Biochemistry Commons^™

Expression Analysis Of Micrornas And Microrna-Like Rnas In Aspergillus Flavus-Infected Aflatoxin Resistant And Susceptible Maize Inbred Lines, Amanda Benton Harper

Theses and Dissertations

Corn (Zea mays) is frequently infected by a soil fungal pathogen Aspergillus flavus. The fungus produces aflatoxins, which cause liver cancer. Maize inbred lines that are resistant to infection by A. flavus have been developed, and these inbred lines provide excellent models for studying molecular mechanisms of maize resistance to the fungus. MicroRNA-like RNAs (milRNAs) recently identified in A. flavus had been found to be correlated with aflatoxin production conditions, suggesting that the milRNAs might play a role in the regulation of aflatoxin production. In this research, small RNAs were isolated from kernels of maize (resistant Mp719 and susceptible Va35) …

The Drug Discovery Of Potential Anti-Cancer Agents To Inhibit The Demethylation Catalytic Activity Of The Jmjc Domain Of Kdm3a, Balgees Alshanqeti

Electronic Theses & Dissertations

Lysine demethylase 3A (KDM3A) is an enzyme that specifically catalyzes the removal of 2 or three methyl groups from lysine 9 of histone 3 (KH3). It belongs to the family of histone demethylases that contain the Jumonji C (JmjC) domain, which means cruciform in Japanese. KDM3A also belongs to a family of hydroxylases that are alpha-ketoglutarate dependent. The role of KDM3A in the cell is not yet understood, however, and based on its demethylation of studies have showed that its expression is elevated in embryonic stem cells in humans and in mice. In general, patterns of elevated expression of KDM3A …

In Silico Development Of An Rna Aptamer Library To Be Use For The Selection Of Rna Aptamer That Target Biomolecules, Nehad Nawfawi

Electronic Theses & Dissertations

The systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the development of high affinity RNA ligands toward and infinite array of target molecules. SELEX is based upon the generation of a randomized population of RNA or DNA molecules followed by a target molecule that selects high affinity ligands from the randomized population followed by the subsequent amplification of the selected molecules. The procedure of selection and amplification is typically carried out through multiple cycles to insure that the identified ligands exhibits the highest affinity toward the target. The procedure is very time- consuming often taking …

Functional And Mechanical Role Of Splice Variant Of Mucin4 (Muc4/X) And Trefoil Factors In Pancreatic Cancer Pathogenesis, Rahat Jahan

Theses & Dissertations

Pancreatic Cancer (PC) is one of the vicious cancers as it ranks third in the race of leading cause of cancer-related death. Lack of early diagnostic marker, poor understanding of molecular mechanism of the disease and failure to conventional chemotherapy makes this disease dreadful.

Mucin 4 (MUC4), a high molecular weight glycoprotein is one of the top differentially expressed molecules in PC while not expressed in normal pancreas. Accumulating evidence from our lab suggested its tumorigenic role in PC by increasing cell proliferation, invasion, chemotherapy resistance, tumor growth, and metastasis. Previously, our lab and other has identified 24 different splice …

Role Of P300 Zz Domain In Chromatin Association And Histone Acetylation, Yongming Xue

Dissertations & Theses (Open Access)

Transcription is strictly regulated by numerous factors including transcription coactivators. The p300 protein and its close paralogue CREB-binding protein (CREBBP, aka CBP) are well-known transcriptional coactivators that have intrinsic lysine acetyltransferase activity. The functions of p300/CBP largely rely on their capabilities to bind to chromatin and to acetylate the histone substrates. However, the molecular mechanisms underlying the regulation of these processes are not fully understood.

Through combination of various biochemical, biophysical and molecular approaches, we show that the ZZ-type zinc finger (ZZ) domain of p300 functions as a histone reader that specifically binds the N-terminal tail of histone H3. Crystal …

Localization Of Sip470, A Plant Lipid Transfer Protein In Nicotiana Tabacum, Shantaya Andrews

Electronic Theses and Dissertations

SABP2-interacting protein 470 (SIP470), a non-specific lipid transfer protein (nsLTP), was discovered in a yeast two-hybrid screening using SABP2 as bait and tobacco leaf proteins as prey. SABP2 is an important enzyme in systemic acquired resistance that converts salicylic acid to methyl salicylate. Localization studies are an important aspect to understanding the biological function of proteins. nsLTPs are generally considered apoplastic proteins and has been localized intracellularly and extracellularly. Transient expression shows highest expression of SIP470-eGFP at 2 days post infiltration into Nicotiana benthamiana. Confocal microscopy showed localization near the periphery of the cell. Subcellular localization using differential centrifugation showed …

Sip-428, A Sir2 Deacetylase Enzyme And Its Role In Biotic Stress Signaling Pathway, Bal Krishna Chand Thakuri

