M.S. - Microbiology

Permanent URI for this collectionhttps://hdl.handle.net/10125/2102

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Quorum sensing between phylogenetically ancient Cyanobacteria and heterotrophic Bacteria in a simulated Mars atmosphere
(University of Hawaii at Manoa, 2023) Handel, Christy Rose; Donachie, Stuart P.; Microbiology
N-acyl homoserine lactone based quorum sensing (AHL-QS), a form of chemical communication and gene regulation in bacteria, was investigated in a Cyanobacteria-heterotroph consortium under contemporary terrestrial and simulated Mars conditions. Co-cultures of a deeply branching, phylogenetically ancient cyanobacterium, Gloeobacter violaceus, and its heterotrophic consortium were maintained in a simulated Mars atmosphere, i.e., atmospheric composition of high CO2-low O2 concentrations. First, the type and relative concentrations of AHLs produced by heterotrophic consortia were determined. Then, differential gene expression of co-cultures by RNA-seq was determined when 1) exogenous AHLs were applied to co-cultures and 2) under simulated Mars atmospheric conditions. The results show that G. violaceus, an early-Earth, photoautotrophic representative, and its heterotrophic consortium that commonly occur in epilithic, Mars-analog environments, 1) have a vast potential for AHL-QS, 2) show differential gene expression in response to Mars atmospheric conditions, as well as exposure to an AHL molecule. Differential gene expression by RNA-seq results reveals that there is significant differential expression of genes involved in cobalt uptake and phage tail assembly by G. violaceus, possibly in response to exogenous application of 3-oxo-C12-homoserine lactone (C12-oxo). This provides context for how microbial consortia in a Mars atmosphere may withstand environmental extremes through inter-species interactions, and informs future work on life’s persistence on Earth and potentially elsewhere in the Solar System. Ultimately, this project has shown that AHL-QS can be linked to an evolutionary timeline of cooperative microbial interactions, particularly in Cyanobacteria.
Microbial Diversity Of The Emperor Seamounts
(University of Hawaii at Manoa, 2022) Lary, Sean Michael; Donachie, Stuart P.; Microbiology
The North Pacific Deep Water (NPDW) current is believed to present a barrier that precludes movement of water, and pelagic and benthic organisms on the Emperor Seamounts between 39° N and 41° N. Different pelagic and benthic communities are thus evident north and south of the NPDW. This study used culture-dependent and culture-independent methods to investigate if any such differences exist in microbial (Archaea, Bacteria and Eucarya) diversity in water and surface sediments, and in mucus produced by Keratoisididae related coral species on seamounts in the same region. Microbial diversity differed significantly by sample type, with coral hosting the lowest diversity in terms of both bacteria (Archaea and Bacteria) and Eucarya, although bacterial diversity alone was greater in deeper corals. Bacteria Amplicon Sequence Variants (ASVs) varied between mucus in different coral genera, but some Bacteria taxa occurred in all mucus samples, including Halomonas, BD1-7 clade, and Sulfitobacter. One Aspergillus-affiliated ASV was also present in all coral mucus samples. The number of ASVs that affiliated with known photoautotrophic chlorophytes and Cyanobacteria was greater in coral mucus than previously reported. Most Dinophyceae-affiliated ASVs were recorded from the sole coral tissue sample, from an uncharacterized coral genus. The 18S rRNA gene in one cultivated nanoflagellate, Cafeterium sp., shared 98.77% nucleotide sequence identity with one abundant bicosoecid-affiliated ASV found in most samples. Multiple Syndiniales group I-affiliated ASVs were also detected at high relative abundances in coral mucus. The findings here suggest zooxanthellae and other photoautotrophs may be part of the deep-sea coral mucus microbiome, even in the absence of photosynthetically available radiation. The abundances of Syndiniales and Bicosoecids in coral mucus suggest grazing and parasitic interactions may exist in the mucus of corals in deep water on the Emperor Seamounts.
