Honors Projects for Molecular and Cell Biology
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ItemThe exocyst complex is an insulin-sensitive regulator of amyloid precursor protein (APP) trafficking and(University of Hawaii at Manoa, 2022)
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ItemThe Role of Trophoblast Lat1 in the Regulation of Placental Function and Fetal Growth: Plasmid(University of Hawaii at Manoa, 2022)
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ItemDietary Intake Mediates Ethnic Differences in Gut Microbial Composition(University of Hawaii at Manoa, 2022)
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ItemANALYZING THE ROLE OF SELENOPROTEIN I IN BONE MARROW DERIVED MACROPHAGES(University of Hawaii at Manoa, 2021)
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ItemAnalysis of the Effect of the Longevity Associated FOXO3 Genotype on Telomerase Activity in a Japanese-Okinawan Cohort(University of Hawai'i at Manoa, 2019)Forkhead box O3 (FOXO3) is one of the few genes that has consistently correlated across multiple long-lived populations to be associated with longevity. 1,2 In a previous study, an analysis of FOXO3 single nucleotide polymorphisms (SNP) identified a specific allele as being associated with less rapid shortening of telomeres, which is a molecular marker of longevity. 3 Telomeres are tandem repeats that shorten after each cell division, and telomerase is an enzyme that adds new tandem repeats, thereby extending the telomeres and the lifespan of the cell. 4 Because previous results have shown FOXO3 protects against telomere shortening, it is expected that telomerase activity will be higher in individuals that carry the longevity-associated allele of FOXO3. It is also expected that telomerase activity will be constant with age in the longevity-associated genotype. In this study, blood was extracted from 100 participants from a Japanese-Okinawan cohort, a notably long-lived population, and peripheral blood mononuclear cells were isolated. The genotype of each sample was obtained and telomeric repeat amplification assays were performed to quantify telomerase activity, as a function of genotype, sex, and age. Results indicate that telomerase activity decreases with age for the TT genotype, but do not decrease, to the same extent, with age for G-carriers. These findings provide insight as to how FOXO3 protects against telomere attrition and link the longevity-associated G-allele to modulated telomerase activity. Keywords: Longevity, FOXO3, telomerase
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ItemTransposon Screen of Marinobacter HI15- 87 Mutants in Dissolved Organic Matter(University of Hawai'i at Manoa, 2019)Marine dissolved organic matter (DOM) represents the largest carbon source in the world. The consumption of DOM by microorganisms results in the production of large amounts of atmospheric carbon dioxide gas, thus playing a significant role in the global carbon cycle. Although some key enzymes involved in the breakdown of phosphonates in DOM have been identified, the specific biochemical pathways of marine microbes to degrade high molecular weight DOM remains largely unconfirmed. Sample isolates of Marinobacter HI15-87 from the waters of Station Aloha, approximately 100km north of Oahu, underwent transposon mutagenesis with the transposon pSMC194 containing three cut sites for the endonuclease HpyCH4IV within Streptomycin and Spectinomycin resistance genes. Approximately 10,000 individual HI15-87 colonies were selected through growth on marine broth containing Streptomycin and Spectinomycin to confirm successful transposon mutagenesis. The selected isolates that did not grow in hydrolyzed DOM were suspected to have received the pSMC194 transposon in a location of its genome that disrupted adjacent genes or pathways essential to degradation of DOM as a carbon source. DNA extraction, HpyCH4IV digestion, and sequencing point to the host cell genes flanking the transposon insert, thereby indicating the HI15-87 genes likely required for DOM consumption. Identification of these genes may allow for better understanding of marine microbial metabolisms and communities.