Electronic Theses and Dissertations

SABP2 (Salicylic Acid Binding Protein 2) plays a vital role in the salicylic acid signaling pathway of plants both regarding basal resistance and systemic acquired resistance against pathogen infection. SIP-428 (SABP2 Interacting Protein-428) is a Silent information regulator 2 (SIR2) like deacetylase enzyme that physically interacts with SABP2 in a yeast two-hybrid interaction and confirmed independently by a GST pull-down assay. We demonstrated that SIP- 428 is an NAD+ dependent SIR2 deacetylase enzyme. Transgenic tobacco plants silenced in SIP- 428 expression via RNAi showed enhanced basal resistance to microbial pathogens. Moreover, these SIP-428-silenced lines also exhibited a robust induction of …

Ca2+ - Induced Structural Change Of Multi-Domain Collagen Binding Segments Of Collagenases Colg And Colh From Hathewaya Histolytica, Christopher Eric Ruth

Graduate Theses and Dissertations

Hathewaya histolytica, previously renamed as Clostridium histolyticum, secretes at least two collagenases, ColH and ColG, that allow for degradation of extracellular matrices of animal tissue. Hathewaya histolytica virulence factors are proposed to undergo domain rearrangement upon secretion from the bacteria to the host. In order to accomplish this, collagenases seek the least ordered regions in collagen to efficiently disassemble the fibril. Two types of domains, Polycystic Kidney Disease-like (PKD) and Collagen Binding Domain (CBD), direct the collagenases ability to disassemble the fibril. Calcium dependent structural change have been reported to increase in thermal stability and in tighter collagen binding for …

The First In Vivo Human Methionine Sulfide Proteome And The Impact Of Smoking, Abdullah Qassab

Graduate Theses and Dissertations

Reactive oxygen species are naturally generated within the human body and they are known to modulate signaling pathway and mediate other physiological activities. However, excessive generation of ROS and the inability of body defense system in detoxifying them results in the so called “oxidative stress”. Methionine has powerful antioxidant properties due to the presence of electronegative sulfur in its structure. Therefore, Met is readily oxidized, and methionine sulfoxide has been linked to several pathological conditions.

The urinary proteome is an attractive candidate for the discovery of biomarkers to diagnose and classify health conditions because of the non-invasive collection procedure. However, …

A Role For Epac1 And Epac2 In Nociceptor Hyperexcitability And Chronic Pain After Spinal Cord Injury, Samantha Berkey

Dissertations & Theses (Open Access)

Chronic pain is a major complaint of those living with spinal cord injury (SCI), affecting 65-80% of the SCI population, but the treatment options remain limited or non-existent. The cAMP sensor EPAC has previously been shown to play a key role in chronic inflammatory and neuropathic pain, though the contribution from each of its two main isoforms, EPAC1 and EPAC2, is unclear. Here I test the hypothesis that both EPAC1 and EPAC2 play a key role in the maintenance of persistent nociceptor hyperexcitability and chronic pain after SCI.

Using both a T9 SCI mouse model and a T10 SCI rat …

Renalase As An Intracellular Metabolite Repair Enzyme, Matthew Robert Hoag

Theses and Dissertations

The human enzyme renalase was discovered in 2005 by nephrologist Gary Desir, who claimed the enzyme is secreted by the kidney into the blood where it was said to catabolize catecholamines in order to modulate blood pressure and heart rate. It has since been shown that the enzyme is expressed in all tissues and does not react with catecholamines. The research detailed in this dissertation led to the discovery that renalase oxidizes two highly toxic isomers of NAD(P)H to form innocuous NAD(P)+. We surmised that such an important cellular function would be pervasive in nature, and our lab was the …

Temperature And Polyunsaturated Fatty Acid’S Effect On Daphnia Magna Reproduction, Mark Albright

Electronic Theses and Dissertations

Organisms adapt to their environments by adjusting their biochemistry and physiology; such adaptation is limited by resource availability and physiological constraints. The freshwater crustacean Daphnia magna inhabits a wide range of environments and must survive and reproduce within a range of temperatures. One limit to low-temperature adaptation is thought to be the availability of unsaturated fatty acids necessary to maintain proper fluidity of cellular membranes. D. magna maintained at 10 ºC on a diet poor in unsaturated fatty acids have been observed to produce clutches that fail to develop. However, this has not been observed on a diet rich in …

Investigation Of The Molecular Mechanisms Of The Shigella Type Iii Secretion System Tip Complex, Abram R. Bernard

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Shigella are bacteria that are responsible for millions of infections and hundreds of thousands of deaths every year. The emergence of antibiotic resistant Shigella adds to the potentially devastating effect that these bacteria can have on human health. Shigella flexneri utilize specialized molecular machinery called the Type III secretion system to infect humans and cause disease. Research of this machinery promises to provide the knowledge, tools, and direction for the development of new avenues to combat shigellosis. This dissertation presents studies of two Shigella proteins, invasion plasmid antigens C and D (IpaC and IpaD). These proteins are part of a …

