Search results

Title: Localization, substrate specificity, and the effect of inhibitors on alkaline phosphatases of Tetrahymena geleii W
Creator: Degenhardt, Edward Frank
Date: 1955
Collection: Electronic Theses & Dissertations

Title: Information-entropy concepts for nutritional systems
Creator: Harper, Jerome Paul, 1948-
Date: 1976
Collection: Electronic Theses & Dissertations

Title: Studies toward the total synthesis of cucurbitacins
Creator: Chenera, Balan
Date: 1984
Collection: Electronic Theses & Dissertations

Title: Corticoid binding in bovine mammary tissue slices and sera
Creator: Gorewit, Ronald C.
Date: 1975
Collection: Electronic Theses & Dissertations

Title: The synthesis, characterization and reactivity of iron-sulfur and molybdenum-iron-sulfur complexes with phenoxide terminal ligands
Creator: Cleland, Walter Edward, Jr.
Date: 1984
Collection: Electronic Theses & Dissertations

Title: Biochemical characterization of the COI1-JAZ receptor for jasmonate
Creator: Katsir, Leron J.
Date: 2008
Collection: Electronic Theses & Dissertations

Title: Disentangling the branched respiratory chain of Shewanella oneidensis MR-1
Creator: Duhl, Kody Lee
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Bacteria possess a wide range of metabolic pathways, allowing them to adapt to an array of environmental changes. Focusing on these different metabolic pathways allows us to observe how bacteria catabolize substrate or use anabolic pathways to generate biomass. A more in-depth look shows that many of these pathways are redundant, meaning a single organism can conduct the same overall reactions differing only by the types of enzymes or intermediates used. Overlapping pathways are common in...
Show moreBacteria possess a wide range of metabolic pathways, allowing them to adapt to an array of environmental changes. Focusing on these different metabolic pathways allows us to observe how bacteria catabolize substrate or use anabolic pathways to generate biomass. A more in-depth look shows that many of these pathways are redundant, meaning a single organism can conduct the same overall reactions differing only by the types of enzymes or intermediates used. Overlapping pathways are common in bacteria and have become a focal point of metabolism research to determine the advantages of conserving redundant pathways throughout evolution. The metal reducing bacterium Shewanella oneidensis MR-1 is a practical model organism for metabolic studies, as it has substantial branching within its respiratory pathways. In this work, we focused on the extensive electron transport chain (ETC) of S. oneidensis MR-1 to understand the importance of seemingly redundant respiratory complexes and their functions during aerobic growth. The S. oneidensis MR-1 genome encodes four different NADH dehydrogenases (NDHs): a proton-pumping Type I NDH (Nuo), two sodium-pumping NDHs (Nqr1 and Nqr2), and one type II ‘uncoupling NDH (Ndh). NDHs oxidize NADH to move electrons into the ETC and generate ion-motive force that drives ATP synthesis, active transport, and motility. We determined that either Nuo or Nqr1 was required for aerobic growth in minimal medium. The presence of theoretically redundant complexes (Nqr2 and Ndh) did not rescue cell growth. Further, we determined that knocking out NDHs led to the inability to properly oxidize NADH. NADH build up inhibited the tricarboxylic acid cycle causing an amino acid synthesis defect and inhibiting growth of the S. oneidensis strain lacking Nuo and Nqr1. Recently, bacterial metabolic models have been developed to explain the use of energetically inefficient pathways during fast growth. Two standout models postulate that energetically inefficient pathways are used to reduce a cell’s proteome cost by eliminating thermodynamic barriers or to reduce dependence on the ETC as cells grow larger. We sought to uncover if these models applied to the respiratory chain of S. oneidensis MR-1 during aerobic growth, as the ETC can vary in energetic efficiency based on the combination of NDH and terminal oxidase used. Our findings indicate that the models apply to S. oneidensis MR-1 in the context of overflow metabolism during growth at higher growth rates, while the structuring of the ETC was not in agreement. Most importantly, determined that both carbon metabolism and the ETC were restructured for adaptive growth under differing conditions. As carbon metabolism became less efficient at faster growth rates, the NDH step of the ETC became more efficient, using complexes with higher coupling efficiencies.
Show less

