2014 REU Students

Amanda Clark

Mentor: Gary Sulikowski
Department:  Chemistry
Home Institution:  Central Michigan University

"Progress Towards the Synthesis of Oxidative Metabolites of Curcumin"

Abstract: Curcumin is the major component of the popular South Asian spice turmeric and is largely responsible for turmeric’s characteristic yellow color. Research has shown that curcumin is the compound that provides turmeric’s desired health benefits including anti-bacterial, anti-inflammatory, antioxidant, and anti-cancer properties. Recent studies have shown that curcumin is not stable past ingestion, but rather undergoes a process of auto-oxidation forming several different compounds. Therefore, it has been reasoned that these metabolites may be the key to curcumin’s various pharmacological effects. This research pursues the synthesis of four curcumin metabolites. Three metabolites are proposed to be accessed by way of a common synthetic intermediate cyclobutene derived from a [2+2] photocycloaddition reaction. A fourth metabolite is proposed to be obtained from a [4+2] thermal cycloaddition (Diels-Alder) reaction.

Corey Hayford

Mentor: Carlos Lopez
Department:  Chemistry and Cancer Biology
Home Institution:  University of Texas at Austin

"Mathematical Modeling of Progression Through the Mammalian Cell Cycle"

Abstract: The life cycle of a cell comprises multiple molecular checkpoints that regulate transitions between different phases of growth, DNA synthesis, and division. When a healthy cell suffers DNA damage, changes in protein dynamics occur that can lead to cell cycle arrest. Depending on damage level, the cell then decides to either engage in DNA repair or execute programmed cell death. In cancerous cells, the cell cycle signaling network becomes dysregulated, causing cells to bypass checkpoints and proliferate unbounded. The damage signal can be disrupted at any stage of the pathway, making it difficult to pinpoint the source of the malfunction and, hence, develop focused treatments. By understanding the molecular mechanisms that underlie cell fate decisions in healthy and carcinogenic cells, we should be able to develop targeted therapies that restore proper functionality or achieve cell death via alternate routes. A mechanistic, predictive understanding of cell-cycle progression and commitment to growth would provide novel insights about cellular proliferation and offer novel drug targets for cancer therapies. Mathematical modeling allows mechanistic hypotheses to be tested and improves understanding of experimental observations. In this project, a model of the entire cell cycle was constructed by merging models of different cell cycle transitions obtained from the literature. The model was constructed in PySB, a Python-based environment for modeling and simulation of biochemical processes. Chemical kinetics simulations were performed to explore molecular interactions under various system perturbations. The model provides a basis for future exploration of cell cycle dynamics under different external conditions.

Gabriel Pajares-Hurtado

Mentor: Larry Marnett
Department:  Biochemistry
Home Institution:  Georgia Institute of Technology

Title TBA

"Oxidative stress propagates methylglyoxal generation via inhibition of glyoxylase 1**"

Oxidative stress propagates the generation of damaging lipid aldehydes, such as 4-hydroxynonenal (HNE) and 4-oxononenal (ONE), and the highly reactive α-oxaldehydes, glyoxal and methylglyoxal (MG). MG has been shown to modify a wide range of biomolecules including DNA, lipids, and proteins, resulting in the generation of advanced glycation end products (AGEs). AGEs have been proposed to further promote both inflammation and oxidative stress, playing a central role in the development of diabetes; however, these mechanisms remain elusive. Glyoxalase 1 (GLO1) serves as the primary mechanism for MG detoxification via thiol-mediated conjugation to glutathione. Here, we examined the effects of the lipid electrophiles HNE on the activity of GLO1. Treatment of HEK293 cells with increased glucose concentrations resulted in a dose-dependent increase in cellular MG, these effects were exacerbated in the presence of HNE. Consistent with these data, click chemistry was utilized with purified GLO1 and ω-alkyl-HNE (aHNE) to reveal a dose-dependent increase in protein adducts. Future studies will aim to confirm these data in a more endogenous setting using RAW264.7 macrophages incubated with increasing concentrations of glucose. Stimulation with the pro-inflammatory agent, Kdo2-Lipid A (KLA), is predicted to result in a further increase in cellular MG production. Together these data serve to support the role of oxidative stress in propagating the generation of MG and ensuing AGEs.

Lidalee Silva

Mentor: David Wright
Department:  Chemistry
Home Institution: University of Puerto Rico at Aguadilla 

"Development of a Catch and Release Diagnostic Platform for the Malarial Biomarker PfLDH"

Abstract: Lateral flow immunochromatographic Rapid Diagnostic Tests (RDT’s) are known for their undemanding applicability in low- resource settings due to their simple interpretation and rapid timeframe for results.  However, RDT’s posses intermittent consistency attributable to their low sensitivity and quantitative character when testing patient blood samples. Previously, a low resource sample processing and extraction method has improved the sensitivity of biomarker identification, but this technique is limited because it captures any target incorporating histidine, thereby reducing specificity. To overcome this obstacle, we developed a catch and release diagnostic platform for Plasmodium falciparum L-lactate Dehydrogenase (PfLDH) malarial biomarker by coupling a Hexa-his tag to its primary antibody. PfLDH is a metabolic enzyme in the malaria parasite that takes part in glycolysis and only inhabits the human body when an infection is present; furthermore it is recognizable in asymptomatic stages. The Hexa-his tag appended to the antibody allows its capture by magnetic Cobalt (II)- Nitriliotriacetic Acid (Co (II)-NTA) beads. An excess concentration of imidazole (500 mM) replaces the histidines bound to (Co (II)- NTA) particles, permitting the elution of the PfLDH-antibody complex after washing. With the concentrated target obtained analysis is performed using the Malstat assay, which detects PfLDH metabolic activity.  At PfLDH concentration of 100nM, purification and concentration was achieved with a 67% recovery of total protein biomarker. This elucidation provides a general platform for the further development of a broad range of low- resource diagnostic tests for developing countries.

Kari Stratton

Mentor: Ned Porter
Department:  Chemistry
Home Institution: Texas A&M University

"Determining the Propagation Rate Constants of Biosynthetic Precursors to Cholesterol"

Abstract:  Cholesterol biosynthesis disorders, such as Smith-Lemli-Opitz, desmosterolosis, lathosterolosis, and Antley–Bixler, are human autosomal recessive syndromes caused by mutations in the genes involved in the biosynthesis of cholesterol. The defects in the biosynthesis lead to elevated levels of cholesterol precursors in affected individuals. Peroxidation of sterols can go through complex mechanistic pathways, generating various oxysterols that may contribute to the pathology of these disorders. Using a linoleate radical clock, the Porter lab has previously reported the propagation rate constants of various biosynthetic precursors to cholesterol such as 7-dehydrocholesterol and 8-dehydrocholesterol, both being among the most oxidizable lipids to date. The focus of this research is to use the linoleate radical clock to determine the rate constants of other cholesterol precursors, such as lathosterol and desmosterol, which are involved in other cholesterol biosynthesis disorders. Another aim of this project is to determine the effect of the C24-unsaturated side chain on the propagation rate constants of the cholesterol precursors in the Bloch pathway as compared to their C24-saturated counterparts in the Kandutsch-Russell Pathway. We found that while desmosterol (15 ± 4 M-1s-1) exhibits approximately the same reactivity as cholesterol (11 M-1s-1) in free radical oxidation, lathosterol (59 ± 4 M-1s-1) is more than five times as reactive. The propagation rate constant of the unsaturated side chain was found to be relatively small (9 ± 2 M-1s-1), suggesting that the reactivity of the sterols mostly depends on the structure and unsaturation degree of the steroidal rings.