Investigating Feral Pigs as Animal Reservoirs for Nontuberculous Mycobacteria in Hawai’i
(University of Hawaii at Manoa, 2022) Hendrick, Haley; Honda, Jennifer R.; Microbiology
Nontuberculous mycobacteria (NTM) are environmentally-acquired opportunistic pathogens that cause recalcitrant pulmonary disease in susceptible individuals. The prevalence of NTM pulmonary disease is increasing globally, and the highest rates within the US are in Hawai'i. Mycobacterium porcinum (Latin, “porcus,” pig) is the fourth most frequently isolated NTM species from the Hawai’i environment and cause lymphadenitis in pigs and wound and pulmonary infections in humans. Because Hawai’i is home to large populations of invasive feral pigs, we hypothesized pigs are animal reservoirs for NTM. This hypothesis was tested with the following aims: (i) Determine the NTM species diversity among Hawai’i feral pigs, and (ii) Assess the growth rate of feral pig, environmental, and respiratory NTM, including environmental and respiratory M. porcinum isolates, and compare survival of these isolates in human macrophages. To accomplish project aims, we collected a matched set of nasal and fecal samples from 50 deceased Hawai’i feral pigs. Samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. A total of 20 NTM species were identified, including Mycobacterium abscessus. Five pig-derived NTM isolates and six M. porcinum isolates were selected for further characterization including tabulating their colony morphology, capacity to form biofilms, and assessing their capacity to survive in human THP-1 macrophages. We found effective control of pig-derived NTM by THP-1 macrophages, but differential production of host cytokines in response to infection. M. porcinum isolates showed significant intraspecies variation with respect to growth rate and macrophage survival. This study confirms feral pigs in Hawai’i harbor pathogenic NTM species and may contribute to the prevalence of NTM in Hawai’i.
Evolution Of SARS-CoV-2 Variants In Geographic Locations With Varying Case Incidence
(University of Hawaii at Manoa, 2022) Fraser, Claire Jisu; Butler, Marguerite A.; Microbiology
The evolutionary dynamics of SARS-CoV-2 over the course of the pandemic are of great concern. Prior studies have identified accelerated protein evolution early in the pandemic, followed by a period of increased selective constraints. While there are expected changes in rates of mutation influenced by viral spread, the translation to selection is not well studied. In addition, new variants are emerging as SARS-CoV-2 continues to evolve and some are spreading more rapidly due to mutations that provide a viral advantage. A comparative analysis between Los Angeles County and Hawai'i, localities with rigorous surveillance sequencing programs and varying case incidence, was conducted to test whether there are evolutionary differences of B.1.1.7, the first identified variant of concern, and B.1.243, a rapidly spreading variant in Hawai'i, across localities. Of these four locality-variant scenarios, only B.1.243 in LA County occurred at low case incidence and the remaining three scenarios were at high case incidence. With the rapid spread of virus, I generally found elevated rates of evolution - both synonymous and nonsynonymous substitution. The two variants had very different histories, with B.1.243 evolving locally in Hawaii before being introduced into LA County in a single migration event, whereas B.1.1.7 travelled between localities repeatedly. Yet, there were great differences in diversity of genome sequences overall and elevation of number of sites under positive and negative selection in Los Angeles County relative to the same variant in Hawaii. Within each local outbreak of each variant, I found little evidence for an early phase of protein adaptation followed by an increase in constraints as found in a previous study across the US. Instead, I find that in general more sites are under negative selection than positive selection at this point of the pandemic. Overall, these results show support for variability in evolutionary dynamics across localities and suggest differences in selective pressures across different populations.