Title: Tunable Fluorescent Organic Salts for Imaging and Therapy
Creator: Broadwater, Deanna May
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Cancer remains a leading cause of death worldwide and many treatments still rely on non-targeted chemotherapy, which has inadequate efficacy and is plagued by toxic side effects. A promising solution is photodynamic therapy (PDT), a noninvasive clinical cancer treatment that combines a light activated photosensitizer (PS) with excitatory light to generate toxic reactive oxygen species (ROS). These photoactive agents can also produce detectable wavelengths of light upon photoactivation, which...
Show moreCancer remains a leading cause of death worldwide and many treatments still rely on non-targeted chemotherapy, which has inadequate efficacy and is plagued by toxic side effects. A promising solution is photodynamic therapy (PDT), a noninvasive clinical cancer treatment that combines a light activated photosensitizer (PS) with excitatory light to generate toxic reactive oxygen species (ROS). These photoactive agents can also produce detectable wavelengths of light upon photoactivation, which has been used clinically to image tumors in cancer diagnostics and image-guided surgery. Having uses as both diagnostic and therapeutic agents, these molecules are known as theranostics. However, current light-activated theranostics are limited by low brightness, poor tissue penetration, and nonspecific cytotoxicity independent of light excitation. Due to these obstacles, PDT is currently limited to precancerous lesions, superficial neoplastic tissue, or palliative care. Therefore, improved theranostic agents are needed. Prevailing efforts to improve existing photoactive agents focus on chemical modifications that cannot independently control electronic properties (which dictate toxicity) from optical properties. To overcome these limitations, work in this dissertation develops a novel counterion pairing platform to modulate the toxicity of organic salts composed of a photoactive cationic heptamethine cyanine (Cy+) and a non-photoactive anion. These counterion-tuned fluorescent organic salts can be designed to be either nontoxic for imaging, or phototoxic for PDT. Organic salts self-organize into nanoparticles with shifted frontier molecular orbital levels dependent on the counterion while the bandgap remains the same. This allows for tuning of electronic properties without affecting optical properties. Improvements in these areas could expand light-activated theranostics into a wider range of cancers and improve patient outcomes. This dissertation will begin with a review of current photoactive agents used in cancer therapy and ongoing challenges to the adoption of PDT as a frontline therapy. Modern PDT regimens and potential combinatorial therapies will be appraised, and recent advances in rational PS design will be highlighted. Initial in vitro studies investigated the optoelectronic tuning capabilities of counterion pairing in human lung carcinoma (A549) and melanoma (WM1158) cell lines. Viability assays establish that pairings with weakly coordinating bulky anions could generate organic salts that are non-cytotoxic and selectively phototoxic, while pairing with standard hard anions yield cytotoxic organic salts. These studies demonstrate that anion pairing can be exploited to shift energy levels and influence ROS generation to either enhance photokilling of cancer cells or improve cell imaging. Organic salts were further investigated in a metastatic breast cancer mouse model to characterize biodistribution, antitumor efficacy within a complex tumor microenvironment, and off-site toxicity. In vivo experiments confirm that counterion tuning can generate a selectively phototoxic antitumor PS which abolishes tumor growth and reduces metastasis without systemic toxicity in a breast cancer mouse model. Overall, this work demonstrates the utility of using counterion tuning to control phototoxicity, and further demonstrates the untapped potential of photoactive theranostic agents for clinical cancer therapy.
Show less

Title: Tropane Alkaloid Biosynthesis in Atropa Belladonna
Creator: Bedewitz, Matthew
Date: 2021
Collection: Electronic Theses & Dissertations
Description: A chemically diverse set of specialized flavorings, fragrances, and medicinal metabolites are produced by plants to modulate their interactions with pollinators, herbivores, and other biotic and abiotic stresses. Tropane alkaloids are one group of these specialized metabolites which are produced in phylogenetically distinct plant families and include the narcotic cocaine and the pharmaceutical compounds hyoscyamine and scopolamine. Scopolamine is produced in the roots of plants in the...
Show moreA chemically diverse set of specialized flavorings, fragrances, and medicinal metabolites are produced by plants to modulate their interactions with pollinators, herbivores, and other biotic and abiotic stresses. Tropane alkaloids are one group of these specialized metabolites which are produced in phylogenetically distinct plant families and include the narcotic cocaine and the pharmaceutical compounds hyoscyamine and scopolamine. Scopolamine is produced in the roots of plants in the Solanaceae family and serves as the precursor to semi-synthetic tropane pharmaceuticals. A set of eleven tissue-specific transcriptomes was generated from the tender perennial Solanaceous plant Atropa belladonna, deadly nightshade, to fill gaps in the biosynthetic pathway from putrescine to scopolamine. This dissertation describes the identification of enzymes which complete three missing portions of the scopolamine biosynthetic pathway. These are the biosynthesis of tropinone, the diversion of phenylalanine into production of phenyllactic acid, and the activation and conjugation of phenyllactic acid with tropine to form littorine, a late pathway precursor to scopolamine. Tropinone is the first metabolite in this pathway with the characteristic 8-azabicyclo[3.2.1]octane tropane core. This pharmacore was synthesized in a classic biomimetic chemical synthesis, but the mechanism for tropinone biosynthesis has remained an open question in all species which produce these compounds. The A. belladonna lateral root transcriptome revealed that tropinone biosynthesis proceeds through an atypical polyketide synthase which uses an imine as its starter, and that the polyketide is cyclized to tropinone by a cytochrome P450. Tropic acid, the specialized acyl group for both hyoscyamine and scopolamine, is produced from the primary metabolite phenylalanine through the intermediate phenyllactic acid. Aromatic aminotransferases are one route by which amino acids can be dedicated to specialized metabolism. Six aromatic aminotransferases are present in the A. belladonna transcriptome, and one of these, ArAT4, is a root-specific phenylalanine aminotransferase required for biosynthesis of phenyllactic acid and ultimately, scopolamine. In contrast to other aminotransferases which equilibrate multiple amino acids, this enzyme is highly directional for the consumption of phenylalanine and production of tyrosine. Littorine, a precursor of hyoscyamine and scopolamine, is the ester of tropine and phenyllactic acid. The mechanism for phenyllactic acid activation and esterification have remained as an open question of tropane alkaloid biosynthesis since the discovery of littorine. Two routes exist for activation and conjugation in plants, through either coenzyme A thioesters or glucose esters. In A. belladonna, littorine biosynthesis proceeds through a glucose ester of phenyllactic acid produced by a glucosyltransferase, UGT84A27, and an acyltransferase, LITTORINE SYNTHASE. This contrasts with cocaine acylation in the Erythroxylaceae, which uses a different route highlighting the repeated, independent origin of tropane alkaloid biosynthesis in plants. The enzymes identified in this dissertation have completed three missing sections of the tropane alkaloid biosynthetic pathway in A. belladonna, resulting in a nearly complete pathway, suitable for engineering.
Show less

