Biochemistry Commons^™

Epitranscriptomic Regulation In Breast Cancer And Pcb-Induced Liver Disease., Belinda Petri

Electronic Theses and Dissertations

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, …

Fbg Αc 389 – 402 Modulates Factor Xiii Crosslinking In The Fibrinogen Αc Region., Francis Dean Orlina Ablan

Electronic Theses and Dissertations

Fibrinogen (Fbg) is a coagulation protein critical for clot formation. Coagulation Factor XIII (FXIII) is a calcium-dependent transglutaminase that crosslinks reactive glutamines (Q) and lysines (K) between fibrin and other anti-fibrinolytic proteins. In the presence of Ca²⁺, FXIII could be activated non-proteolytically (FXIII-A°), or proteolytically by thrombin (FXIII-A*). Significant increases in clot stability and red blood cell retention are linked to FXIII activity in the fibrinogen αC region (Fbg Aα 221 – 610). This region contains several FXIII-reactive glutamines and lysines, as well as a binding site for FXIII-A* (Fbg αC 389 – 402) that includes a key …

Metabolic Foundations Of Exercise-Induced Cardiac Growth., Kyle Fulghum

Electronic Theses and Dissertations

Regular aerobic exercise promotes physiological cardiac growth, which is an adaptive response thought to enable the heart to meet higher physical demands. Cardiac growth involves coordination of catabolic and anabolic activities to support ATP generation, macromolecule biosynthesis, and myocyte hypertrophy. Although previous studies suggest that exercise-induced reductions in cardiac glycolysis are critical for physiological myocyte hypertrophy, it remains unclear how exercise influences the many interlinked pathways of metabolism that support adaptive remodeling of the heart. In this thesis project, we tested the general hypothesis that aerobic exercise promotes physiological cardiac growth by coordinating myocardial metabolism to promote glucose-supported anabolic pathway …

Applications Of Nuclear Magnetic Resonance Spectroscopy: From Drug Discovery To Protein Structure And Dynamics., Mark Vincent C. Dela Cerna

Electronic Theses and Dissertations

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors …

Impact Of Pank1 Deletion On Mitochondrial Acetylation And Cardiac Function During Pressure Overload., Timothy N. Audam

Electronic Theses and Dissertations

Recent studies have associated elevated protein acetylation levels with heart failure in humans. Although mechanisms promoting elevated acetylation levels are not fully known, excess acetyl-CoA may drive enzyme-independent acetylation of cardiac proteins. Accumulation of acetyl-CoA depends on the availability of sufficient CoA, whose production is regulated by pantothenate kinases in the CoA biosynthetic pathway. We show that cardiac proteins are hyperacetylated during heart failure in humans and tested in mice whether limiting CoA abundance would improve ventricular remodeling during pressure overload-induced hypertrophy. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, Pank1 (one of three PANK-encoding …

Investigations Of Elastin Recoil On Molecular And Macroscopic Levels., Nour Mohammad Jamhawi

Electronic Theses and Dissertations

Elastin is one of the most hydrophobic proteins, and it is extremely flexible when hydrated. The driving force for recoil is the decrease in entropy of the protein and/or the hydrating solvent. This dissertation is a study of both mechanisms. Following an introduction (Chapter 1), Chapters 2 and 3 investigate the recoil mechanism on the molecular level in the hydrating solvent and in the protein, respectively. Chapter 4 examines macroscopic properties of recoil by thermomechanics. Conclusions are discussed in Chapter 5. Using double quantum NMR, the deuterated water ordering at the elastin surface was studied quantitatively as a function of …

Investigating A Novel Function For Phosphoserine Aminotransferase 1 (Psat1) In Epidermal Growth Factor Receptor (Egfr)-Mediated Lung Tumorigenesis., Rumeysa Biyik-Sit

