Ph.D. - Microbiology

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    FUNCTIONAL CHARACTERIZATION OF ESSENTIAL BURKHOLDERIA PSEUDOMALLEI VIRULENCE REGULATORS
    ( 2020) McMillan, Ian Andrew ; Hoang, Tung T. ; Microbiology
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    Viral vector construction, production and vector-mediated gene transduction
    ([Honolulu] : [University of Hawaii at Manoa], [May 2013], 2013-05) Wu, Chengxiang
    Until now, viral vectors are considered necessary for gene therapy, and current approaches are prohibited from wide applications mainly due to low efficiency and genotoxicity. The use of optimized vector production systems, the right choice of target cells, and improved transduction protocols may overcome these obstacles. To improve viral vector production, I initially optimized a calcium phosphate-mediated transfection method through inclusion of dextran and combined use of polybrene, and significantly improved the quality and quantity of the produce. Following that, multiple strategies, including a novel E. coli-based recombination system, Taq DNA polymerase treatment, and introduction of a bacteria toxic gene, were established and significantly improved the efficiency of generation of recombinant adenovirus vector. Moreover, multiple molecular manipulative strategies tested to a prototype retroviral vector system improved vector titers by 2-3 logs and led to enhanced transduction of a broad variety of cell types, especially cells of human and mouse haematopoietic and lymphocytic lineages that hold potential for gene therapy against a wide range of inherited and acquired diseases. Furthermore, a series of mutant tRNALys3 genes were constructed and expressed using the optimized viral vector production systems, and showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication through improved priming of HIV-1 reverse transcription from their targeting sites. Transduction of multiple copies of mutant tRNALys3 further enhanced the anti-HIV-1 potency. Lastly, a soluble tumor necrosis factor-α receptor (sTNFR)-Fc fusion protein was designed and expressed to meliorate neurons through neutralizing TNF-α. TNF-α-binding activity of secreted sTNFR-Fc from transduced cells was demonstrated and conditioned medium containing sTNFR-Fc was shown to be protective to neuronal cells from TNF-α-, HIV-1 Tat-, and gp120-mediated neurotoxicity. Overall, this study established multiple strategies and methods for improved viral vector production to facilitate gene therapy tests against HIV/AIDS and other diseases. The mutant tRNALys3-and sTNFR-Fc-based anti-HIV/NeuroAIDS strategies laid the groundwork for development of novel therapeutics against HIV and NeuroAIDS. Particularly, high efficiency transduction of cells of haematopoietic and lymphocytic lineages hold potential of using the genetically modified cells as noninvasive vehicles to deliver therapeutic substances across the blood-brain barrier into the central nervous system.
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    Utilization of invasive algal biomass for bioethanol production and the dynamics of planktonic fungi in the West Pacific
    ([Honolulu] : [University of Hawaii at Manoa], [August 2013], 2013-08) Wang, Xin
    Algae represent the most promising feedstock for biomass derived biofuel production. Certain invasive algae in Hawaii can form dense biomass and are potential feedstocks for bioethanol production. In this study, the biomass from the invasive algae Gracilaria salicornia was used as feedstock for ethanol production using the ethanologenic strain Escherichia coli KO11. The algal hydrolysates were successfully utilized in a two-stage saccharification and fermentation platform, showing no inhibition of its bacterial fermenting ability, and producing 79.1 g ethanol from one kilogram of dry algal mass. Algae contain large quantities of species-dependent polysaccharides that cannot be readily metabolized by current ethanologenic bacteria. To fully explore the potential of microbial conversion of algal biomass and increase the systematic efficiency for ethanol production, culture-dependent and independent methods were applied to identify bacterial candidates fulfilling these purposes. The microbial communities profile associated with selected native and invasive algae were determined, which supplied valuable information in searching for candidates for polysaccharides utilization. Furthermore, microbes that can facilitate consolidated bioprocessing (CBP)--a process that can potentially optimize the systematic efficiency of biomass derived ethanol production--are isolated from various sources. Two bacteria FNP1 and TF2 showed great potential in further engineering for CBP platform development. Collectively, this study supplied valuable information in developing an efficient bioethanol production platform using invasive algal biomass. The dynamics of planktonic fungi in the west Pacific was investigated in part II of the dissertation. This study revealed that planktonic fungi are molecularly diverse and the fungal distribution was related to major phytoplankton taxa and various nutrients including nitrate, nitrite, orthophosphate and silicic acid. Over 400 fungal phylotypes were recovered and nearly half of them grouped into two major novel lineages. Ascomycota and Basidiomycota were found to be dominant groups at majority of the investigated stations. These results suggest that planktonic fungi are an integral component of the marine microbial community and should be included in future marine microbial ecosystem models.