Title: Computational Molecular Design and Innovation : from Drug Discovery to Emerging Contaminants
Creator: Eken, Yigitcan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Computational approaches have found great utility in areas including drug discovery and environmental contamination by investigating protein dynamics, binding and interaction patterns. For drug discovery, in silico biophysical methods serve an important role in reducing the cost of and accelerating the discovery process, as such methods aid in facilitating the identification, optimization and screening of potential drug candidates and in providing important understanding of drug mechanisms of...
Show moreComputational approaches have found great utility in areas including drug discovery and environmental contamination by investigating protein dynamics, binding and interaction patterns. For drug discovery, in silico biophysical methods serve an important role in reducing the cost of and accelerating the discovery process, as such methods aid in facilitating the identification, optimization and screening of potential drug candidates and in providing important understanding of drug mechanisms of actions and structure activity relationships at the atomic level. For computational drug discovery and protein modelling strategies, probable binding conformations of the ligand to its target can be predicted, and these conformations can be further evaluated by using scoring functions, molecular dynamics and free energy calculations to determine binding affinities and understand how a ligand recognizes its host. Despite the utility of computational approaches in areas such as drug design and the study of protein functioning, the choice of methods is not straightforward. Because of this, a series of international blinded host-guest binding prediction challenges are available to identify the most effective approaches to predict a variety of properties. Some of the methods available for calculating free energies include free energy perturbation, replica exchange free energy perturbation and thermodynamic integration approaches, and end-state methods. The later are most promising due to their reduced computational cost and because there is no need for intermediate state simulation.
Show less

Title: Secretory glandular trichomes : analysis and engineering of plant specialized metabolism
Creator: Lybrand, Daniel Benjamin
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Plants produce a wide variety of specialized metabolites that mediate interactions between plants and other organisms or the environment. Specialized metabolic pathways are often unique to a subset of plant species and augment the core metabolic pathways that are nearly universal to all plants. In addition to phylogenetic restriction, specialized metabolic pathways also exhibit compartmentalization within a plant, either at the tissue or cell type level. Many plants possess secretory...
Show morePlants produce a wide variety of specialized metabolites that mediate interactions between plants and other organisms or the environment. Specialized metabolic pathways are often unique to a subset of plant species and augment the core metabolic pathways that are nearly universal to all plants. In addition to phylogenetic restriction, specialized metabolic pathways also exhibit compartmentalization within a plant, either at the tissue or cell type level. Many plants possess secretory glandular trichomes, structures consisting of one or a few cells that protrude from the surfaces of aboveground plant tissues like leaves, stems, and flowers. These trichomes accumulate or secrete specialized metabolites. This work describes the biochemical pathways behind two classes of trichome-localized specialized metabolites: pyrethrins and acylsugars. Metabolic engineering of the pyrethrin pathway in plant hosts and geographic variation of acylsugar phenotypes are also discussed.
Show less