A Biogeographic Analysis of SARS-CoV-2 Variants of Concern in Hawai‘i
(University of Hawaii at Manoa, 2022) Hill, Ethan; Butler, Marguerite A.; Microbiology
In late 2020 and into 2021, the World Health Organization (WHO) began labeling emerging SARS-CoV-2 strains with increased transmissibility and/or virulence as variants of concern (VOCs). Communities, including Hawai‘i, that have limited ICU resources and are geographically isolated and experience frequent international and domestic travel, were very concerned about the potential for these variants to enter through domestic or international travel and implemented strict travel restrictions to reduce the likelihood of introductions. In this study, the dynamics of importation and spread in Hawaiʻi of the Alpha, Gamma Mu, Delta, and Omicron VOCs were investigated. A phylogeographic analysis of these VOCs was conducted using a time-calibrated phylogenetic reconstruction through maximum likelihood and Bayesian methods, followed by a biogeographic analysis using Dispersal-Extinction-Cladogenesis evolutionary models to describe the pattern of spread between counties in the state. This study recovered 1,268 independent introductions into Hawai‘i from international and domestic sources, with only ~30% of those introductions resulting in subsequent community transmission. Nine hundred thirty interisland movements were identified, with ~50% as imports/exports from Honolulu County. It appears that the non-pharmaceutical interventions implemented in Hawai‘i, primarily the pre-travel testing, played a large role in reducing the number of imports, especially early in the pandemic before widespread vaccination of the population. The effect of travel restrictions was confirmed by comparing introductions and community spread before and after the relaxation of these guidelines. Evidence indicates that these policies allowed Hawai‘i’s medical infrastructure to manage severe cases of COVID-19 without being overrun, a particular concern for a geographically isolated population like that in Hawai‘i. More generally, the comparison of sequenced viral genomes with geographic data can reveal movement of the virus between communities at fine spatial and temporal scales, and may be useful for informing public health decisions in future pandemics.
Microbiologically Influenced Corrosion of 1018 Carbon Steel in Static Seawater/Fuel (Petroleum-Based and Renewable) Mixtures
(University of Hawaii at Manoa, 2017-08) Kealoha, Jan A. N.; Microbiology
Microbiologically influenced corrosion of steel was compared to electrochemical corrosion in multiple fuel and seawater combinations. Corrosion rates were higher in Hydro-processed Renewable Diesel (HRD) compared to petroleum-based F-76 (0.035 vs. 0.016 mm/year, respectively) and were higher under aerobic than anaerobic conditions. No significant differences in types of corrosion products, oxygen diffusion, or pH, were observed when comparing natural vs filtered seawater. White carbonates and magnesium hydroxide precipitates were predominantly formed in HRD, whereas red goethite formed in F-76. In the seawater phase, magnetite (black) formed, typically under a layer of orange lepidocrocite. Rust tubercles formed on steel surfaces in the fuel phase of 59% of all samples resulting in corrosion pits on the underlying metal. The HRD and blended fuel contained more rust tubercles, regardless of exposure time. Microbes associated with accelerated corrosion rates were taxonomically assigned on the basis of their partial 16S or ITS1 rRNA gene sequences.
Evidence for sap1 as a Virulence Factor in Burkholderia cepacia Complex
(University of Hawaii at Manoa, 2017-08) Bluhm, Andrew; Microbiology
Burkholderia cepacia complex (Bcc) is a consortium of at least 20 closely related Gram negative species that are a risk factor for cystic fibrosis (CF) patients. Previously in B. pseudomaelli, a hypothetical protein with no known function, was identified to be a novel virulence factor and involved in attachment. In this work, highly conserved homologs in Bcc K56-2 and LO6 were examined in multiple in vitro and in vivo models such as attachment to eukaryotic cell lines, biofilm attachment and formation, Caenorhabditis elegans survival model, Drosophila melanogaster feeding model, and mouse lung infection. We found that the deletion mutants had impaired attachment and biofilm formation, and significantly lower in vivo survival and replication, compared to the wildtype strains. Finally, C. elegans and mice infected with the mutants had better survival compared to wildtype infections, supporting the hypothesis that the protein surface attachment protein 1, or sap1, is a virulence factor.
Utilization of phosphatidylcholine, a lung surfactant component, as a major nurient source during Pseudomonas aeruginosa lung infection
(University of Hawaii at Manoa, 2013-12) Sun, Zhenxin
Pseudomonas aeruginosa can grow to high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. Three different pathways, the betaine, glycerol and fatty acid degradation (Fad) pathways, are involved in the degradation of PC components including a phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs). The Fad pathway still remains largely uncharacterized in P. aeruginosa. During the course of this work, fadBA1,4,5 operons (3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase) were shown to be the most important operons involved in fatty acid degradation through mutational analysis. Various fad mutants and the triple pathway mutant were analyzed extensively by in vitro growth analysis, virulence characterization, and competition study. Defect of growth on PC as sole carbon source was most significant on the triple pathway mutants, as expected. This growth defect translated to in vivo competition disadvantage in BALB/c mice, suggesting the importance of PC as nutrient source in vivo.
