatment showed …

Probing Amyloid-Beta Protein Structure And Dynamics With A Selective Antibody, Shikha Grover

Dissertations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The AD brain is characterized by significant neuronal loss and accumulation of insoluble fibrillar amyloid-β protein (Aβ) plaques and tau protein neurofibrillary tangles in the brain. However, over the last decade, many studies have shown that the neurodegenerative effect of Aβ may in fact be caused by various soluble oligomeric forms as opposed to the insoluble fibrils. Furthermore, the data suggest that a pre-fibrillar aggregated form, termed protofibrils, mediates direct neurotoxicity, and triggers a robust neuroinflammatory response.

Antibodies targeting the various conformation of Aβ are important therapeutic agents to prevent the progression …

Mammalian Dihydropyrimidine Dehydrogenase: Employing Transient State Kinetic Methods To Determine Catalytic And Inhibitory Mechanisms, Dariush Forouzesh

Dissertations

Dihydropyrimidine dehydrogenase (DPD) catalyzes the two-electron reduction of pyrimidine bases uracil and thymine as the first step in pyrimidine catabolism. The enzyme achieves this simple chemistry using a complex cofactor set including two flavins and four Fe4S4 centers. The flavins, FAD and FMN, interact with respective NADPH and pyrimidine substrates and the iron-sulfur centers form an electron transfer wire that links the two active sites that are separated by 56 Å. DPD accepts the common antineoplastic agent 5-fluorouracil (5FU) as a substrate and so undermines the establishment of efficacious toxicity. Though studied for multiple decades, a precise description of the …

Role Of Micrornas In Intestinal Inflammation And Barrier Homeostasis After Alcohol And Burn Injury, Caroline J. Herrnreiter

Dissertations

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression. Within the intestinal epithelium, miRNAs play a critical role in gut homeostasis and aberrant miRNA expression has been implicated in various disorders associated with intestinal inflammation and barrier disruption. In this study, we sought to profile changes in intestinal epithelial cell miRNA expression after alcohol and burn injury and elucidate their impact on inflammation and barrier integrity. In a more targeted approach, we began by focusing on anti-inflammatory miRNAs that, when downregulated, could exacerbate inflammation and result in intestinal barrier disruption. Using a mouse model of acute ethanol intoxication …

Dynamic Control Of The Cardiac Calcium Pump, Sean Robert Cleary

Dissertations

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ into the endoplasmic reticulum of cells to establish a reservoir for Ca2+ signaling. In the heart, the activity of this transporter is tightly controlled via direct interactions with two competing regulatory micropeptides: phospholamban (PLB) and dwarf open reading frame (DWORF). PLB inhibits SERCA, while DWORF activates SERCA. These competing interactions determine cardiac performance by modulating the Ca2+ signals that drive the contraction/relaxation cycle. Previous studies indicated these SERCA-micropeptide interactions are Ca2+-sensitive; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. Here, FRET-microscopy demonstrated that this …

Interactions Of Amyloid Peptides With Lipid Membranes, Yanxing Yang

Dissertations

The aggregation of amyloid proteins into fibrils is a hallmark of several diseases including Alzheimer’s (AD), Parkinson’s, and Type II diabetes. This aggregation process involves the formation of small size oligomers preceding the formation of insoluble fibrils. Recent studies have shown that these oligomers are more likely to be responsible for cell toxicity than fibrils. A possible mechanism of toxicity involves the interaction of oligomers with the cell membrane compromising its integrity. In particular, oligomers may form pore-like structures in the cell membrane affecting its permeability or they may induce lipid loss via a detergent-like effect. This dissertation aims to …

Angiogenic Supports For Microvascular Engineering, Zain Siddiqui

Dissertations

Ischemic tissue disease is caused by a lack of circulation / blood supply to tissue. This can be treated by introducing a number of angiogenic (pro-blood vessel forming) factors into the tissue. This work presents strategies for ischemic tissue treatment utilizing a novel proangiogenic self-assembling peptide hydrogel platform. To demonstrate the utility of this platform, its use alone as an angiogenic therapeutic (both alone as a self-assembling hydrogel and with two-component systems), and its ability to vascularize implants is explored. Due to these angiogenic scaffolds demonstrating efficacy to regenerate microvasculature, this work evaluates diseases that can be treated by the …