Title: Regulation of Chlamydomonas reinhardtii cell cycle during synchronous growth
Creator: Lin, Yang-Tsung
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The regulation of the cell cycle in microalgae has long been a central topic in third-generation biofuel studies, as it relates to biomass accumulation and lipid production, the two determining factors of economically feasible biofuel production. The discovery of Compromised Hydrolysis of TAG 7 (CHT7) in the green alga Chlamydomonas reinhardtii has provided valuable insights into the metabolic status-dependent regulation of the cell cycle. CHT7 belongs to the CHC protein family, members of...
Show moreThe regulation of the cell cycle in microalgae has long been a central topic in third-generation biofuel studies, as it relates to biomass accumulation and lipid production, the two determining factors of economically feasible biofuel production. The discovery of Compromised Hydrolysis of TAG 7 (CHT7) in the green alga Chlamydomonas reinhardtii has provided valuable insights into the metabolic status-dependent regulation of the cell cycle. CHT7 belongs to the CHC protein family, members of which include transcriptional regulators programming the initiation of cell division. A similar role of CHT7 in mediating cell cycle progression has been suggested. However, the regulatory mechanism and specific processes regulated by CHT7 have yet to be determined. In this dissertation, I applied bioinformatics approaches to comprehensively study CHT7-mediated gene regulation during the cell cycle using cell cultures synchronously grown in bioreactors. I found that specific pathways such as DNA replication, chromosome condensation, and spindle assembly are affected by the absence of CHT7. Other affected pathways include cell-wall remodeling and previously uncharacterized putative kinase cascades. In addition, I discovered the presence of two potential cis-regulatory elements near the transcription start site of misregulated genes in cht7, which are potentially linked to homeodomain transcription factors. Moreover, I explored the potential relationship between the CHT7-mediated pathway and the gene network governed by the retinoblastoma protein complex. Lastly, I characterized the function of a novel CHC protein in cell cycle progression and compared it with the role of CHT7. Together, these studies facilitate a better understanding of the cell cycle regulation in microalgae.
Show less

Title: Modeling (Un)Binding Kinetics of Biologically Relevant Systems Using Resampling of Ensembles by Variation Optimization
Creator: Dixon, Thomas
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Conventional drug design optimizes binding affinity when designing molecules to maximize efficacy. However, re cent studies show that taking kinetics into account when designing drugs is necessary in some systems where the drug efficacy does not correlate with binding affinity, instead correlating with residence time (RT). To maximize the RT, knowledge of the kinetic pathway is required, but not currently feasible to determine experimentally due to the instability of the transition state....
Show moreConventional drug design optimizes binding affinity when designing molecules to maximize efficacy. However, re cent studies show that taking kinetics into account when designing drugs is necessary in some systems where the drug efficacy does not correlate with binding affinity, instead correlating with residence time (RT). To maximize the RT, knowledge of the kinetic pathway is required, but not currently feasible to determine experimentally due to the instability of the transition state. Molecular dynamics (MD) allows us to simulatethese pathways with atomic resolution. However, the rare events of interest often occur at timescales as long as milliseconds to hours, and most MD trajectories are computationally limited to the microsecond timescale. In this thesis we use a variant of the Weighted Ensemble (WE) enhanced sampling algorithm, Resampling of Ensembles by Variation Optimization (REVO), to overcome the limitations of MD. This approach is more computationally efficient than conventional MD and does not alter the system’s Hamiltonian nor does it affect the force field parameters used in simulation. We use REVO simulations to produce full binding and unbinding trajectories of biologically relevant systems such as the unbinding of a radioligand bound to Translocator Protein (18kDa) (TSPO), a potential drug target in the treatment of neurodegenerative diseases. We validate these pathways by predicting kinetic rate constants and binding free energies and comparing these results to experiment. Finally, we developed new distance metrics that use experimental data to help guide simulations to a desired conformation. We tested these new distance metrics using Hydrogen deuterium exchange (HDX) data to form the ternary complex between a ligase-proteolysis-targeting chimera (PROTAC) dimer and a target protein.
Show less

Title: Predicting the Properties of Ligands Using Molecular Dynamics and Machine Learning
Creator: Donyapour, Nazanin
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The discovery and design of new drugs requires extensive experimental assays that are usually very expensive and time-consuming. To cut down the cost and time of the drug development process and help design effective drugs more efficiently, various computational methods have been developed that are referred to collectively as in silico drug design. These in silico methods can be used to not only determine compounds that can bind to a target receptor but to determine whether compounds show...
Show moreThe discovery and design of new drugs requires extensive experimental assays that are usually very expensive and time-consuming. To cut down the cost and time of the drug development process and help design effective drugs more efficiently, various computational methods have been developed that are referred to collectively as in silico drug design. These in silico methods can be used to not only determine compounds that can bind to a target receptor but to determine whether compounds show ideal drug-like properties. I have provided solutions to these problems by developing novel methods for molecular simulation and molecular property prediction. Firstly, we have developed a new enhanced sampling MD algorithm called Resampling of Ensembles by Variation Optimization or “REVO” that can generate binding and unbinding pathways of ligand-target interactions. These pathways are useful for calculating transition rates and Residence Times (RT) of protein-ligand complexes. This can be particularly useful for drug design as studies for some systems show that the drug efficacy correlates more with RT than the binding affinity. This method is generally useful for generating long-timescale transitions in complex systems, including alternate ligand binding poses and protein conformational changes. Secondly, we have developed a technique we refer to as “ClassicalGSG” to predict the partition coefficient (log P) of small molecules. log P is one of the main factors in determining the drug likeness of a compound, as it helps determine bioavailability, solubility, and membrane permeability. This method has been very successful compared to other methods in literature. Finally, we have developed a method called ``Flexible Topology'' that we hope can eventually be used to screen a database of potential ligands while considering ligand-induced conformational changes. After discovering molecules with drug-like properties in the drug design pipeline, Virtual Screening (VS) methods are employed to perform an extensive search on drug databases with hundreds of millions of compounds to find candidates that bind tightly to a molecular target. However, in order for this to be computationally tractable, typically, only static snapshots of the target are used, which cannot respond to the presence of the drug compound. To efficiently capture drug-target interactions during screening, we have developed a machine-learning algorithm that employs Molecular Dynamics (MD) simulations with a protein of interest and a set of atoms called “Ghost Particles”. During the simulation, the Flexible Topology method induces forces that constantly modify the ghost particles and optimizes them toward drug-like molecules that are compatible with the molecular target.
Show less