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    Molecular epidemiology of seasonal and pandemic influenza A (H1N1) in Hawaiʻi
    ([Honolulu] : [University of Hawaii at Manoa], [May 2013], 2013-05) Nelson, Denise Cynthia
    Influenza is a viral infection causing seasonal outbreaks, periodic epidemics and global pandemics in humans, the latest being the 2009 pandemic. The State of Hawaiʻi is particularly vulnerable to the spread of influenza due to its unique geographic position in the Pacific Ocean with heavily trafficked passenger and freight patterns. By combining epidemiological data on case occurrences with their laboratory-derived viral sequences, we are able to trace viral strain origins based on phylogenetic relationships between isolates. In collaboration with the Hawaiʻi Department of Health State Laboratories Division, we present a study in which seasonal, or pandemic, H1N1 influenza A viral isolates collected from infected individuals in Hawaiʻi were extracted, hemagglutinin and neuraminidase genes were amplified and sequenced, and examined for evolutionary relationships and spatio-temporal patterns. Implications of molecular data are also supported by epidemiologic information and statistical support of summary transmission data. Phylogenetic analysis suggests that Hawaiʻi acts as both a source and sink population for type A influenza virus: in some instances Hawaiʻi isolates represented the earliest instance of a strain subsequently seen elsewhere; in other instances Hawaiʻi isolates clustered with strains observed earlier in other countries or geographic regions. Through the continued usage of molecular methods, we hope to develop an improved understanding of influenza dynamics in Hawaiʻi. Targeting an area of geographic importance additionally assists in depicting how location and population distribution play a role in the spread of infectious disease. Enhanced comprehension as a result of these analyses may help to improve efficiency and effectiveness of preparation and response efforts, and reduce the impact of influenza on Hawaiʻi and the continental United States.
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    The epidemiology and entomological interactions associated with dengue transmission in Ang Mo Kio GRC, Central Singapore
    ([Honolulu] : [University of Hawaii at Manoa], [December 2013], 2013-12) Henry, Amy Beth
    Dengue is arguably the most important arboviral disease of humans, having increased dramatically in geographic range and prevalence over the last 25 years. Dengue virus has two main vectors, Aedes aegypti and Aedes albopictus. For decades both vectors have also been increasing their geographic range on regional and global scales. This study took place in Singapore, where dengue fever is a major public health threat despite a successful vector control program. Similar to other hyperendemic countries, local dengue transmission dynamics in Singapore are not well understood: where dengue transmission is occurring, the relative contribution of the two dengue vectors, and the ability to correlate traditional vector surveillance methods to transmission risk remains controversial. In collaboration with the Program of Infectious Diseases at Duke-NUS Graduate Medical School, Singapore Ministry of Health, Singapore National Environmental Agency, and Ang Mo Kio Town Council an adult Aedes female fixed position vector surveillance program was established that detailed temporal and spatial Ae. aegypti and Ae. albopictus distribution and abundance in Ang Mo Kio, Central Singapore. This surveillance method yielded similar results to standard surveillance techniques over a range of habitats and time points. Furthermore, sensitivity of the adult surveillance method presented here is uniquely increased by placing traps on the second floor of Housing Development Board (HDB), government subsidized multistory residential buildings, as opposed to ground level; average Ae. aegypti catch rate of the ground floor was 0.09 and average Ae. aegypti catch rate of the second floor was 0.42. Starting on the second floor a very strong inverse relationship between Ae. aegypti catch rate and floor height (Pearson linear correlation r=-0.91, t=-4.47, df=4, p=0.01) was also identified. In addition, intensive entomological investigations, in focal areas with varying levels of Aedes abundance, identified by the fixed position surveillance system, uncovered details about mosquito ecology and "hotspots" at a local scale that can improve our understanding of dengue transmission dynamics. Dengue transmission is believed to primarily occur in residential units but host seeking Ae. aegypti and Ae. albopcitus were collected at similar frequencies in congregation areas on the ground floors of, HDBs and at greater abundance than inside residential units. Improving knowledge on the focal nature of dengue transmission is critical to designing more targeted and cost-effective surveillance and control strategies in the future, both in Singapore and urban areas elsewhere.
