Biochemistry Commons^™

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 …

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 …

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 …

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 …

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 …

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 …

Ferrocenium Salt Aided Substitution Reactions And Synthesis Of Glycosylated Curcumin Derivatives, Deva Saroja Talasila

Dissertations

Organic synthesis has been significantly advanced with the employment of transition metal complexes. The discovery of transition metal catalysts provided the synthetic community with powerful tools for accelerating reactions and making them more selective and efficient. Many chemical reactions do not happen without a catalyst.

Iron-based catalysts have several advantages for the chemical industry because it is a non-toxic and ecologically friendly metal. Our group previously found that ferrocenium cations with a 3+ oxidation state of iron-catalyzed propargylic substitution reactions at low temperatures. The sandwich structure of ferrocenes allows substituents to be introduced on the cyclopentadienyl rings, which allows for …

Structural And Transient Kinetic Analysis On Mechanism-Based Inactivators Of Human Ornithine Aminotransferase, Arseniy Butrin

Dissertations

HCC, Hepatocellular carcinoma, MBI, Mechanism-based inactivator, OAT, Ornithine Aminotransferase

Immune Cell-Protein Interactions In Alzheimer’S Disease, Kapur Dhami

Dissertations

Misfolded protein aggregates are one of the significant contributing factors in many neurogenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and others. My PhD research project was to study various aspects of amyloid-β peptide, a 40-42-residue peptide and the primary component of the senile plaques found in Alzheimer’s disease (AD) brains. One objective of my research was to purify and characterize the intermediate Aβ42 species using an array of biophysical techniques like size exclusion chromatography, fluorescence assays, circular dichroism, cellular assays, and protein assays. The other major research thrust of my project was to study the role of microglial …

Mechanism Of Sila- And Germafluorenes For Biological Applications, Shelby Jarrett

Dissertations

2,7-disubstituted silafluorenes and germafluorenes, originally designed for OLED applications, are a class of fluorescent dyes that have gained recent interest as probes for bioimaging and as biosensors to monitor cellular dynamics and interactions. Desirable biological probes absorb in the visible region, have high extinction coefficients, high quantum yield and excellent photostability. Here, their spectral properties are investigated under aqueous conditions for relevant biological applications. These molecules display intense blue fluorescence in the solid state and in solution, have high extinction coefficients, and exhibit appreciable solubility in aqueous solution. To better understand potential applications, the mechanism of fluorescence was investigated. It …

Bodipy-Based Light-Responsive Materials, Zhaoxiong Wan

Dissertations

The energy required for life on our planet is provided by light. In science, light is an important and powerful tool for measuring and modifying substances. For example, under the effect of illumination, light-responsive materials can change their physical and chemical properties. Photochemistry is a branch of research to study the effects of light irradiation on chemical structures and reactions. Some photochemical changes could lead to chemical bond dissociation (such as in photosynthesis) and structure isomerization (such as in the retina). Photochemistry has a strong relationship with human lives and it is essential in many ways. Among photophysics, fluorescence emission …

Investigating The Role Of Fun30, A Chromatin Remodeler, In Dna Repair, Mehwish Iqbal

Dissertations

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. DSB repair needs to take place within the complex organization of the chromatin, and this requires changes in the chromatin structure adjacent to DSB sites. These changes occur through covalent histone modifications that alter histone-DNA contacts as well as by the action of ATP-dependent chromatin remodelers. Many chromatin remodelers, including Fun30, are involved in DSB repair. Fun30 facilitates DNA end resection at DSB site during the homologous recombination repair pathway. Apart from its role in DNA repair, Fun30 promotes gene silencing at heterochromatic loci such as telomeres, …

Abl2 Promotes Alcohol-Associated Liver Disease Via Ppar Gamma Regulation, Gregory Malnassy

Dissertations

Alcohol-associated liver disease (AALD) is an umbrella term for a spectrum of diseases resulting from chronic alcohol (e.g. ethanol) abuse ranging in severity from reversible conditions such as alcohol-induced steatosis to advanced and largely irreversible liver pathologies including alcoholic steatohepatitis (ASH), alcoholic hepatitis (AH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). AALD is one of the primary causes of chronic liver disease worldwide and accounts for 44% of liver disease deaths in the United States. Drinking rates, both in the United States and globally, have increased year over year for the past three decades, a trend which has resulted in significantly …

Bodipy Based Photodynamic Therapy And Potassium Probes, Xiangshan Liu

Dissertations

Boron dipyrromethene (BODIPY) has become more and more popular in photophysical and biological applications. BODIPY based fluorescence compounds have numerous advantage properties including low toxicity, high absorption extinction coefficients, high fluorescence quantum yields, sharp fluorescence emissions, and resistance to photobleaching. All the good properties lead to the wide applications of BODIPY based compounds. In this research, two different kinds of BODIPY are synthesized. Firstly, BDBrC containing carbazole and BODIPY core is synthesized. Cellular uptake research shows BDBrC can target on lysosome and singlet oxygen generation research result indicates that BDBrC can be used in photodynamic therapy. BDBrC also shows pH-sensitive …

Granulins In Norm And Neurodegenerative Pathologies, Anukool Bhopatkar

Dissertations

Granulins (GRNs) are small, cysteine-rich modules produced from the proteolytic cleavage of the precursor protein called progranulin (PGRN). GRNs are present in the form of seven tandem repeats within the precursor and are known to be produced in the extracellular and in lysosomal environments. In physiology, PGRN and GRNs plays pleiotropic roles such as neuronal growth and differentiation, immunomodulation, wound healing. Recent studies have implicated pathological role for PGRN in Alzheimer disease (AD) and frontotemporal dementia (FTD) but specific mechanism(s) remains unclear. However, potential interactions between GRNs and Ab42 and TDP-43 seem like a plausible underlying mechanism. Studies presented here …

Complexation Of Glycoalkaloid Α- Tomatine With Sterols And Its Potential Application As An Anti-Cancer Drug, Bishal Nepal

Dissertations

Glycoalkaloids (GAs) are secondary metabolites found mostly in higher plant species and some marine invertebrates. They are known to form complexes with 3β-hydroxy sterols such as cholesterol causing membrane disruption. So far the visual evidence showcasing the complexes formed between glycoalkaloids and sterols has been mainly restricted to some earlier studies using Brewster angle microscopy. This study aimed to develop a method for topographic and morphological analysis of sterol-glycoalkaloid complexes. Langmuir-Blodgett (LB) transfer of monolayers comprising of glycoalkaloid tomatine, sterols, and lipids in varying molar ratios onto mica followed by AFM examination was performed. The AFM method used required minimal …