Title: DEORPHANIZATION AND CHARACTERIZATION OF SEA LAMPREY OLFACTORY TRACE AMINE-ASSOCIATED RECEPTORS
Creator: JIA, LIANG
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The sense of smell plays an important role in mediating diverse behaviors in the animal kingdom. Odor detection in the sea lamprey is mediated by a limited number of odorant receptors (ORs) and trace amine-associated receptors (TAARs). Upon binding with odorants, the receptors are activated and subsequently activate the downstream neuronal signaling cascade that transforms the chemical information into electrophysiological signals. Odorous biogenic amines, when enriched in biological...
Show moreThe sense of smell plays an important role in mediating diverse behaviors in the animal kingdom. Odor detection in the sea lamprey is mediated by a limited number of odorant receptors (ORs) and trace amine-associated receptors (TAARs). Upon binding with odorants, the receptors are activated and subsequently activate the downstream neuronal signaling cascade that transforms the chemical information into electrophysiological signals. Odorous biogenic amines, when enriched in biological excretions, stimulate TAARs of the main olfactory epithelium and evoke innate behaviors in animals. I hypothesized that these biogenic amines are potent ligands for lamprey TAARs, and characterized the structural basis for amine recognition in these receptors. Chapter 1 describes discovery that spermine, an odorous polyamine in semen, serves as a sex pheromone in sea lamprey. Spermine potently stimulates the lamprey olfactory system, activates TAAR348 receptor, and attracts ovulated females. A novel antagonist to this receptor inhibits olfactory and female behavioral responses to spermine. This discovery elucidates a mechanism that male animals recruit mates through the release of chemical cues in ejaculates. In chapter 2, I demonstrated that two clades of independently evolved TAARs, represented by sea lamprey TAAR365 (sTAAR365) and mouse TAAR9 (mTAAR9), share a similar response profile. The results suggest a conserved mechanism whereby independently evolved TAAR receptors utilize convergent structural bases to detect various biogenic polyamines. In chapter 3, I found that a cadaverine-responsive sea lamprey TAAR receptor, TAAR346a, exhibits high basal activity when heterologously expressed in HEK293T cells. Triethylamine serves as an inverse agonist for TAAR346a that can specifically attenuate its high basal activity. These data support a model in which the inverse agonist recognizes only one of the two orthosteric sites used by the agonist as it elicits its inhibitory effect on the basal activity of the receptor. Further evidence was provided to highlight the importance of interhelical interactions in modulating ligand-independent activation of TAAR346a. Thus, this thesis contributes to a better understanding of sea lamprey olfaction and the structural basis of TAARs for amine recognition in vertebrate animals.
Show less

Title: PARTNERSHIP AND PREDATION IN PLANT-BIOTIC INTERACTIONS : THEORY, METHODS, AND EVIDENCE
Creator: Rowe, Shawna L.
Date: 2022
Collection: Electronic Theses & Dissertations
Description: As sessile organisms, plants face an endless series of encounters with other living organisms in their environment. Ranging from the beneficial to the harmful, the pressures of these biotic interactions force plants to rapidly adapt to survive and thrive. This dissertation is aimed at addressing questions about the metabolism of plant interactions with herbivores and nutritional symbionts in legumes. Despite spanning different systems and methods, these questions reflect my interest in...
Show moreAs sessile organisms, plants face an endless series of encounters with other living organisms in their environment. Ranging from the beneficial to the harmful, the pressures of these biotic interactions force plants to rapidly adapt to survive and thrive. This dissertation is aimed at addressing questions about the metabolism of plant interactions with herbivores and nutritional symbionts in legumes. Despite spanning different systems and methods, these questions reflect my interest in understanding the biochemistry underlying ecological and evolutionary function and my desire to develop tools that enable the investigation of such questions. To this end, the work in this dissertation is aimed at building conceptual and methodological tools to enable more thorough investigations of plant symbioses and, more broadly, plant-biotic interactions across levels of biological organization.First, I present a framework for making predictions on evolutionary trajectories and origins of plant--microbe communication systems. By highlighting the prevalence of coercive interactions in plant--microbe interactions, I demonstrate the plausibility of such interactions types to be an evolutionary precursor to seemingly stable signaling mechanisms. This work aims to provide useful evolutionary context for investigations concerning the evolutionary stability and exploitation of signaling mechanisms in established biological relationships. Second, I present collaborative work in which we developed and applied a cost-effective, high-throughput protocol for quantifying multiple biochemical defense responses from small quantities of plant tissue using spectrophotometric techniques. This protocol was then applied on two distinct populations of the legume Medicago polymorpha to investigate how changes defensive traits in responses to altered selective pressures have manifested over the course of novel range expansion. Our work demonstrated the feasibility and potential of assessing defense responses across plant populations. Further, the work documents a shift in herbivore preference of plants from familiar and unfamiliar ranges by demonstrating a herbivore preference for plant tissues with pre-induced defenses over uninduced tissues from an unfamiliar geographic range. Finally, I demonstrate the creation, construction, and validity of a novel microcosm system for assessing nutrient exchange in the symbiotic mutualism between plants and arbuscular mycorrhizal fungi (AMF). The novel system is reliable, biologically-relevant, durable, and sufficiently simple and cost-effective to deploy. I demonstrate the validity of the microcosm system and discuss in-progress work which demonstrates its potential to rigorously investigate unknown aspects of the plant-AMF mutualism. Taken together, these developments and suggestions contribute to the growing set of methods and frameworks developed for improving our understanding a various plant-biotic interactions.
Show less