Natural selection and the genetically encoded amino acid alphabet
(University of Hawaii at Manoa, 2013-05) Ilardo, Melissa Ann
Current science has advanced far beyond Crick's 'frozen accident' interpretation of the origin of the standard genetic code. Codon assignments can and do change, and new amino acids can be added to the code. Combined with the simple observation that the complex molecular machinery responsible for the standard code is a product of considerable evolution, it becomes legitimate and important to ask what else explains how and why one particular genetic code emerged within LUCA that still dominates the staggering diversity of life on our planet. Put another way, once we recognize the code as an evolvable phenomenon, we can ask what evolutionary forces shaped the emergence of the particular codon assignments found within the standard genetic code. Biological thinking has coalesced around three major ideas: the Adaptive Hypothesis, the Stereochemical Hypothesis, and the Biosynthetic or Co-Evolutionary Hypothesis. Assessing the validity of all three theories (and any further estimation of their relative contributions) depends upon further investigations of two fundamental assumptions. These assumptions relate to the two previously mentioned chemical languages between which the genetic code acts as an interface: nucleotides and amino acids. A plethora of nucleotides and amino acids formed through biotic and abiotic processes were available in abundance during the earliest stages of life's evolution, as will be addressed in detail in Chapter 2. For the purpose of concluding this review of ideas regarding the evolution of the standard genetic code, what matters is to notice that any estimates made as to the relative importance of the theories described in this chapter build from the assumption of four nucleotides to encode twenty amino acids.
HetP and its three homologues: regions necessary for function of HetP and requirement of homologues for fixation of nitrogen in the filamentous cyanobacterium Anabaena sp. strain PCC 7120
(University of Hawaii at Manoa, 2013-08) Hurd, Kathryn Lynn
The filamentous cyanobacterium Anabaena sp. strain PCC 7120 is a Gram-negative prokaryote that performs oxygenic photosynthesis. In addition to being an obligate phototroph, Anabaena is capable of differentiating specialized nitrogen-fixing cells called heterocysts. The development of terminally-differentiated heterocyst cells occurs in the absence of fixed nitrogen and forms a one-dimensional pattern along the filament of vegetative cells. The exchange of intercellular signals controls the regulated spacing of the heterocyst cells that on average arise every tenth cell along the filament (Figure 1). The formation of heterocyst cells effectively separates the oxygen-labile nitrogenase complex from oxygen-evolving photosynthesis that occurs in vegetative cells. Heterocysts and vegetative cells are mutually interdependent. Heterocyst cells lack photosystem II and the capacity to fix carbon and must rely on the vegetative cells for sources of reductant. In return, heterocysts supply the filament with fixed nitrogen (Cumino et al. 2007; Marcozzi et al 2009). The development of two distinct cell types in a simple one-dimensional pattern makes Anabaena a simple example of cellular differentiation and pattern formation.