Electronic Theses and Dissertations

Phosphoserine aminotransferase 1 (PSAT1) catalyzes the second enzymatic step within the serine synthetic pathway (SSP) and its expression is elevated in numerous human cancers, including non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutant NSCLC is characterized by activating mutations within its tyrosine kinase domain and accounts for 17% of lung adenocarcinomas. Although elevated SSP activity has been observed in EGFR-mutant lung cancer cells, the involvement of PSAT1 in EGFR-mediated oncogenesis is still unclear. Here, we explore a putative non-canonical function for PSAT1 using biochemical approaches to elucidate unknown interacting proteins and genomic RNA-seq profiling to identify cellular …

Investigating The Disorder And Compaction Of Designed Minielastin Using Nuclear Magnetic Resonance., Ma Faye Charmagne Aquino Carvajal

Electronic Theses and Dissertations

Minielastins are elastin-based proteins with alternating hydrophobic and cross-link modules similar to tropoelastin. Tropoelastin is the ~70 kDa soluble monomeric precursor of elastin. The extracellular matrix protein, elastin provides elasticity to tissues and organs such as lungs, arteries and ligaments. The elastic properties of natural elastin are believed to be entropic in origin. In vivo, the elastin matrix is approximately 50% water by weight. Without water, elastin is brittle and hard. Minielastins, like tropoelastin, undergo a liquid-liquid phase transition upon an increase in temperature. Factors such as hydrophobicity, chain length and concentration affect the coacervation temperature, T_c. The …

The Retinoblastoma Protein Mediates Metabolic Reprogramming In Lung Cancer., Lindsey R. Conroy

Electronic Theses and Dissertations

Lung cancer is among the most frequently diagnosed cancers and is the leading cause of cancer-related deaths worldwide. One of the hallmark events in lung cancer pathogenesis is deregulation of the cell cycle. The retinoblastoma protein (pRb) is a tumor suppressor that is deleted, mutated, or inactivated in most lung cancer cases. Canonically, pRb functions to regulate cell cycle progression by repressing the transcriptional activity of the E2F family of transcription factors, inhibiting S phase entry. Although the cell cycle functions of pRb have been well established, recent studies have highlighted a functional role for pRb in controlling cellular metabolism. …

The Role Of Slc7a11 In Controlling Extracellular And Intracellular Redox Environments Of Lung Fibroblasts - Potential Targets For Intervention In Aging And Idiopathic Pulmonary Fibrosis., Yuxuan Zheng

Electronic Theses and Dissertations

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition by fibroblasts. Aging and oxidative stress increase the susceptibility to IPF. Redox couples, cysteine/cystine (Cys/CySS) and glutathione/glutathione disulfide (GSH/GSSG), and their redox potentials (E_h) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular E_h(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system X_c^-, an antiporter that imports CySS and exports glutamate. The first aim of this dissertation is to investigate the mechanistic link between Slc7a11 …

Investigation Of Phosphoserine Aminotransferase 1(Psat1) In Breast Cancer Progression., Stephanie Metcalf

Electronic Theses and Dissertations

This dissertation describes my research into the involvement of phosphoserine aminotransferase 1 (PSAT1) in breast cancer progression; specifically, in triple negative breast cancer (TNBC) metastasis and endocrine resistance in estrogen receptor positive breast cancer (ER+BC). Breast cancer is the most common tumor diagnosis among women. While the overall 5-year survival for breast cancer is reaching 90%, the 5-year survival for metastatic disease is only 22%. Metastasis and endocrine resistance combined can affect over 50% of patients. One of the proteins and pathways implicated in both metastasis and endocrine resistance in breast cancer is phosphoserine aminotransferase 1 (PSAT1) and the serine …

Relationships Of Protein Biomarkers Of The Urokinase Plasminogen Activator System With Expression Of Their Cognate Genes In Primary Breast Carcinomas., Seth B. Sereff

College of Arts & Sciences Senior Honors Theses

Background: Urokinase plasminogen activator (uPA), its receptor uPAR and serine protease inhibitors PAI-1 or PAI-2 play key roles in tissue membrane remodeling and invasion of basement membranes by induction of a fibrinolytic pathway. Earlier studies reported that uPA and PAI-1 protein levels in breast carcinomas assist in prediction of response to chemotherapy. Our goal is to develop molecular signatures of candidate genes and identify novel relationships with these four protein biomarkers that demonstrate clinical utility for assessment of breast carcinoma outcomes.

