Honors Projects for Molecular and Cell Biology

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    Expression of Aromatase in Escherichia coli
    (University of Hawaii at Manoa, 2016-05) Zhang, Shalin ; Ng, Ho Leung ; Molecular Cell Biology
    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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    Studying 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) Xu, Zhaotong ; de Couet, Heinz Gert ; Molecular Cell Biology
    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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    HIF-1 Regulated Variant Splicing of CaMKIIγ in the Hypoxic Heart
    (University of Hawaii at Manoa, 2016-05) Pinell, Blake ; Shohet. Ralph ; Molecular Cell Biology
    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.
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    Assessing the Clinical Significance of HPV Quantitation in HIV-Associated Anal Dysplasia
    (University of Hawaii at Manoa, 2016-05) Go, Kendrick ; Shiramizu, Bruce ; Molecular Cell Biology
    Approximately 90% of anal cancers and nearly 100% of cervical cancers are associated with human papillomavirus (HPV) infection of epithelial cells. Although most sexually transmitted HPV infections are asymptomatic and are cleared by the immune system within 1-2 years. Unresolved infections can present themselves as genital warts or anal neoplasia as lowgrade squamous cell intraepithelial lesions (LSILs), atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells of undetermined significance but can’t rule out HSIL (ASC-H), and high-grade squamous cell intraepithelial lesions (HSILs) resulting from an anal cytology screen. Human immunodeficiency virus, type 1 (HIV)-positive men and women have a 52 times and 14 times higher risk of increased risk of invasive anal cancer and should therefore receive annual anal cytology screening tests as part of their routine health maintenance. The objective of this study was to assess the clinical significance of HPV quantitation in HIV-associated anal dysplasia, which could possibly be incorporated into the current anal cancer diagnostic algorithm. Anal cytology samples were used in this study to detect and quantitate high-risk HPV subtypes 16 and 18, which are associated with roughly 70% of anal dysplasia and anal cancer cases in the United States. Preliminary results indicate that HPV 16 is significantly associated with atypical cytology (p= 0.0298) but HPV 18 is not significantly associated with atypical cytology (P=0.9984). We observed in a higher median HPV 16 copy number in HSIL and ASC-H and a lower median HPV 16 copy number in ASCUS and NEG. The parent study (RMATRIX) is continuing specimen collection to increase the sample size and determine the statistical significance of HPV 16 and 18 copy number according to cytology grades.
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    Detection of HIV Genomes in Distinct Monocyte Subsets from Virally Suppressed HIV-Infected Individuals Implicates their Role as Cellular HIV Latent Reservoirs
    (University of Hawaii at Manoa, 2015-05) Leung, Eddy ; Ndhlovu, Lishomwa ; Molecular Cell Biology
    HIV compromises the human immune system and establishes life long infection. While HIV can be effectively suppressed with anti‐retroviral combination therapy (cART) in blood to undetectable levels, the establishment of the latent reservoir in immune cells stands as one of the greatest obstacles in developing a cure for HIV. The resilience of this latent cellular HIV reservoir is due to its ability to adopt a dormant provirus form in immune cells, from which HIV may emerge to re‐establish an active infection if cART is stopped. It is well understood that CD4+ T cells are the chief cellular sanctuaries for the latent reservoir. However, current evidence points to an alternative reservoir in the form of monocytes and macrophages. In this study, we sought to understand the role of monocyte characteristics that are critical in the establishment of the HIV reservoir. We obtained highly purified monocyte sub-populations from the blood of HIV-infected participants on virally suppressive cART and evaluated the viral content and composition using flow cytometric and PCR technologies. We observed unique HIV viral signatures among the monocyte populations. Comparisons drawn between the viral reservoir and cellular characteristics may lend to new insights on the role of monocytes in seeding and maintaining the latent HIV reservoir.