Title: DISRUPTIONS IN HEPATIC ONE CARBON METABOLISM AND THE GUT MICROBIOME DURING THE PROGRESSION OF NON-ALCOHOLIC FATTY LIVER DISEASE
Creator: Fling, Russell Ryan
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and...
Show moreThe etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and central metabolism. In a AhR-dependent manner, mice orally gavaged with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) exhibit steatosis progressing to steatohepatitis with fibrosis akin to NAFLD progression. NAFLD and hepatocellular carcinoma (HCC) is also closely correlated with dysregulation of central metabolism e.g., hepatic one carbon metabolism (OCM), and gut dysbiosis contributing to NAFLD progression and worsening prognosis. This report investigates mechanisms involved in the dysregulation of the gut microbiome and OCM associated pathways relevant to NAFLD progression through comparisons of molecular analyses of TCDD-treated mice to human NAFLD and HCC. OCM describes the biosynthesis, homeostasis, and utilization of the cell’s main methyl donor, S-adenosylmethionine (SAM) including high flux anabolic biosynthesis of polyamines, phosphatidylcholine and creatine. In later stages of NAFLD, OCM is dysregulated with altered OCM gene expression as well as SAM and s-adenosylhomocysteine (SAH) levels. To assess TCDD-elicited effects on OCM, mice were orally gavaged with TCDD every 4 days for 28 days. Serum and livers collected at early (8 days) and late (28 days) time points were subjected to metabolomic analyses with integration of chromatin immunoprecipitation sequencing, transcriptomics and protein levels. Results from these studies suggest AhR-mediated repression of OCM required prolonged repeated TCDD-treatment and indirect effects elicited by AhR activation e.g., oxidative stress. Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with NAFLD and recapitulated in TCDD-treated mice. Similar to NAFLD, TCDD also increases systematic levels of secondary bile acids. These microbial transformed secondary bile acids are involved in modulation of host bile acid signaling pathways relevant to NAFLD. To investigate the effects of TCDD on the gut microbiota, the cecum contents of TCDD-treated mice were subjected to shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species, notably Lactobacillus reuteri. Top enriched species were also associated with increased abundances of bile salt hydrolase sequences, responsible for the initial deconjugation reaction in secondary bile acid metabolism. L. reuteri levels were also attributed to enrichment of mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis genes, a pathway that was also elevated in cirrhosis patients. These results extend the role of Lactobacilli in the AhR/intestinal axis and NAFLD progression as well as highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD. Collectively, these studies evaluated TCDD-elicited mechanisms involved in disruptions in host and microbial metabolism, highlighting the AhR’s role in NAFLD progression.
Show less

