Biochemistry, Biophysics, and Structural Biology Commons^™

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 …

Reaction Pathways Initiated By One-Electron Oxidation Of Guanine: Reaction Pathways Leading To Stable End Products, Zach Cutright

Undergraduate Honors Theses

8-hydroxyguanine (8-oxoG) is one of the most important products resulting from the oxidation of guanine in DNA. 8-oxoG is known as a biomarker of oxidative stress such as lung cancer. 2,5-diamino-4H-imidazol-4-one (Iz) and its hydrolysis product 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)-amino]-2,5-dihydrooxazol-5-one (Oz) are also important products of oxidation of guanine in DNA. Recently, novel types of dimer intermediates related to 8-oxoG and Iz have been identified in our research lab. Using high performance liquid chromatography (HPLC), LC-MS, and NMR coupled with educated hypotheses, it is possible to identify the products, X1 and X2, resulting from the oxidation of guanine and decipher the mechanisms leading …

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. …

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 …

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 …

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 …

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 …

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 …

Oxidative Quenching Of Photoexcited Ru(Ii)-Bipyridine Complexes By Oxygen, Danielle Rebecca Latham

Undergraduate Honors Theses

An experimental approach was taken in determining the quantum yield of Ruthenium(II) with Oxygen using two different Ru complexes. This reaction results in Ruthenium(III) and O2-. The Coulombic interactions caused by a carboxylate functional was found to increase the yield of charge separation. This was done using a diode to measure the intensity of the completed reaction over a certain time frame. The intensities were turned into concentrations. The concentration over time was used to determine the quantum yield. This information is useful in creating more efficient light emitting diodes.

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 …

In Vitro Investigation Of The Effect Of Exogenous Ubiquitin On Processes Associated With Atherosclerosis, Chase W. Mussard

Undergraduate Honors Theses

Atherosclerosis, characterized by the build-up of cholesterol, immune cells and cellular debris within arterial walls, is accelerated following myocardial infarction by poorly understood mechanisms. Ubiquitin, a small, well-studied intracellular protein involved in protein turnover via the proteasome pathway, has recently been shown to exert extracellular effects on cardiac myocytes, in vitro, and in mice undergoing myocardial remodeling. This study investigates the potential role of extracellular ubiquitin in atherosclerosis by determining its effects on two critical atherosclerotic processes: the migration of vascular smooth muscles cells and the uptake of modified LDL by monocyte/macrophages in foam cell formation. In the presence …

Identification Of N-Acylethanolamine Hydrolyzing Enzyme In Solanum Lycopersicum, Derek A. Stuffle

Undergraduate Honors Theses

N-acylethanolamines (NAEs) are fatty acid derivatives that occur naturally in plant and animal systems. In mammals, they regulate physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, and cell proliferation. NAEs are metabolized by fatty acid amide hydrolase (FAAH), which belongs to the amidase signature family. It is hypothesized that putative FAAH functions as the catalyst in the metabolism of N-acylethanolamine in tomato plants. To test the hypothesis, FAAH protein homologs were identified in tomato via in silico analysis. Among the six homologs identified, FAAH1 and FAAH2 were selected for further validation. This study …

Genetic Variation In Long-Term And Short-Term Physiological Changes In Daphnia Magna During Acclimation To High Temperature, Bret L. Coggins

Undergraduate Honors Theses

The aquatic zooplankton crustacean Daphnia magna must be able to tolerate thermal stress in order to survive their native shallow ponds that are susceptible to drastic seasonal and diurnal temperature fluctuations as well as to globally increasing temperatures. Survival in such variable environments requires plastic responses that must include fundamental aspects of Daphnia biochemistry and physiology. Adaptive response to selection favoring such plastic phenotypes requires the presence of genetic variation for plastic response in natural populations. Adverse effects of elevated temperature on aquatic organisms are diverse and so are their plastic responses; among the most severe challenges aquatic organisms face …

