Biochemistry Commons^™

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 …

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 …

Nicotinamide Riboside And Beta-Hydroxybutyrate Activate Parallel Pathways For C. Elegans Lifespan Extension, Mckenzie Peters

Undergraduate Honors Theses

Supplementation with nicotinamide riboside (NR), a form of vitamin B3 and a precursor of nicotinamide adenine dinucleotide (NAD⁺) extends lifespan in the nematode C. elegans and delays aging-related pathologies in mammals. During aging, levels of NAD⁺ decline causing metabolic dysfunction and oxidative damage. Studies in C. elegans found that when NR was administered during larval development it induced the mitochondrial unfolded protein response (UPR^mt), which is frequently associated with lifespan extension. Both calorie restriction (CR) and ketogenic diets (KD) have been shown to extend lifespan, in part through increasing NAD⁺ and through increasing levels …

Severe Hypoxia Up-Regulates Gluconeogenesis In Daphnia, Morad C. Malek

Undergraduate Honors Theses

Hypoxia is a significant low oxygen state that has complex and diverse impacts on organisms. In aerobes, various adaptive responses to hypoxia are observed that vary depending on the level of oxygen depletion and previous adaptation, hence the continued attention to hypoxia as an important abiotic stressor. Adaptive responses to hypoxia are primarily governed by the hypoxia-inducible factors (HIFs), which activate downstream genetic pathways responsible for oxygen transport and metabolic plasticity. In aquatic habitats, oxygen availability can vary greatly over time and space. Therefore, aquatic organisms’ adaptation to hypoxia is likely pervasive, especially in genotypes originating from waterbodies prone to …

Vitamin D And Its In Vitro Therapeutic Action Mediated Through Vdr Rather Than Pdia3, Jaeden Pyburn

Undergraduate Honors Theses

Brain calcification is a common occurrence in the aging process, with >20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of either PDIA3 or VDR transduce vitamin D mediated suppression of calcification in …

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 …

Therapeutic Drug Monitoring Of Apixaban Using Chromogenic Kits, Brooke Vogel

Undergraduate Honors Theses

Apixaban is a novel oral anticoagulant that prevents clotting by directly inhibiting Factor Xa in the coagulation cascade. Due to its different pharmacokinetics, previous standards for testing anticoagulant concentrations are ineffective at measuring apixaban. In this study, Hyphen Biomed Biophen Direct Xa Inhibitor and Biophen Heparin chromogenic kits from Aniara Diagnostica were used along with a NanoDrop™ One/One^C Microvolume UV-Vis Spectrophotometer to see if either of these kits provide acceptable precision and accuracy for the quantification of apixaban in plasma samples, as well as if there is a significant difference in these two kits at varying concentrations of apixaban. …

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’ …

Effects Of Nicotinamide Riboside And Beta-Hydroxybutyrate On C. Elegans Lifespan, Jeffery Peters

Undergraduate Honors Theses

The nicotinamide riboside (NR) form of vitamin B₃and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B₃are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD⁺decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD⁺levels. BHB is naturally produced by the body when individuals fast …

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 …

Modification, Verification Of Sequence And Optimization Of Expression Of P297f An Inactive Mutant Of Flavonol Specific Glucosyltransferase From Grapefruit (Cp3gt), Sarah Fox

Undergraduate Honors Theses

Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed …

Subcellular Localization Of Tobacco Sabp2 Under Normal And Stress Conditions, Sanjeev Das

Undergraduate Honors Theses

Subcellular Localization of Tobacco SABP2 under Normal and Stress Conditions

Salicylic acid (SA), a phytohormone, plays an important role in plant physiology. SA mediated innate immune pathway is an important pathway for plant immunity against pathogens. Plants resisting pathogen infection synthesize higher levels of Methyl Salicylate (MeSA), which is then converted to SA by the esterase activity of Salicylic Acid Binding Protein 2 (SABP2). The high level of the converted SA leads to enhanced pathogen resistance. The study of subcellular localization of a protein is critical in explaining its potential biochemical functions. SABP2 tagged with eGFP was expressed transiently in …

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 …

The Chelation Of Metal Ions By Vicibactin, A Siderophore Produced By Rhizobium Leguminosarum Atcc 14479, Joshua Stinnett

Undergraduate Honors Theses

Vicibactin is a small, high-affinity iron chelator produced by Rhizobium leguminosarum ATCC 14479. Previous work has shown that vicibactin is produced and secreted from the cell to sequester ferric iron from the environment during iron-deplete conditions. This ferric iron is then transported into the cell to be converted into ferrous iron. This study uses UV-Vis spectroscopy as well as ion trap-time of flight mass spectroscopy to determine that vicibactin does form a complex with copper(II) ions, however, at a much lower affinity than for iron(III). Stability tests have shown that the copper(II)-vicibactin complex is stable over time. The results of …

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 …

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 …

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 …

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 …

Determination Of The Substrate Specificity Of Citrus Paradisi Flavonol Specific 3-O-Glucosyltransferase Mutant D344p, Nathan R. Spaulding

Undergraduate Honors Theses

Plants produce a vast array of secondary metabolites. A group of phenolic compounds, the flavonoids, are metabolites ubiquitous among plants and are known to aid in processes such as plant reproduction, UV defense, pigmentation and development. In relation to human health, flavonoids have been found to possess anti-inflammatory, anti-cancer, and antioxidant properties. Flavonoid’s ability to participate in so many interactions is due in part to their subclass variation and further chemical modification. One such modification is glucosylation, where a glucose molecule is added to the flavonoid substrate. The enzymes that catalyze these reactions are known as glucosyltransferases (GT). Citrus paradisi …

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) …

The Effect Of R382w Mutation On Citrus Paradisi Flavonol Specific 3-O-Glucosyltransferase, Kathleen King

Undergraduate Honors Theses

Flavonoids are a class of plant metabolites with C6-C3-C6 structure responsible for many biological functions, including coloration and defense. Citrus paradisi, grapefruit, contains a wide variety of flavonoids which are grouped by the extent of modification, examples of which are flavonols, flavones, and flavanones. A major modification is the addition of glucose by glucosyltransferases (GTs) to stabilize the structure and provide ease of transport. Glucosyltransferases can be highly substrate and regiospecific. With Cp3OGT, glucose is added at the 3-hydroxy position. This 3GT only accepts flavonols as its substrate; however, a Vitis vinifera (grape) 3-GT can accept both flavonols and …