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ItemExpression of Aromatase in Escherichia coli(University of Hawaii at Manoa, 2016-05)Human aromatase is an enzyme necessary for estrogen biosynthesis from androgen precursors, and is often the target for chemotherapy drugs that fight against breast cancers with estrogen receptor-positive breast cancer cells. Tamoxifen, a drug that acts as an estrogen receptor antagonist, was the primary method of treatment for women with hormone receptor-positive breast cancer cells in the twentieth century and is still widely used today. More recent forms of chemotherapy drugs for breast cancer include aromatase inhibitors, which directly act on aromatase to inhibit the enzyme. Aromatase inhibitors are effective and highly selective for aromatase, but are limited by resistance issues, and therefore motivate researchers to produce drugs that will overcome those resistance mechanisms. Recent studies have shown that tamoxifen metabolites may be working to block estrogen synthesis through a second pathway; tamoxifen metabolites inhibit aromatase through noncompetitive inhibition. Although aromatase has been crystallized, there are no crystal structures that have elucidated the presence of these allosteric sites. The objective of this project is to express enough functional aromatase in E. coli cells to be able to perform ligand-binding studies on aromatase and identify allosteric sites with protein crystallography. Structural studies of aromatase and its allosteric sites will provide information on the obscure interactions of aromatase with other compounds and possibly shed light for a new generation of chemotherapy drugs.
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ItemStudying the Role of Prenylated Rab Acceptor 1 Domain Family, Member 2 (PRAF2) in Drosophila melanogaster by Knockdown and Gain-of-Function Analysis(University of Hawaii at Manoa, 2016-05)Prenylated Rab acceptor family (PRAF) proteins are highly conserved among multicellular organisms and several paralogous genes of PRAF exist. PRAF proteins are expressed in a variety of tissues and are associated with cellular transport and endo/exocytic vesicle trafficking via Rab protein interactions. Currently, the function of a new PRAF protein, PRA1 domain family, member 2 (PRAF2) is still not understood. However, PRAF2 is upregulated in cancerous cells of the breast, colon, lung, and ovaries and serves as a candidate prognostic marker for neuroblastoma. This study focuses on studying the role of PRAF2 in Drosophila melanogaster by knockdown and gain-of-function analysis. In the first part of the experiment, a knockdown of PRAF2 in Drosophila ommatidia was achieved using RNAi methodologies. Results for the PRAF2 knockdown showed that PRAF2 expression at 25°C was sufficient in causing structural abnormalities in Drosophila ommatidia. When flies were grown at 28°C, results were inconclusive because the specific and non-specific effects of the GMR-GAL4 driver and PRAF2-RNAi could not be distinguished. In the second part of the experiment, PRAF2 was overexpressed in Drosophila. Fly eye specimens were then obtained for further examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Results showed that PRAF2 overexpression correlated with increased spacing between photoreceptor cells in Drosophila ommatidia, which also affected ommatidia shape. Further research must be conducted on PRAF2 to better understand its mechanisms in vesicular trafficking, which might provide insight on how abnormalities in PRAF2 function can contribute towards the development of cancerous cells.
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ItemHIF-1 Regulated Variant Splicing of CaMKIIγ in the Hypoxic Heart(University of Hawaii at Manoa, 2016-05)Hypoxia-inducible factor 1 (HIF-1) is an oxygen-labile transcription factor that plays a crucial role in the regulation of cellular processes associated with hypoxia in the heart. In this study, we evaluated the effects of HIF-1 on variant RNA splicing of calcium/calmodulindependent protein kinase II gamma (CaMKIIγ). CaMKIIγ is known to play an important role in calcium signaling and heart function. Previously collected data indicated that expression of an oxygen-stable HIF-1 altered the relative abundance of three CaMKIIγ splicing variants in the hearts of transgenic mice. Expression of the full-length variant appeared to be down-regulated whereas variants 2 and 3 increased in abundance following HIF-1 induction. Based on this preliminary data, we chose to examine the mechanism of HIF-mediated splicing using two different models of physiological hypoxia: cell culture and a mouse MI surgical model. My hypothesis was that the altered splicing pattern seen in the HIF transgenic mice would be recapitulated in the physiological settings. We have found that in cells, expression levels of all three transcript variants initially decreased when exposed to hypoxia but that the expression levels of variant 3 returned to normoxic levels, and thus greater relative abundance relative to the other isoforms by 24 hours. In our in vivo model of infarction, CaMKIIγ variant 1 expression decreased 3 days after surgery, as was seen in the transgenic model. These results support the hypothesis that HIF-1 is involved in the alternative splicing of CaMKIIγ and could contribute to the loss of cardiac contractility observed after hypoxic injury.