Biochemistry Commons^™

Flavonol Glucosylation: A Structural Investigation Of The Flavonol Specific 3-O Glucosyltransferase Cp3gt, Aaron S. Birchfield

Electronic Theses and Dissertations

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin …

Synthesis Of Bacterial Glycerophospholipids For Biomembrane Model Studies: A Means To Advanced Biofuels, Felix Adulley

Electronic Theses and Dissertations

To reduce reliance on fossil fuels, sustainable biofuels are being pursued, especially advanced biofuels like 1-butanol that have higher energy content and greater compatibility with existing infrastructure than ethanol. A persistent challenge is the yield-limiting toxicity of biofuels and process solvents, such as tetrahydrofuran, to the microbes that ferment biomass into biofuel. The cell membrane is a focal point of toxicity, and understanding how it interacts with fuels and solvents is key to improving yield. Phospholipid bilayers are the core of biomembranes, and model biomembranes of defined composition provide the ideal platform for biophysical studies. To this end, glycerophospholipids characteristic …

Lipidomic Analysis Of Various Developmental Stages Of Physcomitrium Patens, Deepshila Gautam

Electronic Theses and Dissertations

Lipids maintain fluidity of the cell membrane during the lifetime of all organisms. The moss Physcomitrium patens, an early land plant, enters reproductive phase under cold (15°C) conditions relative to its gametophytes (22°C). Thus, we hypothesized that their lipid content and composition would be distinct. Using ESI-MS/MS, we showed that the content and acyl composition of 11 lipid classes varied during development. Galactolipids were abundant in gametophytes but insignificant in sporophytes; among phospholipids, phosphatidylcholine was predominant in both phases. Although, sporophytes contained around five-fold less lipids than the gametophyte, their phosphatidic acid content, which accumulates during stress, was 18-fold …

The Relationship Between Cell-Free Dna And Resistance Training, Henry Lang

Electronic Theses and Dissertations

The primary purposes of this dissertation were to explore relationship between cell free DNA (cf-DNA), creatine kinase (CK), C-reactive protein (CRP), vertical jump testing delayed onset muscle soreness (DOMS) in response to a high-volume resistance training protocol, and to assess the sensitivity of cf-DNA to different resistance training volume loads. The secondary purpose was to examine the relationship between cf-DNA and relative strength. Study 1 was an exploratory attempt to discover relationships between cf-DNA, CK, CRP, delayed onset muscle soreness, and performance variables. Seventeen resistance trained males were recruited, 9 were randomly assigned to receive BCAAs while 8 received a …

Understanding The Implications Of Anandamide, An Endocannabinoid In An Early Land Plant, Physcomitrella Patens, Md Imdadul Haq

Electronic Theses and Dissertations

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ …

A Novel Mode Of Action Of C-Reactive Protein In Protecting Against Streptococcus Pneumoniae Infection And Synergy With Antibiotics, Donald Ngwa

Electronic Theses and Dissertations

C-reactive protein (CRP) is a part of the innate immune system, is synthesized in the liver, its blood level increases in inflammatory states, and it binds to Streptococcus pneumoniae. The conformation of CRP is altered under conditions mimicking an inflammatory milieu and this non-native CRP also binds to immobilized/aggregated/pathogenic proteins. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection. For protection, CRP must be injected into mice within two hours of administering pneumococci, thus, CRP is protective against early-stage infection but not against late-stage infection. It is unknown how CRP protects …

Protection Against Atherosclerosis By A Non-Native Pentameric Crp That Shares Its Ligand Recognition Functions With An Evolutionarily Distant Crp, Asmita Pathak

Electronic Theses and Dissertations

C-reactive protein (CRP) is an acute phase protein of the innate immune system that has been evolutionarily conserved. Human CRP is known to exist in two different pentameric conformations; native CRP and non-native CRP that possess differential ligand recognition functions. The structure of CRP evolved from arthropods to humans, in terms of subunit composition, disulfide bonds, and glycosylation pattern. Along with change in structure, the gene expression pattern of CRP also evolved from a constitutive protein in lower invertebrates to an acute phase protein in humans. The objective of this study was to determine the function of a non-native pentameric …

Exploration Of Ataxia Telangiectasia And Rad3-Related’S (Atr’S) Role In Cell Death Regulation: Implications In Development, Cancer, And Stroke, Brian Cartwright

Electronic Theses and Dissertations

From gametogenesis until death an organism’s genome is under constant bombardment from endogenous and exogenous sources of DNA damage. To maintain genomic integrity amid this damage, cells have evolved responses which allow them to either preserve viability for recovery or initiate self-destructive pathways depending on the severity of DNA damage. One protein involved in initiating and carrying out these responses is the protein kinase ataxia telangiectasia and Rad3-related (ATR). ATR is known primarily for its regulatory role in initiating the checkpoint-signaling cascade following DNA damage and replicative stress. These signaling events lead to cell cycle arrest, DNA repair, or apoptosis …

Does Thermotolerance In Daphnia Depend On The Mitochondrial Function?, Rajib Hasan