Methods: This retrospective study used de-identified biomarker results and clinical outcomes from primary breast cancers that were stored in …

Coagulation Factor Xiiia: Biochemical Properties Underlying Physiological Function., Boris Anokhin

Electronic Theses and Dissertations

Factor XIIIA (FXIIIA) is a transglutaminase that crosslinks intra- and extracellular protein substrates. The oligomeric state of active FXIIIA remains controversial, and the present work commenced with addressing this issue. The results of size exclusion chromatography and analytical ultracentrifugation confirmed a dimeric state for zymogen and, for the first time, indicated a monomeric state for the active FXIIIA in solution. Comparing sedimentation properties of proteolytically and nonproteolytically activated FXIIIA suggested conformational and functional differences between the two forms. Those differences were further assessed in a series of catalytic activity studies. Kinetic analysis revealed affinity for the glutamine substrate was higher …

Functional Consequence Of Psat1 Association On Pkm2'S Inherent Enzymatic Activity., Alexis Avidan Vega

Electronic Theses and Dissertations

Pyruvate kinase M2 (PKM2) is predominantly found in tumors, where it allows the cancer cell to adapt to metabolic conditions through allosteric regulation of its activity. We recently discovered that phosphoserine aminotransferase 1 (PSAT1) associates with and activates PKM2. Here, I sought to affirm PSAT1's ability to increase PKM2 activity through kinetic and association studies of wild-type or mutant PKM2 enzymes. I demonstrate that His-tagged WT and mutant PKM2 enzymes are active, exhibit different kinetics, yet cannot be activated by PSAT1. Comparison studies using untagged WT PKM2 suggest that inclusion of the His-tag disrupts PSAT1 association. In support, pull-down strategies …

Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle

Electronic Theses and Dissertations

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences …

Bayesian Analytical Approaches For Metabolomics : A Novel Method For Molecular Structure-Informed Metabolite Interaction Modeling, A Novel Diagnostic Model For Differentiating Myocardial Infarction Type, And Approaches For Compound Identification Given Mass Spectrometry Data., Patrick J. Trainor

Electronic Theses and Dissertations

Metabolomics, the study of small molecules in biological systems, has enjoyed great success in enabling researchers to examine disease-associated metabolic dysregulation and has been utilized for the discovery biomarkers of disease and phenotypic states. In spite of recent technological advances in the analytical platforms utilized in metabolomics and the proliferation of tools for the analysis of metabolomics data, significant challenges in metabolomics data analyses remain. In this dissertation, we present three of these challenges and Bayesian methodological solutions for each. In the first part we develop a new methodology to serve a basis for making higher order inferences in metabolomics, …

Egfr Signaling From The Early Endosome., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue …

The N-Terminal Methyltransferase Homologs Nrmt1 And Nrmt2 Exhibit Novel Regulation Of Activity Through Heterotrimer Formation., Jon David Faughn

Electronic Theses and Dissertations

Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered through formation of complexes with close homologs. This work involves the N-terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels and in a tissue-specific manner. They are both nuclear methyltransferases with overlapping target consensus sequences …

Functional And Structural Impact Of The Loss Of The Leucine-Rich Repeat Protein Lrit1 In The Mouse Retina., Catherine Ann Cobb

Electronic Theses and Dissertations

Mutations in genes encoding the leucine-rich repeat (LRR) proteins nyctalopin and LRIT3 lead to complete congenital stationary night blindness because they are critical to depolarizing bipolar cell function in the retina. LRIT3 has two closely related family members, LRIT1 and LRIT2. In silico analyses of publicly available RNA-Seq data showed that Lrit1 was highly expressed in the retina. Here I describe the expression pattern and impact of loss of LRIT1 on retinal function. To enable these studies, we used CRISPR/Cas9 technology to create an Lrit1^-/- mouse line. Retinal morphology and morphometry analyses showed no gross changes in retinal structure …