Detection of discordant isolates of drug resistant mycobacterium tuberculosis
(University of Hawaii at Manoa, 2013-05) Hoshide, Matt Kaneo
Early diagnosis of mycobacterium tuberculosis is critical for proper treatment. With a regimen of rifampin and isoniazid, a patient with active tuberculosis can become non-infectious within 2 weeks (5). However, as resistance to these two drugs develops, second line antibiotic treatment must be used which lasts an extra 6 to 18 months and patients remain infectious for a longer period of time, which can further propagate the spread of mycobacterium tuberculosis (6). Testing for antibiotic resistance in M. tuberculosis is a lengthy process due to its long generation time. The WHO standard guideline for the drug susceptibility test of M. tuberculosis is inoculating the bacteria through dilution on Löwenstein Jensen agar with a set concentration of test antibiotic and incubating it at 37 degrees Celsius. Colonies are then counted on the 28th day and a proportion is calculated by comparing the colony count of the test medium to a control. If the proportion exceeds a critical proportion or if no colonies appear in the lowest drug concentration medium with the highest inoculum, the isolate is determined to be resistant or sensitive, respectively. However, if neither criteria are matched, the incubation must continue until the 40th day where the final results are read through the same process (7). The development of drug resistance strains in mycobacterium tuberculosis ultimately relies on exposure to the resistant drug because the presence of drug resistance mutations does not confer any selective advantage over strains lacking those mutations until exposure occurs (15). Within each geographical region, strains of M. tuberculosis may be under unique and various selective pressures to develop specific types of polymorphisms. Therefore, it can be hypothesized that there are genetic differences in the drug resistant strains of M. tuberculosis found in different regions and that the proportion of polymorphisms in genes associated with drug resistance will be different between geographic locations. Study Objectives 1. To sequence the hot spot regions of nine genes (rpoB, inhA promoter, katG, ahpC promoter, gyrA, gyrB, rrs, eis promoter, and tlyA). 2. To identify discordant isolates through the comparison of drug susceptibility data and sequence data. 3. To further define the resistance patterns found in M. tuberculosis within and between different geographical regions represented by the widely dispersed study sites
Development of a non-enzyme based capillary dipstick assay for the detection and purification of environmental pathogens
(University of Hawaii at Manoa, 2010-12) Luu, Van Phi
Novel mAbs were produced with a wide range of anti-Dickeya binding reactivities. A mAb line produced from the first fusion generated a BOX-PCR type B,C and D binding antibody (MAb Pine-1). A second mAb line had broad spectrum specificity that reacts to BOX-PCR type A through E and most of the Dickeya reference strains (MAb Pine-2). Phylogenetic analysis based on gyr-B gene expression and BOX-PCR types are consistent with the mAb reactivities. Also a field dipstick assay was developed for the detection of live environmental pathogens that is able concentrate the organism for further analysis and was able to detect S. enterica serotype Typhimurium at 105 CFU/ml, which is within the infective dose of most Salmonella species. The dipstick is able to isolate and purify S. enterica serotype Typhimurium from a mixed culture of 100:1 S. marcescens to S. enterica serotype Typhimurium, with a final purity of greater than 80% determined by colony counts, which is culturable to streak for identification.
Numerous fatty acyl-CoA synthetase homologues are involved in fatty acid degradation in pseudomonas aeruginosa
(University of Hawaii at Manoa, 2011-05) Zarzycki-Siek, Jan Bozydar
The goal of this research is to characterize the newly identified fatty acyl-CoA synthetase homologue, PA3860 (fadD3), which is thought to take part in Fad though its exact contribution to fatty acid metabolism in P. aeruginosa is unknown. Deciphering the role of fadD3 in fatty acid degradation will broaden our knowledge of the β-oxidation pathway in this bacterium. This was achieved by completing the following specific aims: 1. Purification of FadD3 and performance of biochemical analysis of its acyl-CoA synthetase function 2. Genetical characterization of fadD3 (PA3860) of P. aeruginosa through mutational analysis and gene fusion studies During the course of this research additional discoveries about the β-oxidation pathway of P. aeruginosa were made: 1) additional acyl-CoA synthetase homologues were found, i.e. fadD4, fadD5, and fadD6 2) and their role in Fad was determined along with fadD3.
Bioprospecting for thermostable glycoside hydrolases: a metatranscriptomic approach
(University of Hawaii at Manoa, 2012-12) Lyford, Jeffrey Ross
The conversion of cellulosic material from linear form to monomer is the rate-limiting step in current biofuels production technologies from lignocellulosic material. The renewed focus on clean, sustainable, carbon-neutral fuels has resulted in increased interest in novel cellulolytic enzymes and microbial strains, with the aim to increase the efficiency of the above conversion. However, the availability of suitable cellulolytic enzymes has been restricted by the limited number of cellulolytic microbial strains in which these enzymes can be procured. One approach to increase efficiency has been to bioprospect novel thermophilic microbial strains, the logic being that an increase in production temperature will result in increased rates of lignocellulosic hydrolysis. Unfortunately, microbiologists have had limited success in cultivating cellulolytic thermophiles with only a small number of strains isolated. The goal of this project was to circumvent the need to cultivate cellulolytic thermophiles by applying a metatranscriptomic approach to discover new thermostable cellulases commonly referred to as glycoside hydrolases (GH-enzymes that hydrolyze the glycosidic linkages between sugar molecules). This was achieved by employing a novel in situ enrichment technique to enrich for thermostable GHs directly from a geothermal environment. These GHs were being actively expressed by the resident microbial population to hydrolyse lignocellulose. This approach not only eliminated the need for cultivation, but also selected for the actively expressed GHs unregulated in response to the lignocellulosic material feedstock. This strategy removed the emphasis on identifying potentially relevant and substrate-active GHs from genomic data via genomic analysis of a cultivated microorganism or from environmental metagenomic surveys.