Electronic Theses and Dissertations

Thermotolerance limit in aquatic organism is set by the ability to sustain aerobic scope to sudden temperature shifts. This study tested the genetic and plastic differences in thermotolerance of Daphnia that can be explained by the differences in the ability to retain mitochondrial integrity at high temperatures. Five genotypes with different biogeographic origins were acclimated to 18^ᵒC and 25^ᵒC. We developed a rhodamine 123 in-vivo assay to measure mitochondrial membrane potential and observed higher fluorescent in heat damaged tissues as the disruption of the mitochondrial membrane potential. Significant effects on temperature tolerance were observed with CCCP …

The Distinct Expressions Of Integrins Αdβ2 And Αmβ2 Differently Regulate Macrophage Migration In 3d Matrix In Vitro And In Tissue During Inflammation, Kui Cui

Electronic Theses and Dissertations

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins α_Dβ₂ and α_Mβ₂ to macrophage migration and development of chronic inflammation.

We found that integrin α_Dβ₂ is upregulated on M1-macrophages in vitro and …

Preparation Of A Flavonol Specific Glucosyltransferase Found In Grapefruit And Site-Directed Mutants For Protein Crystallization, Aaron Birchfield

Electronic Theses and Dissertations

This research was designed to determine the conditions necessary to remove c-myc and 6x-His tags from a flavonol specific glucosyltransferase found in grapefruit (CP3GT) using thrombin in preparation for crystallization. X-ray crystallography of CP3GT crystals may elucidate structural features that account for flavonol specificity in some glucosyltransferase enzymes. A thrombin cleavage site was inserted into WT CP3GT and one mutant. Recombinant CP3GT was expressed in yeast and purified. Optimal conditions for thrombin digestion were explored. Digestion with 100U of thrombin for 2 hours at 4^o C was optimal for removing tags from CP3GT. Storage at 4^o C for …

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 …

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 …

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 …

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 …

Metabolic Plasticity In The Cellular Stress Response, Ying Li

Electronic Theses and Dissertations

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that ¹³C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained …

Quantitative Studies Of Amyloidogenic Protein Residue Interaction Networks And Abnormal Ammonia Metabolism In Neurotoxicity And Disease, Jeddidiah Griffin

Electronic Theses and Dissertations

Investigating similarities among neurological diseases can provide insight into disease processes. Two prominent commonalities of neurological diseases are the formation of amyloid deposits and altered ammonia and glutamate metabolism. Computational techniques were used to explore these processes in several neurological diseases. Residue interaction networks (RINs) abstract protein structure into a series of nodes (representing residues) and edges (representing connections between residues likely to interact). Analyzing the RINs of monomeric forms of amyloidogenic proteins for common network features revealed similarities not previously known. First, amyloidogenic variants of lysozyme were used to demonstrate the usefulness of RINs to the study of amyloidogenic …

Role Of Cannabinoid Receptor Type 2 (Cb2) In Late Stage Atherosclerosis, Makenzie Fulmer

Electronic Theses and Dissertations

Atherosclerosis is a chronic inflammatory disorder of medium and large vessels. Immune signaling and dyslipidemia are two of several processes which influence lesion development in atherosclerosis. Cannabinoids, such as those found in marijuana, exert their effects through two cannabinoid receptors, CB1 and CB2. Recent studies using CB2 knockout mice and CB2-selective ligands have shed light on a protective role of CB2 in early stages of atherosclerosis. However, the role of CB2 in advanced stages of atherosclerosis remains unclear. To determine if CB2 plays a role in advanced atherosclerotic lesion composition and progression, we investigated the effects of systemic CB2 gene …

A Novel Method For Synthesis Of Hydroxytyrosol, Emmanuel Onobun

Electronic Theses and Dissertations

Hydroxytyrosol, 3,4-dihydroxyphenolethanol, a naturally occurring polyphenol most common in olive tree (Olea europaea), is one of the most effective member of the polyphenols family, because of its remarkable antioxidant activity, its ability to inhibit oxidation of low density lipids (LDL), and its protection against DNA oxidative damage. Hydroxytyrosol, which is widely used in cosmetics and food supplements industries, can be purchased as an olive oil extract that contains low concentration of hydroxytyrosol besides other polyphenols. The price and low natural abundance of hydroxytyrosol make alternative synthetic sources very attractive. In this research, a novel method for the synthesis of pure …

A Novel Role Of Human Dna Damage Checkpoint Protein Atr In Suppressing Ca2+ Overload-Induced Parp1-Mediated Necrosis, Hui Wang-Heaton

Electronic Theses and Dissertations

Ataxia telangiectasia and Rad3-related (ATR) is well known for its regulatory role in DNA damage responses (DDR) as a checkpoint kinase that phosphorylates hundreds of protein substrates. However, its role in cellular non-DNA damage stress responses (NDDR) is unknown. Necrosis is one form of cell death and traditionally has been regarded as a passive and uncontrolled cell death. Recently, evidence has emerged to support the concept that necrosis also may occur in a programmed manner and that PARP1 can be a mediator. Active poly (ADP-ribose) polymerase 1 (PARP1) hydrolyzes nicotinamide adenine dinucleotide (NAD⁺) to produce poly (ADP-ribose) (PAR) …