Type Ix Secretion System : Characterization Of An Effector Protein And An Insight Into The Role Of C-Terminal Domain Dimeration In Outer Membrane Translocation., Lahari Koneru

Electronic Theses and Dissertations

Porphyromonas gingivalis and Tannerella forsythia are two of the primary pathogens that are associated in the etiology and progression of chronic periodontitis. In T. forsythia, KLIKK proteases are the recently identified group of proteolytic enzymes that are secreted through Type IX secretion system (T9SS). Among, these KLIKK proteases a synergistic relationship was observed between karilysin and mirolysin in invading the host complement system for the survival of the bacteria. Since, karilysin has been already characterized, in this study we propose to study about mirolysin through structural, biochemical and biological characterization. The obtained results from the experiments has shown the …

Characterizing Reactive Glutamines In Fibrinogen And Elucidating Factor Xiii Substrate Specificity., Kelly Njine Mouapi

Electronic Theses and Dissertations

Fibrinogen is the most abundant protein involved in blood coagulation and has been associated with many pathological implications in cardiovascular disease. At the final stages of blood clot formation, the transglutaminase Factor XIIIa introduces γ-glutamyl-ε-lysinyl covalent bonds between reactive glutamines and lysines in fibrin, which results in a tighter clot network that is resistant to fibrinolysis. Factor XIIIa crosslinks specific reactive glutamines on fibrinogen, selecting more reactive glutamines in the αC region of fibrinogen than any other chain. Although crosslinking pairs in the αC region have been identified, little is known about the extent of crosslinking and the role played …

Mechanism Investigation Of Pseudouridine Synthases Trub And Rlua With Rna Containing 5-Fluorouridine And 4-Thiouridine., Uyen T. Duong

Electronic Theses and Dissertations

Pseudouridine synthases (Ψ synthases) are the enzymes that catalyze the isomerization of uridine (U) to pseudouridine (Ψ), which is the most prevalent post-transcriptional modification of RNA. The Ψ synthases fall into six different families that share no significant global sequence similarity; however, they all involve a conserved aspartic acid residue which is absolutely essential for activity. Tyrosine is a conserved residue in the active site in five of the six families of Ψ synthases (phenylalanine in the TruD family) and was hypothesized as the general base for the isomerization reaction. To confirm the function of Tyr-96, Y96F RluA was assayed …

Role Of Mir-29b-1 And Mir-29a In Endocrine-Resistant Breast Cancer., Penn Muluhngwi

Electronic Theses and Dissertations

Therapies targeting estrogen receptor α (ERα) including selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM); selective estrogen receptor downregulators (SERDs), e.g., fulvestrant (ICI 182,780); and aromatase inhibitors (AI), e.g., letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited as ~ 40% of breast cancer patients will eventually acquire resistance to them. The role of miRNAs in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This dissertation begins with a review on miRNAs implicated …

Role Of Microrna-21 In Atherogenesis., Rihab Hamed-Berair

Electronic Theses and Dissertations

MicroRNA-21 (miR-21) is an evolutionarily conserved microRNA, abundant in most cardiovascular tissues. It has been implicated in the pathogenesis of several cardiovascular diseases including restenosis, myocardial infarction, and heart failure. However, little is known about the contribution of miR-21 in atherosclerosis. My data show that expression of miR-21 is increased by >1.5-fold in murine atherosclerotic lesions and by 1.5-2.0-fold in the macrophages of Western diet (WD)-fed LDLR-KO mice (for 12-20 weeks). In vitro, LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-4-fold and down-regulated its target protein, PDCD4, in bone marrow-derived macrophages. Basally, macrophages isolated from miR-21-KO …