In silico and in vitro characterization of ten putative RsbR-like proteins in Saprospira grandis
(University of Hawaii at Manoa, 2012-05) Kim, Sun Ae
Globin-coupled sensors (GCSs) are heme-binding proteins that consist of an N-terminal sensor globin domain and a C-terminal transducer domain. Their functions are varied depending on the C-terminal domain and can be classified into two groups such as aerotactic and gene regulation groups. The gene regulating group is further divided into DNA-binding, 2nd messenger and protein-protein interaction group. So far, aerotactic transducers: HemAT-Hs from Halobacterium salinarum and HemAT-Bs from Bacillus subtilis are well characterized in a molecular level and a cellular level. In addition, GCSs from the 2nd messenger group within the gene regulating group such as BpeGreg (Bordetella pertussis), EcGreg (E. coli), CvGreg (Chromobacterium violaceum) and AvGreg (Azotobacter vinelandii) are recently characterized as well. Recently, genome sequencing of Saporospira grandis has been completed and revealed in-depth genomic information that related to interesting traits of this organism such as gliding motility, production of rhapidosome and ixotrophic nutrient obtaining mechanisms. Moreover, the initial machine annotation showed that there are ten putative GCSs which contain C-terminal STAS domains. Based on the machine annotation results, we have ten genes that were predicted to be GCSs and have a C-terminal STAS (Sulfur transporter and anti-sigma antagonist) domain (Table 1.1). The putative function of these proteins is an anti-sigma factor antagonist like SpoIIAA, RsbR and RsbS in B. subtilis which involves in nutritional, physical and environmental stress responses. The protein named RsbR1 (SGRA_3210) within S. grandis was first discovered and its neighbor genes rsbS,-T,-V,-X and rsbU shared similarities with rsbR and its adjacent genes (rsbS,-T,-U,-V,-W, sigB, rsbX) in B. subtilis. Also, the structure of RsbR consists of the N-terminal non-heme globin domain and the C-terminal STAS domain. All these genes and their encoded proteins are related to activate more than 150 stress response genes upon physical stresses through releasing a sigma factor B. Based on these similarities, we postulate RsbR1 and nine other proteins are globin-coupled sensors that are involved in the stress response. In this study, in silico and in vitro characterization of ten GCSs were carried out on both the N-terminal globin and the C-terminal STAS domains. Multiple alignments of the globin domains showed that only SGRA_0571 (RsbR2), SGRA_3210 (RsbR1) and SGRA_3852 (RsbR3) aligned their histidine residue with the proximal histidine residue in known globin domains. Two phosphorylation sites from RsbR and its paralogs in B. subtilis were aligned with nine GCSs except SGRA_3852 and YtvA which is known not to have any phosphorylation site, instead it has a GTP binding motif. Two amino acid residues (D and G) from the GTP binding motif (DXXG) are all aligned in all GCSs. The 3D structure alignments of the globin domain of Geobactor sulfurreducens and RsbR1, RsbR2 and RsbR3 showed around 23% identity but the proximal histidine, where it held the heme, were all aligned with the histidine residue of three GCSs. For in vitro characterization, all ten genes were cloned, expressed and purified. The purified proteins were dialyzed and carried UV spectroscopic analysis. Only three GCSs (RsbR1, RsbR2 and RsbR3) exhibited oxygenated myoglobin like peaks. This shows that these proteins are able to bind oxygen. RsbR1 was undergone for crystallization. Initial crystallization screening showed some red crystals. Initially we found ten GCS genes from the annotation of the genome. In silico results predicted them all as GCSs. Multiple alignments of the globin domains and STAS domains of ten GCSs showed only RsbR1, RsbR2 and RsbR3 had the conserved proximal histidine residues which were vital for heme-binding. The STAS domains of ten GCSs were shared high homology with the STAS domains of RsbR and its paralogs from B. subtilis including two conserved phosphorylation sites and possible GTP-binding motifs. However, RsbR3 did not show the conserved phophrylation sites. All ten GCS genes were cloned, expressed and purified. RsbR1, RsbR2 and RsbR3 displayed oxygenated myoglobin-like absorption spectra and they were the only proteins that were validated as GCSs.