Title: DEVELOPMENT OF CARBOHYDRATE BASED CONJUGATE VACCINES USING Qβ VIRUS LIKE PARTICLES WITH ANTI-BACTERIAL OR ANTI-CANCER PROPERTIES
Creator: Rashidijahanabad, Zahra
Date: 2021
Collection: Electronic Theses & Dissertations
Description: ABSTRACTDEVELOPMENT OF CARBOHYDRATE BASED CONJUGATE VACCINES USING Qβ VIRUS LIKE PARTICLES WITH ANTI-BACTERIAL OR ANTI-CANCER PROPERTIESByZahra RashidijahanabadChimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) are exciting directions to harness the power of the immune system to fight cancer. Chapter 1 is focused on GD2 ganglioside and the mucin-1 (MUC1) protein, two important tumor associated carbohydrate antigens, and latest advances in CAR T cells and bispecific...
Show moreABSTRACTDEVELOPMENT OF CARBOHYDRATE BASED CONJUGATE VACCINES USING Qβ VIRUS LIKE PARTICLES WITH ANTI-BACTERIAL OR ANTI-CANCER PROPERTIESByZahra RashidijahanabadChimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) are exciting directions to harness the power of the immune system to fight cancer. Chapter 1 is focused on GD2 ganglioside and the mucin-1 (MUC1) protein, two important tumor associated carbohydrate antigens, and latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce “on target, off tumor” side effects are introduced. Besides CAR T cells and bispecific antibodies, carbohydrate-based vaccines hold great promise for a number of diseases, which will be the focus of the rest of this dissertation. Several challenges are associated with carbohydrate antigens in regard to inducing specific and protective antibodies as they are poorly immunogenic and the resulting antibodies induced by immunizing with carbohydrates only, typically have low affinity. Currently, developing carbohydrate-based vaccines requires covalent conjugation of the carbohydrate antigen with a protein carrier for optimal antibody response. Thus, generated antibodies have higher affinity against glycan structures. In chapter 2, a potential conjugate vaccine was developed by linking O-specific polysaccharide (OSP) antigen purified from Vibrio cholerae Inaba with Qβ virus like particles (VLPs) efficiently via squarate chemistry as one of the first examples of polysaccharide conjugation to VLPs. The Qβ-OSP conjugate was characterized with mass photometry on the whole particle level. Pertinent immunologic display of OSP was confirmed by immunoreactivity of the conjugate with convalescent phase samples from humans with cholera. Mouse immunization with the Qβ-OSP conjugate showed that the construct generated prominent and long-lasting IgG antibody responses against OSP, and the resulting antibodies could recognize the native lipopolysaccharide from Vibrio cholerae Inaba. This was the first time that Qβ was conjugated with a bacterial polysaccharide for vaccine development, broadening the scope of this powerful carrier. Tumor associated carbohydrate antigens (TACAs) are another class of attractive carbohydrate antigens for the development of anti-cancer immunotherapy with respect to monoclonal antibodies and vaccines. Tetrasaccharide sialyl-Lewisa is an attractive therapeutic target for cancer therapy since it is widely expressed on epithelial tumors of the gastrointestinal tract. The overexpression of sLea appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis. In chapter 3, sialyl-Lewisa conjugate vaccine with Qβ was developed. The resulting construct, Qβ-sLea, induced antibody production in vivo and the resulting antibodies showed high selectivity for sLea antigen in in vitro studies and effectively reduced tumor growth in mice.
Show less

Title: THE EPIGENETIC MECHANISMS OF POLYCOMB AND TRITHORAX PROTEINS IN STEM CELL MAINTENANCE AND LEUKEMOGENESIS
Creator: Aljazi, Mohammad
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Functionally, Polycomb repressive complex 2 (PRC2) mediates transcriptional repression of differentiation genes critical for mouse embryonic stem cell (mESC) maintenance. Culturing mESCs in 2i serum-free medium inhibits FGF/ERK signaling and activates the Wnt/β-catenin pathway, which induces a naive cell state characterized by a reduced expression of lineage-specific genes. Interestingly, in naive mESCs, both PRC2 chromatin occupancy and the repressive histone3 lysine 27 trimethylation ...
Show moreFunctionally, Polycomb repressive complex 2 (PRC2) mediates transcriptional repression of differentiation genes critical for mouse embryonic stem cell (mESC) maintenance. Culturing mESCs in 2i serum-free medium inhibits FGF/ERK signaling and activates the Wnt/β-catenin pathway, which induces a naive cell state characterized by a reduced expression of lineage-specific genes. Interestingly, in naive mESCs, both PRC2 chromatin occupancy and the repressive histone3 lysine 27 trimethylation (H3K27me3) modification they mediate are largely depleted. To explore the molecular mechanism attributing to the transcriptional changes in naive cells, we performed RNA-sequencing for mESCs cultured in serum versus 2i medium. Gene expression analysis revealed reduced Jarid2 levels in naive mESCs. Reactivation of FGF/ERK signaling caused elevated Jarid2 expression, whereas ERK1/2 deletion decreased its expression levels. Ectopic expression of ERK depleted cells restored Jarid2 expression, showing that Jarid2 expression dependent on ERK signaling. Using ChIP-seq analysis, we observed reduced occupancy for Jarid2 and PRC2 and decreased H3K27me3 levels in both naive and Erk1/2 depleted mESCs. Expression of Jarid2 in Erk1/2 depleted cells reestablished PRC2 occupancy and H3K27me3 modifications. Taken together, these results reveal the molecular mechanism associated with FGF/ERK signaling and PRC2 recruitment in mESCs.The TrxG (Trithorax) group member ASH1L serves as a regulator of cell development. However, its functional role in MLL-rearranged leukemia initiation and maintenance is not well understood. Using an Ash1L conditional knockout mouse model, we demonstrated that ASH1L in hematopoietic progenitor cells (HPCs) impaired initiation of MLL-AF9-induced leukemic transformation in vitro. Ablation of ASH1L in the MLL-AF9-transformed leukemia cells impeded maintenance in vitro and leukemia progression in vivo. Furthermore, Ash1L depleted cells expressing wild-type ASH1L rescued MLL-AF9-induced leukemia transformation, while the transformation of cells expressing enzymatically inactive ASH1L inhibited their maintenance. Implementation of RNA-sequencing analysis revealed that ASH1L controls the expression of MLL-AF9 target genes by occupying their promoters and depositing H3K36me2 marks at these sites. Altogether, these results demonstrate that the enzymatic activity of ASH1L is crucial for MLL-AF9-induced leukemic transformation and maintenance. In addition, our study identifies a potential therapeutic target in MLL-AF9-induced leukemias. Histone post-translational modifications are vital for epigenetic mediated gene regulation. While past studies have characterized the functional role of many histone H3 lysine residues modifications, the post-translational modification of histone H3 lysine 37 and the factors contributing to these modifications remain undefined in mammals. Using in vitro methyltransferase assays, we found that SMYD family member 5 (SMYD5) catalyzes mono-methylation of H3 lysine 36 and 37 (H3K36/K37me1). Mutation of the conserved histidine within the catalytic SET domain abolished SMYD5 methyltransferase activity in vitro. Additionally, loss of Smyd5 in mESCs reduces the global histone H3K37me1 level in cells. Thus, our data functionally identifies SMYD5 as an H3 specific methyltransferase that mediates H3K36me/H3K37me1 in vitro. It also reveals that SMYD5 serves as one of the histone methyltransferases catalyzing histone H3K37me1 in vivo.
Show less