Virulence Regulation In Pseudomonas Aeruginosa Via The Alginate Regulators, Algu And Algr, The Posttranscriptional Regulator, Rsma, And The Two-Component System, Algz/R, Sean Stacey

Electronic Theses and Dissertations

Pseudomonas aeruginosa is a Gram-negative bacillus able to colonize a wide variety of environments. In the human host, P. aeruginosa can establish an acute infection or persist and create a chronic infection. P. aeruginosa is able to establish a niche and persist in human hosts by using a wide array of virulence factors used for: movement, killing host cells, and evading immune cells and antibiotics. Understanding virulence factors and their regulation has proved to be an important means of combating the morbidity and mortality of P. aeruginosa as well as the ever-increasing threat of drug resistance. By targeting virulence factors …

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 …

Fatty Acid Amides And Their Biosynthetic Enzymes Found In Insect Model Systems, Ryan L. Anderson

USF Tampa Graduate Theses and Dissertations

A fatty acid amide is precisely as the name suggests: A fatty acid (CHn-COOH), in which the hydroxyl group of the carboxylic acid is displaced by an amine functional group from a biogenic amine (R-NH2), ultimately forming an amide bond. Furthermore, these fatty acid amides can be composed of a variety of different acyl chain lengths donated by the fatty acid and a myriad of different biogenic amines. Thus, these molecules can be subdivided in a number of different ways including the separation of short chain (acetyl to heptanoyl) and long chain (palmitoyl to arachidonoyl) and also based off the …

Investigating The Roles Of Fucosylation And Calcium Signaling In Melanoma Invasion, Tyler S. Keeley

USF Tampa Graduate Theses and Dissertations

Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, …

Identifying Functional Components Of The Endoplasmic Reticulum Quality Control And Degradation Factor Edem1, Lydia Lamriben

Doctoral Dissertations

The ER Degradation-Enhancing Mannosidase-Like protein 1 (EDEM1) is a critical endoplasmic reticulum (ER) quality control factor involved in identifying and directing non-native proteins to the ER-associated protein degradation (ERAD) pathway. However, its recognition and binding properties have remained enigmatic since its discovery. Here we provide evidence for an additional redox-sensitive interaction between EDEM1 and Z/NHK that requires the presence of the single Cys on the α-1 antitrypsin ERAD clients. Moreover, this Cys-dependent interaction is necessary when the proteins are isolated under stringent detergent conditions, ones in which only strong covalent interactions can be sustained. This interaction is inherent to the …

Structural Studies Of Acid Alpha Glucosidase And Pompe Disease, Derrick Deming

Doctoral Dissertations

Acid α-glucosidase (GAA) is required for the degradation of lysosomal glycogen. Pompe disease is an autosomal recessive disorder caused by reduced GAA activity, resulting in the accumulation of glycogen within lysosomes. The most severe form of the disease is characterized by a progressive deterioration of cardiac and skeletal muscle leading to death before two years of age. An intense interest from both the academic and pharmaceutical communities led us to determine the crystal structure of GAA. The structure provides insight into Pompe disease by examining how over 200 disease-associated point mutations perturb GAA function. To aid in the development of …

Role Of The Facial Triad In Factor Inhibiting Hif (Fih): Ligand Binding, Substrate Selectivity, And Coupling, Vanessa Chaplin

Doctoral Dissertations

Alpha-ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O₂ for varied reactions. While most of these enzymes contain a non-heme Fe bound by a His₂Asp facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O₂. The enzyme “factor inhibiting HIF” (FIH) contains a His₂Asp facial triad and selectively hydroxylates polypeptides, however removal of the Asp ligand in the D201G variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of …

Feronia-Related Receptor Kinase 7 And Feronia And Their Role In Receiving And Transducing Signals, David Vyshedsky

Masters Theses

Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter …

A Comprehensive Catalog Of Post-Translational Modifications Of Drosophila Melanogaster Hox Protein, Sex Combs Reduced, Anirban Banerjee

Electronic Thesis and Dissertation Repository

During formation of the anterior-posterior axis, Homeotic selector (HOX) proteins determine the identity of Drosophila body segments. HOX proteins are transcription factors that regulate gene expression during development. Besides a highly conserved DNA-binding homeodomain (HD), HOX proteins also contain functionally important, evolutionarily conserved small motifs. These short motifs found in HOX proteins may be Short Linear Motifs (SLiMs). SLiMs are proposed to be sites of phosphorylation and this may regulate the activity of HOX proteins. The primary aim of this work was to develop a comprehensive catalogue of the sites of phosphorylation and other post-translational modifications (PTMs) for Fushi tarazu …