Modulation of TNF-α production by small RNA in dengue virus infected human monocytic cells
(University of Hawaii at Manoa, 2012-08) Hammond, Kimberly Elaine
The immunopathology of dengue virus (DENV) infection is associated with increased TNF-α production. In this study, small RNA-mediated regulation of TNF-α and the effect of TNF-α knockdown during DENV infection were analyzed. This provides insight into the role of TNF-α during DENV infection, both in terms of its contribution to immunopathogenesis and its regulatory mechanism by miRNAs. Utilizing a lentiviral expression system, human monocytic U937 cells that express short hairpin RNAs designed to target TNF-α mRNA were established. TNF-α expression was downregulated in these monocytes, and upon DENV infection they showed decreased endothelial cell activation ability. This demonstrates an overall decrease in the proinflammatory response upon TNF-α knockdown during DENV infection. To analyze the role of microRNAs (miRNAs) in the TNF-α response, miRNAs that potentially target the 3' UTR of TNF-α were predicted. Many of the miRNAs were differentially regulated during DENV infection. miR-320a and miR-592 were among those downregulated, and chosen for further analysis. TNF-α post-transcriptional regulation by miR-320a and miR-592 was confirmed utilizing a TNF-α 3'UTR luciferase reporter. U937 cells transfected with miR-320a and miR-592 mimics followed by DENV infection displayed decreased TNF-α expression and their culture supernatants demonstrated decreased ability to activate endothelial cells. It is concluded that one function of these miRNAs is to negatively regulate TNF-α, and their downregulation contributes to the immunopathogenesis of DENV infection.
Effective concentration and detection of human enteric viruses in Hawaiian environmental waters
(University of Hawaii at Manoa, 2012-05) Connell, Christina Noelle
Health risks associated with sewage-contaminated recreational waters are of important public health concern. Reliable water monitoring systems are therefore crucial. Current recreational water quality criteria rely predominantly on the enumeration of bacterial indicators, while potentially dangerous viral pathogens often remain undetected. Human enteric viruses have been proposed as alternative indicators; however, their detection is often hindered by low viral concentrations present in the aquatic environment. Reported here are novel and effective laboratory protocols for enhanced enteric virus detection in Hawaiian environmental waters. First, a fine-tuned, highly optimized assay for the detection of enterovirus, an important enteric virus subset, was developed by comparatively evaluating eighteen published enterovirus primer pairs for detection sensitivity. The primer set exhibiting the lowest detection limit under optimized conditions, EQ-1/EQ-2, was validated through testing urban wastewater, and then utilized in a field survey of 22 recreational bodies of water located around the island of Oahu, Hawaii. Eleven sites tested positive for enterovirus, indicating fecal contamination in a significant portion of Hawaiian waters. Additionally, the filter-feeding phenomenon of indigenous bivalve mollusks was explored as a natural bioconcentration technique to infer microbial quality of the surrounding waters. Shellfish were collected from 12 coastal locations and dissected for subsequent nucleic acid extraction from internal tissues. Optimized RT-PCR/PCR protocols were then applied to test for the presence of various enteric viruses, including enterovirus, adenovirus, norovirus genogroups I and II, and F-specific RNA coliphage. Shellfish collected from around the island tested positive for several enteric virus types, indicating that these animals are indeed natural and competent bioindicators of water quality. The extremely sensitive and innovative techniques implemented here are valuable resources to aid accurate reflection of microbial contamination in Hawaii's environmental waters.
