Honors Projects for Microbiology

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    Intrauterine Growth Restriction in Babies Born to Cameroonian HIV-Positive Women: Identification of Possible Mechanisms Responsible for Low Birth Weight
    (University of Hawaii at Manoa, 2015-08) Rasay, Shayne Janne Jossef Dumalanta ; Taylor, Diane W. ; Microbiology
    HIV increases the risk of intrauterine growth restriction (IUGR), which results in the delivery of low birth weight (LBW) babies, who are 40 times more likely to die during their first year of life. However, the mechanism(s) for IUGR among HIV-exposed newborns remain unknown. In addition to socioeconomic and cultural factors, several hypotheses have been proposed to explain the biological component of IUGR. These include reduced 1) placental angiogenesis and vasculogenesis, 2) production of fetal growth hormones, and 3) transportation of nutrients. The first two hypotheses were examined in this investigation. To evaluate altered vessel formation, placental plasma levels of angiopoietins, ANG-1 and ANG-2, and galectin-13 were measured. Levels of insulin-like growth factor-1 (IGF-1) and one of its binding proteins, IGFBP-1, were measured to evaluate growth hormone dysregulation. In this case-control study, 21 HIV-positive and 30 HIV-negative pregnant mothers were recruited at delivery in the Central Hospital of Yaoundé, Cameroon, Africa. Results showed that biomarker levels in HIV-negative women were similar to literature values for healthy adults; but no significant differences were observed among the different groups. However a statistically significant (p=0.028) declining pattern of galectin-13 levels was noted as the severity of HIV infection increased. Overall, our results suggest that decreased angiogenesis and reduced production of growth factors may not cause LBW, but the dysregulation of maternal vascular development may play a role. Further studies using additional biomarkers are needed to identify the combination of social, economic and biological risk factors that increase the risk of HIV-associated LBW babies.
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    Enhancing the immunogenicity of a P. falciparum MSP1-19 malaria vaccine using a putative MSP1-33 T helper epitope
    (University of Hawaii at Manoa, 2015-05) Hokama, Acasia ; Hui, George ; Microbiology
    Developing an efficacious vaccine for the vector-borne infectious disease, Malaria, remains a top priority for disease control. The P. falciparum Merozoite Surface Protein 1(MSP1) is a leading blood-stage malaria vaccine candidate, and anti-MSP1 antibodies have been found to be important in providing protection against blood infections. MSP1 (195 kDa) goes through a number of proteolytic cleavage events to produce a 42-kDa fragment, which is further cleaved into 33-kDa (MSP1-33) and 19-kDa (MSP1-19) fragments. Although antibodies against MSP1-19 are protective, the MSP1-19 molecule alone cannot induce broad immune responsiveness due to lack of T helper epitopes on this protein fragment. We have recently identified four putative T epitopes on MSP1-33 that may enhance the quantity and quality of antibody responses to MSP1-19. We hypothesize that selective inclusion of one or more of these epitopes will have a measurable effect on the immunogenicity of MSP1-19. As proof of principle, we chose one of these epitopes to construct a new MSP1 vaccine by linking it to MSP1-19 via recombinant protein expression approach. The ability of this epitope to enhance the immunogenicity of MSP1-19 was examined in outbred mice in terms of production of high antibody titer, parasite growth inhibitory antibodies, and broad vaccine responsiveness. The recombinant protein tested produced a higher percentage of antibody responders than the native protein. The levels of Il-4 produced by antigen stimulation inferred that a TH2 response was also stimulated. These studies will hopefully lead to a rational approach to develop a more effective human malaria MSP1-42 vaccine.
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    Visualization of the tissue-specific promoter activity of the genes encoding protein disulfide isomerase-7, -12 and -13 in Arabidopsis thaliana
    (University of Hawaii at Manoa, 2014-09-26) Wong, Katharine ; Christopher, David ; Microbiology
    For proteins to properly carry out their functions, they must be folded correctly. Protein disulfide isomerases (PDIs) assist protein folding by catalyzing the formation and/or rearrangement of disulfide bonds between cysteine residues in newly- synthesized polypeptides. The goal of the project is to characterize the expression patterns of three PDI genes in the model plant, Arabidopsis thaliana. The Arabidopsis PDI (AtPDI) family consists of 13 members, the majority of which are found in the endoplasmic reticulum (ER), possess two catalytic domains, but lack transmembrane domains. In contrast, AtPDI7, AtPDI12 and AtPDI13 are unusual PDIs because they are primarily located at the Golgi apparatus, and possess two transmembrane domains and only one catalytic domain. Using the β-glucuronidase (GUS) reporter system, these three PDI genes were determined to have distinct tissue-regulated expression patterns. The AtPDI7 promoter was primarily active in developing tissues, while the AtPDI12 promoter was active in pollen, stipules, developing seeds, and the vasculature of roots. The AtPDI13 promoter activity was restricted to pollen and stipules. Interestingly, the AtPDI12 promoter was also inducible at the root tip by the plant hormone, auxin. The variable GUS staining patterns imply that there may be partial functional specificity between AtPDI7, AtPDI12, and AtPDI13. Further characterization of the tissue-specific expression of these PDIs would contribute in the understanding of how the different members of the PDI family each contribute to the process of protein folding in organisms.