The 2Β Insert Perturbs Folding, Stability And Hydrophobic Exposure Of Stromal Interaction Molecules, Steve Chung

Electronic Thesis and Dissertation Repository

Stromal interaction molecule (STIM)1 and 2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels through oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil domains couple to PM Orai1 protein subunits to form Ca2+ released activated Ca2+ (CRAC) channels that facilitate store-operated Ca2+ entry (SOCE). One splice variant of STIM2, STIM2β, contains an extra 8-residue (2β insert) located within the coiled-coils and inhibits SOCE through an unresolved mechanism, adding another layer of complexity to Ca2+ regulation in mammals. I hypothesize that the 2β insert perturbs the coiled-coil conformation and dynamics commensurate with …

Allosteric Regulation Of Pyruvate Carboxylase, Yumeng Liu

Dissertations (1934 -)

Pyruvate carboxylase (PC; E.C.6.4.1.1) is a multifunctional, biotin-dependent enzyme that catalyzes the MgATP-dependent carboxylation of pyruvate to oxaloacetate. The overall reaction is accomplished by the coupling of two half reactions occurring at two spatially distinct catalytic domains by the translocation of a carrier domain, resulting in a net transfer of CO2 from bicarbonate to pyruvate. PC activity is regulated by multiple allosteric effectors with acetyl CoA serving as an activator in most species and L-aspartate serving as an inhibitor for microbial PC. The kinetic characterization of PC from different species have revealed that PC homologs are subject to divergent degrees …

Regulation Of Beta-Catenin By The Ctlh Complex, Christopher Chiasson

Electronic Thesis and Dissertation Repository

RanBPM is a highly conserved protein that has been shown to impact numerous cellular processes including migration, proliferation, and apoptosis. RanBPM exists in a multi-protein complex called the CTLH complex, which functions as an E3 ubiquitin ligase. A putative member of the CTLH complex, WDR26, has been implicated in the Wnt pathway through regulating β-catenin protein levels. This study investigated whether β-catenin regulation is performed by the CTLH complex. WDR26 was confirmed as part of the CTLH complex, and this complex is interacting with Axin1. CTLH members RanBPM and RMND5A both regulate beta-catenin levels in a proteasome dependent manner. Additionally, …

The Distinctive Regulatory Mechanisms Of Bacterial Acetyl-Coa Carboxylase, Alexandra Leigh Evans

LSU Doctoral Dissertations

Metabolic Regulation is a complex system used to control cellular metabolism in response to conditions in the cell’s environment. For most enzymes, the cell can rely upon a minimal amount of regulation; however, critical enzymes, such as acetyl-CoA carboxylase, must be regulated at multiple levels. Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis. In bacteria, acetyl-CoA carboxylase forms a complex of three subunits–biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase–which catalyze the carboxylation of acetyl-CoA to form malonyl-CoA via two half-reactions. In the first half-reaction, biotin covalently linked to biotin carboxyl carrier protein is carboxylated by biotin …

Towards An Atomic Level Model Of The Structure And Calmodulin Mediated Activation Of Eef-2k, Nathan E. Will

Dissertations, Theses, and Capstone Projects

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM) dependent member of the a-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific residue (Thr-56), decreasing its affinity for the ribosome and reducing the rate of peptide chain elongation during protein translation. In contrast to the “release-of-inhibition’ mechanism operative in most CaM-dependent proteins kinases, the activation of eEF-2K is proposed to occur through a two-step process subsequent to the engagement of CaM and involves (1) auto-phosphorylation on T348 and (2) engagement of an allosteric site by phospho-T348 leading to a state with the highest activity towards the substrate eEF-2. …

A Novel Exo-Proteomic Approach To The Study Of Traumatic Brain Injury, Ron B. Moyron

Loma Linda University Electronic Theses, Dissertations & Projects

Concussions and Traumatic Brain Injuries (TBI) are significant health concerns and affect a wide cross section of society. Current diagnostic criteria and modalities, such as brain imaging and subjective measures of consciousness such as the Glasgow Coma Scale (GCS) score, are insufficient to properly diagnose the full spectrum of head injuries. Assessment of injury severity and outcome are further complicated by the vast array of symptoms, many of which mimic those displayed by other disorders. It is important to possess a better diagnostic tool for head injury triage and outcome prediction. One current line of inquiry seeks to discover a …

Supercharged Models Of Intrinsically Disordered Proteins And Their Utility In Sensing, Peter J. Schnatz

Dissertations, Theses, and Capstone Projects

In this thesis I show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this I first modified the designed helical bundle hemoprotein H4 using simple molecular modeling, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. Due to the high surface charge density, ligand binding to this protein is allosterically activated by low concentrations of divalent cations and the polyamine spermine. …