Honors Projects for Biochemistry

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    Hyperphosphorylation of the Parkinson’s Disease protein α-Synuclein increases the rate of neurotoxic fibril formation in vitro
    (University of Hawaii at Manoa, 2014) Minami, Remy ; James, Nicholas ; Department of Cell and Molecular Biology and Chemistry
    Parkinson’s disease is a neurological disease that affects approximately 3% of the population over the age of 60 and is the second most common neurodegenerative disorder. PD is characterized by reduced levels of dopamine in the brain due to neuronal death in the substantia nigra. The surviving neurons contain spherical inclusions called Lewy Bodies (LBs) that are composed of a dense aggregation of proteins and lipids. α-Synuclein (aSyn) is the primary protein component of LB’s. Alterations in the expression of aSyn have been shown to affect neurotransmitter (such as dopamine) release. Also, when incubated at 37° C aSyn spontaneously forms fibril structures demonstrating the importance of this protein in LB formation. Hyperphosphorylated aSyn is the major fraction of protein found in LBs. This research project investigated the role of phosphorylation in aSyn aggregation in order to gain insight into the molecular basis of PD. S129E aSyn is a mutation that mimics phosphorylation at serine 129. In this project, wild-type aSyn and S129E aSyn were expressed and purified, and the rate of fibril formation was compared using a Thioflavin T fluorescence assay. The Thioflavin T assay showed a threefold increase in the rate of fibril formation in the S129E aSyn, consistent with a disease model in which hyper-phosphorylated aSyn increases the likelihood of formation of neurotoxic fibrils. The confirmation of the importance of phosphorylation in LB formation provides a molecular basis into the pathogenesis of PD and suggests further research on protein kinases that phosphorylate aSyn as a promising area of future research.
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    Optimizing Aromatase Expression and Uncovering Novel Allosteric Inhibitors for Breast Cancer Treatment
    (University of Hawaii at Manoa, 2017-05) Hu, Michelle ; Ng, Ho Leung ; Biochemistry
    Breast cancer occurs in 1 of 8 women while 2,600 new cases of breast cancer are expected in men during 2016 alone1 . Aromatase, a cytochrome P450 enzyme that interconverts androgens into estrogens, is linked to hormonal breast cancer development2 . Aromatase inhibitors are currently used to treat breast cancer, but the mode of binding for some inhibitors remains unknown. The objective of this project is to optimize aromatase recombinant expression in E. coli and discover novel allosteric inhibitors. While screening possible new inhibitory compounds using an activity assay, we identified AR11 and AR13, which produced IC50 values of 25.35 μM and 0.41 μM respectively. We have not yet been successful in increasing recombinant expression of mutant-type aromatase, despite adjusting induction time, incubation temperature, and cell strain. Although optimization of aromatase expression was not achieved, possible inhibitors were uncovered which will be used in future screening of protein crystallization conditions once expression is improved. These crystal screens can then be used to generate new structures of aromatase:inhibitor complexes, leading to improved inhibitor potency and reduced toxicity.
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    Molecular Taxonomic Identification of the Oriental Fruit Fly (Bactrocera dorsalis) and the Melon Fly (Bactrocera cucurbitae): How Similar Are They at the DNA Level?
    (University of Hawaii at Manoa, 2015-05) Freitas, Uyen ; Haymer, David ; Biochemistry
    Two species of fruit flies inflict massive amounts of agricultural damage throughout the Asia-Pacific region, including Hawaii. These are the oriental fruit fly, Bactrocera dorsalis, and the melon fly, Bactrocera cucurbitae. Many programs use chemical insecticides to control and eliminate the damage caused by these species, but biologically based methods are considered desirable alternatives. The first requirement of any of the biological methods is to make accurate and reliable species identifications. Unfortunately, in many cases, these species can be difficult to properly identify when traditional methods relying on morphological characters are used. The traditional methods depend almost entirely on the use of adult characters, but many specimens are captured at pre-adult stages that are difficult or impossible to identify at the species level using this approach. Fortunately, taxonomic methods based on the use of DNA characters, known as molecular taxonomy, have several advantages for identification of species, even for those found within closely related groupings known as species complexes. This method uses specific genetic markers found in an organism's DNA to identify it and characterize it as belonging to a particular species. One of the markers most commonly used for this purpose is the cytochrome oxidase I (COI) gene from the mitochondrial DNA. This project proposes to use molecular taxonomic methods using data from the COI gene to show the extent to which specimens of Bactrocera dorsalis can be discriminated from Bactrocera cucurbitae using DNA level markers. This will augment the use biologically based control measures, allow for more robust agricultural development, and create a stronger foundation for more sustainable farming in Hawaii as well as in other parts of the world. Another major goal of this project is collect and analyze specimens from these two species of fruit flies from Vietnam. Although these flies are known to occur throughout Southeast Asia, to date there have been no reports analyzing the genetic makeup of populations in Vietnam.