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    The prevalence and public health significance of human pathogenic vibrio species (v. cholerae, v. vulnificus, v. parahaemolyticus, v. alginolyticus) in Hawaiʻi's diverse tropical coastal water environments
    ([Honolulu] : [University of Hawaii at Manoa], [May 2011], 2011-05) Vithanage, Gayatri
    Studies on the prevalence and ecology of Vibrio species in tropical areas, such as Hawaii, is limited, and up to now, there have been no studies conducted in Hawaii to determine the prevalence of these pathogens in our coastal waters. The major goals of this study was to determine the prevalence of the four human pathogenic Vibrio spp. (V. cholerae, V. vulnificus, V. parahaemolyticus, V. alginolyticus) in coastal water environments of Hawaii (islands of Oahu and Hawaii), and to determine the public health significance these pathogens have to people who use these coastal water for recreational purposes. The study showed that water salinity and temperature affected the four human pathogenic Vibrio spp. V. vulnificus and V. parahaemolyticus were prevalent in low salinity sites that were impacted by land run-off but not detectable in high salinity, non-impacted swimming sites. Both species were also prevalent at low salinity swimming ponds on the island of Hawaii. V. alginolyticus was prevalent in all sites regardless of salinity. In addition to low salinity, high water temperature also had an impact. High temperature, low salinity ponds located on the Island of Hawaii were shown to select for V. vulnificus, V. parahaemolyticus and V. alginolyticus. These ponds have shown past evidence of infection and death due to V. vulnificus associated with the use of these ponds. Isolates recovered from these thermal ponds may potentially be more virulent as they have been adapted to survival at temperatures similar to that of human body temperature. V. cholerae was not recovered in either impacted or non-impacted sites. The prevalence of pathogenic Vibrio spp. in sediments followed a similar trend to what was seen with coastal beach samples. V. alginolyticus was prevalent in both primary and secondary beach sediment while V. vulnificus and V. parahaemolyticus were only prevalent in secondary beach sediment. Thus, apparently sediments from secondary coastal waters can spread pathogenic Vibrio species into the water column. Data from this study also showed that V. vulnificus and V. parahaemolyticus were sporadically present in raw and primary treated sewage from three different wastewater treatment plants, while V. cholerae was consistently recovered in raw and primary treated sewage from all three treatment plants. V. vulnificus can cause severe wound infections, which can rapidly lead to death. Thus, this species poses a public health significance. In summary, data gathered from this study was able to provide basic information, that was lacking, regarding the distribution of the four main human Vibrio pathogens in a tropical area such as Hawaii. This data was then used to make a basic assessment of the potential public health significance these pathogens have on humans who use Hawaii's coastal waters for recreational purposes, and to determine if and when warning signs would be warranted to notify the public of the potential risk for infection.