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    Vibrio coralliilyticus strain OCN014 is the Etiological Agent of Acropora White Syndrome at Palmyra Atoll
    (University of Hawaii at Manoa, 2014-09-26) Poscablo, Donna ; Callahan, Sean ; Microbiology
    Corals play an important role in marine ecosystems by providing a habitat for many members of the reef community. Threats such as coral disease have pushed many reefs past the point of recovery and are lost forever. Investigation of coral disease outbreaks at Palmyra Atoll in 2010 and 2011 resulted in the isolation of Vibrio sp. OCN014 from diseased Acropora cytherea. OCN014 was proposed as the pathogen responsible for the disease Acropora white syndrome (AWS), which affects Acropora spp. throughout the atoll. In this study, Koch’s postulates were applied to demonstrate OCN014 as the etiological agent of AWS. OCN014 was determined to be a strain of Vibrio coralliilyticus, a species of bacteria previously shown to cause disease in coral. Under laboratory conditions, infections by OCN014 were found to be temperature dependent, with infections observed at 29 °C and none at 25 °C. The genome of OCN014 was sequenced and compared to a previously published genome of the V. coralliilyticus strain type strain BAA-450, which has also been shown to be temperature sensitive. A genetic system was developed to screen for genes which are differently expressed at 25 and 29 °C to identify potential regulators of OCN014 virulence.
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    Investigating the Role of Epigenetics on the Anti-Cancer Activity of Noni (Morinda citrifolia)
    (University of Hawaii at Manoa, 2014-09-26) Avilez, Kekaihalai ; Maunakea, Alika ; Microbiology
    Inherent in the traditional native Hawaiian concept of health is the understanding that environmental factors, including nutrition and diet, trans-generationally impact health outcomes. Epigenetic mechanisms now explain the molecular links between these environmental factors and health outcomes. Noni (Morinda citrifolia) has been employed for centuries by Kahuna lāʻau lapaʻau (medical practioners) to address many health ailments, including cancer. Modern studies abound validate the efficacy of noni for cancer treatment. Consistent with the traditional concept, we hypothesize that epigenetic mechanisms underlie the well-established anti-cancer effects of noni. Thus, we propose to examine the global and genome-wide epigenomic patterns of the colon cancer cell line HCT-116 before and after treatment with noni. Using Western blotting techniques, we will measure global levels of DNA methylation and histone modifications in treated and untreated cells. We will next employ state of the art technologies to identify, characterize, and integrate genome-wide DNA methylation and gene expression alterations in HCT- 116 cells in response to noni. We anticipate that epigenetically labile sites, in particular hyper-methylated tumor-suppressor gene promoters, will be responsive to noni treatment, which could lead to changes in gene expression. We will confirm cases of noni-induced DNA demethylation by comparing these data with that of HCT-116 cells genetically deficient for DNA methylation. Results from this study will establish, for the first time, a link between the anti-tumor effects of noni and epigenetic gene regulation, demonstrating that the traditional native Hawaiian concept of health included a mechanistic rationale for the role of the environment on physical health.
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    The Characterization Of Chromatin Modifying Genes In Dengue Virus Infected Human Monocytic Cells: Implication In Modulating Inflammation
    (University of Hawaii at Manoa, 2014-09-26) Acker, Zachary ; Verma, Saguna ; Microbiology
    Epigenetic control of gene expression is a complex mechanism and involves DNA methylation and histone acetylation mediated by opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) enzymes. Epigenetic modifications are well characterized in Multiple Sclerosis, cancer, diabetes and asthma. Altered HDAC activity is linked to cytokines production in bacterial and fungal infections. However, the data on the epigenetic modifications in virus infections are not well characterized. Dengue virus has become a significant public health concern and the antibody-dependent enchancement (ADE) phenomenon further complicates the dengue pathogenesis by potentiating inflammation. The objective of this study was to determine the expression profiles of complete panel of histone modifier genes in human monocytic cells (THP1) following dengue infection. THP1 cells were infected with DEN2-NGC strain directly or under ADE conditions (4G2 antibody). Total RNA from mock and infected cells was used for Human Epigenetic Chromatin Modification Enzyme for RT2 ProfilerTM Array to detect the expression of 84 chromatin modifying enzymes. Further, THP1 cells were treated with HDAC (TSA) and HAT (Anacardic Acid) inhibitors and virus titers and cytokine levels were measured. Our results show that Dengue infection caused changes in 10-20% of genes involved with chromatin modification. Differences in the expression profiles were also observed between Dengue infection with and without ADE conditions. Experiments are ongoing to analyze effects of inhibiting HDACs and HATs on the expression key inflammatory cytokines associated with dengue pathogenesis, IL- 8 and TNF - α This is the first report describing changes in complete panel of histone modifying enzymes and associated transcription factors in Dengue infection. Based on the literature indicating a strong association between changes in HDAC/HAT levels with production of inflammatory cytokines in other inflammatory diseases, we speculate that inflammatory response induced during Dengue infection is at least partially modulated by epigenetic mechanisms.