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    In vitro tubule formation of Yop1 expressed in E. Coli
    (University of Hawaii at Manoa, 2015-05) Kurasaki, Kellie ; Ng, Leung Ho ; Molecular Cell Biology
    The endoplasmic reticulum has three distinct regions: sheets of the nuclear envelope, peripheral ER sheets, and peripheral ER tubules. ER tubules are spread throughout the cell giving them contact with other organelles, which may allow for the transfer of lipids or for calcium signaling. The protein Yop1, found in S. cerevisiae, is one of several proteins necessary to maintain these ER tubules. For this project, Yop1 was conventionally cloned into pMCSG28 and then transformed into E. coli because of its ease of growth and genetic manipulation. The focus of this project is to determine an optimal expression and purification method of Yop1 from E. coli, while still yielding functional protein. Yop1 was purified with nickel affinity chromatography and size exclusion chromatography. Yop1 was tested for functionality by using transmission electron microscopy to see if it was able to form tubules in vitro.
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    Resolving Genetic Variation Among Populations of the Melon Fly, Bactrocera cucubitae, Using DNA Markers
    (University of Hawaii at Manoa, 2014-01-15) Golez, Inah ; Haymer, David ; Molecular and Cell Biology
    DNA barcoding is a relatively new technique employed by researchers to identify and classify species based on short segments of DNA in the mitochondrial genome. Although the idea of DNA barcoding has many potential benefits, it is not clear if it can be used to differentiate between morphologically similar cryptic species such as those found in many insect orders, including those in the family of true fruit flies known as the Tephritidae. To compare closely related populations or species using DNA barcodes, sequence relationships can first be determined by looking for single nucleotide polymorphisms (SNPs). In addition, the GenBank database from NCBI can be searched using tools such as BLAST and ORF Finder to make comparisons of sequence information for different species.
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    Improving the Transposition Activity of Genie Plasmids
    (University of Hawaii at Manoa, 2012-05-04) Campbell, Scott ; Urschitz, Johann
    The ability to stably integrate a gene of choice into mammalian genomes has applications in modern medical research and clinical therapies. Many studies involving the transfer of recombinant DNA, especially gene therapy research and transgenic animal production, require a vector that can quickly, efficiently, and safely direct the integration of genetic material into a genome in vivo. Transposon-mediated gene insertion is one of the most effective systems currently available for this purpose. In this study we designed and constructed pmGENIE-4, a novel vector for efficient mammalian transgenesis and gene therapy, based on the pmGENIE-3 vector created at the University of Hawaii Institute for Biogenesis Research. Unlike donor-helper vector systems that require co-transfection with a donor plasmid containing the transgene and a helper plasmid containing the transposase gene, pmGENIE-3 contains a single piggyBac transposase gene and transgene cassette on a single helper-independent plasmid. Another unique feature of pmGENIE-3 is that the transposase is self-inactivating after excision of the transgene from the plasmid. piggyBac excises the transgene cassette from the plasmid at terminal repeat elements (TREs), one of which is located in an intron of the piggyBac coding sequence. The transposase then inserts the fragment into TTAA sites in the host by the cut and paste mechanism typical of type II transposases. These self-inactivating constructs work well in transfecting cell lines and have proven competent for the production of transgenic mice. The piggyBac transposase is better suited for transgenesis than other transposases such as Sleeping Beauty, Tol2, and Mos1, due to its higher transpositional activity and larger cargo capacity. The pmGENIE-3 plasmid can also be converted into a BAC for large transgene insertions. To further improve upon the 2 transposition efficiency of the pmGENIE-3 vector, a new vector (pmGENIE-4) was constructed that carries two copies of the piggyBac gene. Two active transposases functioning simultaneously could potentially double the efficiency of the system, which is an important feature for applications such as transgenesis, where only a few copies of the plasmid may reach the nucleus. We successfully constructed the pmGENIE-4 plasmid and sequenced it to verify integrity. Quantitative real-time PCR (qRT-PCR) was performed and Western blot analysis will be performed to compare piggyBac mRNA and protein expression levels from pmGENIE-4 relative to pmGENIE-3. Finally, we will compare transpositional activities by colony count. If a higher transposition efficiency is achieved with the new plasmid, research in gene therapy and transgenesis will have a more effective tool at its disposal for the integration of recombinant DNA into host genomes.