Title: EXPLORING THE INTRICACIES OF NOVEL MIO-DEPENDENT AMINOMUTASES AND EXPANDING THEIR USE THROUGH REPURPOSING AND SCALE-UP
Creator: Walter, Tyler
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Aminomutases (AMs) catalyze the isomerization of α-amino acids to their corresponding β-amino acids. A unique class of aminomutases utilizes a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group formed via post-translational modification of three consecutive residues within the active site. These MIO-dependent aminomutases likely descend from ammonia lyases, responsible for catalyzing the removal of ammonia from α-amino acids to produce the corresponding acrylates, which are...
Show moreAminomutases (AMs) catalyze the isomerization of α-amino acids to their corresponding β-amino acids. A unique class of aminomutases utilizes a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic group formed via post-translational modification of three consecutive residues within the active site. These MIO-dependent aminomutases likely descend from ammonia lyases, responsible for catalyzing the removal of ammonia from α-amino acids to produce the corresponding acrylates, which are important metabolites in a variety of hosts. MIO-dependent enzymes catalyze this isomerization for aromatic amino acids (i.e. Phe, Tyr, His), the products of which are vital components to several antibiotics, antifungals, and anticancer therapeutics.In 2013, a MIO-dependent tyrosine aminomutase (TAM) from Japanese rice (Oryza sativa) was discovered while searching for defense induced metabolites. The enzyme responsible for the increased β-Tyr levels they observed, OsTAM, was cloned into E. coli and studied to elucidate its isomerization mechanism and confirm the product stereochemistry. However, during the course of this investigation, we found several interesting characteristics of OsTAM that completely changed the current dogma within MIO-dependent enzyme research including (1) OsTAM releases nearly 25% p-coumarate, the corresponding acrylate of Tyr, which is over 2-fold higher than other AMs; (2) OsTAM is the first TAM derived from plants, presenting a new opportunity to compare AMs across species; and (3) OsTAM is the first TAM to catalyze the isomerization of Phe, which paves the way for further study of the residues involved in substrate selectivity. Further study of the residues near the phenyl ring of the substrate showed two key residues, Y125 and N446, that controlled both substrate selectivity and the intrinsic activity of MIO-dependent enzymes. Arguably the most studied MIO-dependent PAM, TcPAM, lies along the biosynthetic pathway towards paclitaxel, a potent chemotherapeutic. Several studies within the lab showed that TcPAM has transamination activity when supplied an amine donor and acceptor acrylate. Recently, TcPAM was shown to catalyze the ring-opening amination of exogenously supplied cinnamate epoxides to produce phenylserine, key cores of numerous biologically active compounds. Interested in the potential of scale-up of this biocatalytic scheme, we developed a batch bioreactor method to increase cell densities of TcPAM-expressing cells and therefore increase the yield of the expressed enzyme. To increase the control and convenience of this process, we adapted autoinduction media developed by Studier in a batch bioreactor system. To the best of our knowledge, this has never been optimized for overexpression of an enzyme for later biocatalysis. With this batch bioreactor system, we turned our attentions towards scale-up of phenylserine, purification from the aqueous reaction mixture, and eventual cross-coupling to create biphenyl scaffolds aimed at recent discovery of a potent inhibitor of tumor growth with this molecular architecture. Initial studies allowed us to increase phenylserine production to the low milligram scale and develop a laboratory scale method for purification using C18 resin.
Show less

MSU Libraries

Digital Repository

Enabled Filters

Pages

Pages

MSU Libraries

Digital Repository

You are here

Search results

Pages

Pages