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    Methods to Develop and Validate a Culturally and Youth Relevant Survey on Smoking Among Filipina Girls in Hawaii
    (University of Hawaii at Manoa, 2014-01-15) Lim, Matthew ; Sy, Angela ; Microbiology
    Asian American (AA) youth have low tobacco use rates, but Filipino youth report much higher use of tobacco than other AA youth, and Filipina girls have an especially high prevalence. In Hawaii, aggregated data from 2005, 2007, and 2009 Youth Tobacco Survey indicate that 20.1% (N=4200) of Filipina high school girls smoked, compared to only 5.6% and 5.3% of Japanese and Chinese girls respectively. While smoking prevalence fell for all high school girls in Hawaii between 2003 and 2005, it rose for Filipina girls from 12.4% to 13.7%. A pilot descriptive study with Filipina girls in Hawai‘i revealed that the family may influence tobacco use behaviors.
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    Theraputic Effect of Human Stem Cell Treatment on Cardiac Function and Myocardia Perfusion
    (University of Hawaii at Manoa, 2014-01-15) Chow, Alice ; Microbiology
    My project focuses on comparing the efficacy of treatment with human bone marrow or human adipose-derived stem cells (ASCs) on a myocardial infarction in a mouse model. Cardiovascular disease kills about 17.1 million people each year. There are many types of cardiovascular disease, but my project focuses on myocardial ischemia. Clinical trials have shown improvements of cardiac function following the treatment of myocardial infarction with bone marrow treatments. Human bone marrow and ASCs were injected into infarcted areas of hearts in a mouse model. Echocardiography was performed before infarction and after 30 days of treatment to determine the function of the heart. Sections of embedded hearts were stained to enable capillary density counting as an indicator of reperfusion of the scarred myocardium in the peri-infarct region. We found no significant difference in the heart function between treatment with human ASCs and human bone marrow stem cells on myocardial reperfusion. However, hearts receiving human ASC showed a significant increase in reperfusion compared to the hearts receiving human bone marrow stem cells.
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    Degredation of Pyrene
    (University of Hawaii at Manoa, 2013-07-16) Sanchez, Shannon
    Polycyclic aromatic hydrocarbons (PAHs) are known to be toxic, carcinogenic, and mutagenic. They are formed as a result of incomplete combustion of organic materials. These pollutants are known to affect organisms including humans and are currently listed as a major environmental contaminant in the United States and surrounding territories. Research on biodegradation has been performed using microorganisms to degrade these materials to safer compounds. Mycobacterium species M. crocinum czh-3, M. rutilum czh-117, and M. gilvum czh-101 are known to degrade the PAH, pyrene. Although there have been studies using these strains to observe degradation of various PAHs and their mixtures, no study has been performed on the effects a bacteria consortia would have on a single PAH’s degradation. Faster degradation is hypothesized for a consortium of bacteria. To test this hypothesis, degradation rates of single PAHs by bacterial consortia were determined. Through the use of in vitro culturing methods, high-pressure liquid chromatography (HPLC) and mass spectrometry, the amount of bacterial growth and PAH degradation was monitored. The data were used to determine the synergistic/antagonistic effects of the respective bacteria. One of the future goals is to use the bacterial strain of Mycobacterium in bioaugmentation or introducting a particular bacterial strain to treat contaminated soil, and to see how and if they degrade contaminants in situ, as well as in vitro.
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    Insights into the Mechanistic Basis of the Irukandji Syndrome by evaluating the Hematologic and Immunologic Responses in Whole Blood
    (University of Hawaii at Manoa, 2011-04-25) Lee, Amanda ; Yanagihara, Angel
    The Hawaiian Box jellyfish, Alatina mordens (previously classified as Carybdea alata), aggregates on certain lee shores of Oahu 7-10 days after each full moon. This animal's appearance and the composition of its venom present areas of interest for the tourism industry and local beachgoers because of the painful stings they inflict. This stinging capability is mediated by potent venom, which contains pore-forming proteins (porins) that are introduced into the blood stream of the sting victim through thousands of stinging cell tubules that pierce the skin tissue. We have shown that venom porins assemble to create large pores in the membrane of the blood cells and cause the release of molecules from the cell. We hypothesize that host molecules released from cells may account for a variety of symptoms that include, the life threatening, Irukandji syndrome.