Venom Peptide Induced Inhibition Of Escherichia Coli Atp Synthase, Sofiya Azim

Undergraduate Honors Theses

ATP is the main cellular energy generated by the enzyme ATP synthase in almost all organisms from bacteria to vertebrates. While malfunction of the ATP synthase complex is responsible for several disease conditions, the enzyme itself can be used as a potent molecular drug target to combat many diseases including microbial infections, cancer, tuberculosis, and obesity. Recent widespread escalation of antibiotic resistant microbes in general and E. coli in particular demands novel alternative approaches to combat microbial infections. Inhibition of ATP synthase by inhibitors such as peptides is known to deprive microbes of required energy, resulting in microbial cell death. …

Redesign Of Trans-Splicing Molecules For The Correction Of Dystrophia Myotonica Type 1 Toxic Rna Transcripts, Eleanor G. Harrison

Undergraduate Honors Theses

Dystrophia myotonica (DM1), one of the most common forms of muscular dystrophy, is caused by a repeated trinucleotide expansion in the DMPK gene. This mutation results in the accumulation of toxic cellular RNA transcripts. Spliceosome-mediated RNA trans-splicing (SMaRT) technology is a form of gene therapy that possesses the potential to correct these toxic RNA transcripts and thus cure the disease. Despite its promise, prior research applications of SMaRT technology to DM1 have been hampered by poor efficiency and have not been validated in a relevant model of the disease. In order to improve the efficiency of trans-splicing, this study examined …

Expression Of Human Neutrophil Cathepsin G In Pichia Pastoris, Evan T. Perry

Undergraduate Honors Theses

Cathepsin G (CatG), a serine protease found in the azurophil granules of neutrophils, participates in killing engulfed microorganisms. CatG is a poorly understood enzyme, in part because it can only be obtained as mature enzyme purified from human blood, and because it seems to have dual specificity for chymotrypsin-like and trypsin-like substrates. Therefore, yeast Pichia pastoris was used to express immature recombinant human CatG to provide a source of the enzyme free of biohazards and to allow study of its dual specificity and C-terminal processing. To avoid potential cleavage by a yeast kexin protease, the amino sequence was modified without …

The Effect Of Ultraviolet Light On Cell Viability, Dna Damage And Repair In Hutchinson-Gilford Progeria Syndrome And Bj Fibroblasts., Mckayla Johnson

Undergraduate Honors Theses

Patients of Hutchinson-Gilford Progeria Syndrome (HGPS) display a rate of aging up to ten times that of normal human populations. It might be expected that HGPS cells would have a decreased ability to repair DNA damage through the cell cycle as compared to normal cells such as those of the BJ cell line since DNA damage accumulation is a hallmark phenotype of aging. On earth, we are exposed to far more ultraviolet-B (UV-B, 280-315 nm) and UV-A (315-400 nm) than UV-C (100-280 nm) radiation, since the latter is filtered-out by the atmospheric ozone layer. The relative sensitivity of prematurely aging …

The Effects Of The Vitamin E Isomers Gamma Tocopherol And Gamma Tocotrienol On The Nfkb Pathway In The Pc-3 Cell Line., Brittney Rudder

Undergraduate Honors Theses

Regions along the Mediterranean and Southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of vitamin E (tocopherols and tocotrienols). This study examines the potential of gamma tocopherol (GT) and gamma tocotrienol (GT3) to reduce prostate cancer proliferation by examining their effects on the NFκB pathway. NFκB is known to inhibit apoptosis in cancer cells. Our data shows that both GT and GT3 are capable of down regulation of NFκB precursors and up regulation of Caspase …

Effects Of Vitamin E Isomer, Gamma Tocotrienol (Gt3), At Inhibiting Cell Growth And Inducing Apoptosis In Colon Cancer Cell Line Hct-116., Havya Dave

Undergraduate Honors Theses

Colorectal cancer is the third most prominent cancer world-wide and it is the second leading cause of cancer deaths in the United States. Many dietary components affect the risk of developing colorectal cancer, such as Vitamin E. Of the eight isomers of Vitamin E, four have a tocotrienol structure. Tocotrienols are found at highest concentrations in palm oil, which is ingested more in areas of Asia where the incidence of colorectal cancer is the lowest, suggesting a role of tocotrienols in the prevention of colorectal cancer. The metabolism of Arachidonic acid pathway produces a host of pro-inflammatory metabolites either by …