Modulation Of Cell Death Signaling And Cell Proliferation By The Interaction Of Homoserine Lactones And Paraoxonase 2., Aaron Mackallan Neely

Electronic Theses and Dissertations

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule that functions to facilitate bacteria-bacteria communication. C12 has also been reported to affect many aspects of human host cell physiology, including evoking cell death in various types of cells. However, the signaling pathway(s) leading to C12-triggerred cell death remains unclear. To clarify cell death signaling induced by C12, we examined mouse embryonic fibroblasts (MEFs) deficient in one or more caspases. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in cells, probably through the direct induction of mitochondrial membrane permeabilization. Previous studies indicate that …

Co-Treatment With Conjugated Linoleic Acid And Nitrite Modulates Mitochondrial Respiration And Electron Transport Chain Activity In Vivo And Attenuates Mitochondrial Dysfunction During Cardiac Injury., Patrick Van Hoose

Electronic Theses and Dissertations

Cardiovascular disease and subsequent complications, such as myocardial infarction (MI), is the leading cause of death within the United States. Clinical intervention to reduce cardiac damage following myocardial ischemia is limited. Polyunsaturated fatty acids have been linked to an overall beneficial effect on cardiovascular health and function. Conjugated linoleic acid (cLA) is an 18:2 unsaturated fatty acid found within the diet in ruminant meat and dairy products. The cis-9 trans-11 isomer of cLA is the most prominent isomer within the diet and cLA has been linked to favorable outcomes in numerous disease states. The mechanism mediating the effects of cLA …

Coordinate Regulation Of The Wnt And Hippo Pathways By The Novel Ras Effector Nore1a., Marvin Lee Schmidt Jr.

Electronic Theses and Dissertations

Ras is the most frequently activated oncogene found in human cancer, but its mechanisms of action remain only partially understood. Ras activates multiple signaling pathways in order to promote transformation, but can also exhibit a potent ability to induce growth arrest and death. NORE1A (RASSF5) is a direct Ras effector that acts as a tumor suppressor promoting senescence and apoptosis. Its expression is frequently lost in human tumors and its mechanism of action remains unclear. Here I show that NORE1A forms a direct, Ras regulated, complex with β-TrCP, the substrate recognition component of the SCF-β-TrCP ubiquitin ligase complex. NORE1A also …

Role Of C-Kit In The Growth, Migration And Differentiation Of Human Cardiac Progenitor Cells., Bathri Narayan Vajravelu

Electronic Theses and Dissertations

The belief that adult mammalian heart lacks regenerative potential was challenged by the identification of c-kit positive cardiac progenitor cells (CPCs) in the heart. A recent phase I clinical trial (SCIPIO), has shown that autologous c-kit positive CPCs improve cardiac function and quality of life when transplanted into ischemic heart disease patients. c-Kit is a type III receptor tyrosine kinase and a common stem cell antigen. Stem cell factor (SCF) is the only known ligand for c-kit. Although c-kit is extensively used as an invariable marker of resident CPCs and shown to be important in the context of different cell …

Biophysical And Computational Investigations Into G-Quadruplex Structural Polymorphism And Interaction With Small Molecules., Huy Tuan Le

Electronic Theses and Dissertations

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to …

Genetic And Mechanistic Analysis Of Rat Mammary Cancer Susceptibility., Jennifer Sanders

Electronic Theses and Dissertations

Breast cancer is a complex disease, which is influenced by genetic, epigenetic and environmental components. Genetic susceptibility to breast cancer is made up of high, moderate and low penetrance alleles. High and moderate penetrance alleles are rare and constitute only a small percentage of the genetic susceptibility. Most variation in genetic susceptibility is controlled by low- penetrance, common polymorphisms. Comparative genetics uses model organisms to study human disease. Rat strains exhibit different susceptibility phenotypes to chemical induced carcinogenesis. The Wistar-Furth (WF) rat strain is susceptible to chemically induced mammary carcinogenesis, while the Wistar-Kyoto (WKy) and Copenhagen (COP) rat strains are …