Investigation Of Novel Functions For Dna Damage Response And Repair Proteins In Escherichia Coli And Humans, Benjamin A. Hilton

Electronic Theses and Dissertations

Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER …

Characterization Of A Putative Phospholipase D ´ Like Gene As A Lipid Signaling Modulator And Its Role In Salicylic Acid Mediated Defense Pathway In Nicotiana Tabacum, Phillip T. Dean

Electronic Theses and Dissertations

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed …

Regulation Of C-Reactive Protein Gene Expression And Function, Avinash N. Thirumalai

Electronic Theses and Dissertations

Human C-reactive protein (CRP) is the prototypic acute phase protein whose serum concentration increases rapidly during inflammation. CRP is also associated with atherosclerosis; it is deposited at lesion sites where it may interact with modified lipoproteins. There are 2 major questions regarding CRP: 1. How is the serum concentration of CRP regulated? 2. What are the functions of CRP in atherosclerosis?

Our first aim was to determine the role of the constitutively expressed transcription factor Oct-1 in regulating CRP gene expression. We found that Oct-1 overexpression inhibited (IL-6+IL-1β)- induced CRP gene expression; maximal inhibition required the binding of Oct-1 to …

Characterization Of A Putative Sir2 Like Deacetylase And Its Role In Sabp2 Dependent Salicylic Acid Mediated Pathways In Plant, Md I. Haq

Electronic Theses and Dissertations

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant …

Mechanisms Of The Anti-Pneumococcal Function Of C-Reactive Protein, Toh B. Gang

Electronic Theses and Dissertations

Human C-reactive protein (CRP) increases survival of and decreases bacteremia in mice infected with Streptococcus pneumoniae. Such protection of mice against pneumococcal infection is seen only when CRP is administered into mice 6 hours before to 2 hours after the injection of pneumococci, but not when CRP is given to mice at a later time. Our first aim was to define the mechanism of CRP-mediated initial protection of mice against infection. It was proposed that CRP binds to phosphocholine (PCh) moieties present in the cell wall and activates the complement system on the pneumococcal surface that kills the pathogen. …

An Examination Of The Inhibitory Effects Of Antibiotic Combinations On Ribosome Biosynthesis In Staphylococcus Aureus, Justin Beach

Electronic Theses and Dissertations

Bacteremia initiated by Staphylococcus aureus infections can be a serious medical problem. Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. Combination therapy for certain infections has been used to reduce the emergence of resistance when a single agent has become ineffective. We hypothesize that the use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects resulting from indirect effects on ribosomal subunit synthesis.

To determine this we measured the effects of single and multiple antibiotics on cell growth …

Bioengineering The Expression Of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, And C-Reactive Protein In Yeast, Eliot T. Smith

Electronic Theses and Dissertations

The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human …

Synthesis Of Resveratrol Esters And Aliphatic Acids., Stanley Mofor Jing

Electronic Theses and Dissertations

Resveratrol (RV) is a naturally occurring phytoalexin of the stilbenoid family produced by some plant species, and present in grape skin, peanuts, and red wine. It has been found to exhibit anti-cancer, anti-inflammatory, anti-viral, anti-aging, cardio protective, and anti-oxidant properties. Bioavailability is a huge setback that limits the potentials of RV. As a result, efforts have been made to design and synthesize RV esters and aliphatic acids in an attempt to increase its bioavailability, solubility in water, and possibly improving its biological activities. Resveratrol esters, 3,5,4'-triacetyloxystilbene (2) and Methyl 1,1',1''- (3,4',5-stilbenyl)-1,6-hexanedioate (3) have been synthesized. Compound 3 is a new …

Biochemistry Of Hemolysin Toxin Activation By Fatty Acylation: Characterization Of An Internal Protein Acyltransferase, Michael S. Trent

Electronic Theses and Dissertations

Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD . The A gene product, nontoxic hemolysin (proHlyA) is made toxic by post-translational fatty acylation of two internal lysine residues. HlyC, C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. ProHlyA and …

The Influence Of A Human Repetitive Dna On Genome Stability, Eugenia L. Posey

Electronic Theses and Dissertations

A uniquely human interspersed repetitive DNA sequence family, the L2Hs, are highly polymorphic in human genomes. Several features of interspersed repeated DNA may contribute to the instability observed. Certain motifs (direct repeats, palindromes, and inverted repeats) comprising L2Hs elements may adopt unusual secondary structures such as cruciforms or hairpins. These motifs have been associated with features of genome instability in recombination, insertions and deletions. The L2Hs elements also are AT-rich (76%) compared to the bulk of human DNA (52%). That their dynamic nature (i.e. polymorphisms) may arise from recombination, insertions and deletions has led to the hypothesis that the L2Hs …