Characterization of biocontaminants in biodiesel fuels and potential roles in the formation of microbially induced corrosion
(University of Hawaii at Manoa, 2012-12) Ching, Travers Han-ming
Biological contamination of renewable biodiesel fuels is a recognized problem that requires detailed investigations (Passman, 2001). Increased demand for energy independence and viability as a fossil fuel alternative has rapidly expanded use of biodiesel globally. However, there are several problems associated with biodiesel. One such problem is biological contamination, which reduces fuel stability and induces corrosion. At the start of this project, it was hypothesized that metal corrosion and fuel degradation by microorganisms are cometabolically enhanced by biodiesel in blended fuels. To investigate this hypothesis, experiments were designed to characterize microbial contaminants in biodiesel fuels and study their potential roles in microbial degradation, microbially induced corrosion and cometabolism.
Metagenome recruitment of the global ocean survey dataset to four closely-related SAR11 genomes
(University of Hawaii at Manoa, 2014-05) Brucks, Eric Snodgrass
The free-living marine bacterial clade known as SAR11 is thought to be one of the most abundant lineages of organisms on our planet. It is also one of the smallest free-living organisms, and possibly the ancestor of the very successful endosymbiont, mitochondria. Members of this group of bacteria possess an incredibly small genome that displays a high degree of synteny and high DNA recombination rate, which represents the upper limit for bacteria. This study examines the global metagenomic read recruitment of the genomes of four closely related SAR11 strains, HIMB4, HIMB5, HIMB83, and HIMB140, in publicly available coastal and open ocean metagenome libraries. Recruitment plots were used to identify and analyze clusters of genes with unusual recruitment patterns, and utilized 2,797,042 open ocean and 2,131,159 coastal high quality reads. It was found that strain HIMB83 possessed the most highly recruited genome and had the highest coastal preference of the four. A hypervariable region previously identified in SAR11, HVR2, was identified in all four strains, and was characterized by a large number of genes coding for cell wall and membrane components as well as several enzymes involved in carbohydrate metabolism. HVR1 was also present in all examined genomes where it was dominated by 12 proteins involved in the type II secretion and type IV pilus pathway. The recruitment of the proteorhodopsin gene present in each of the strains was similar, with a greater degree of conservation on the 3' end of the gene. A phage integrase and extra DNA polymerase was found within the genome of HIMB4, suggesting a possible lysogenic virus present within the genome. This study adds to the current understanding of SAR11 subgroup 1a in a global context, and provides a workflow for generating metagenome recruitment plots for future studies.
Lipid body function as non-classical calcium stores in immunocytes
(University of Hawaii at Manoa, 2014-12) Phan, Nolwenn Kathryn
Lipid bodies, found in most eukaryotic cells, are intracellular lipid storage organelles. The role of lipid bodies has been most studied in adipocytes and hepatocytes due to their role in energy storage. Comparatively, little is known of the role they play in immunocytes. Both in adipocytes/hepatocytes and in cells of the immune system, lipid body numbers are dynamically regulated and can accumulate to pathophysiological levels (steatosis). In both locales this steatotic state can be induced by nutrient overload and metabolic stress, and in the latter also under certain conditions of infection. The over-arching goals of this project are (1) to study the composition (lipid and protein) and functional contributions made by lipid bodies in immunocytes, and (2) to assess the impact of altered lipid body numbers upon cellular function. The work presented in this thesis describes three areas of progress towards these goals. First, we developed a microaspiration method for isolating highly purified lipid bodies, allowing for their ex vitro manipulation and study of lipid/protein content using microscopy. This technique was validated using fluorescence microscopy of the neutral lipid dye Oil Red O in microaspirated lipid bodies. Second, we assessed the impact of the accumulation of lipid bodies (steatosis) on transcytoplasmic calcium signaling, a major activation pathway in the model immune cell system studied here. Third, we tested a new hypothesis arising from our work on transcytoplasmic calcium signaling. The apparent ability of lipid bodies to act as long term loci for calcium accumulation, coupled with recent studies showing that lipid bodies may contain mitochondria and endoplasmic reticulum, led us to hypothesize their potential role as bona fide calcium stores. Our data revealed that the lipid body population within mast cells is not homogenous. However, some LB exhibit the ability to sequester calcium and to release it in the manner of a bona fide calcium store. These observations represent a potentially novel role for lipid bodies and indicate the possibility of their contribution to calcium dynamics in immune cells under both physiological and pathophysiological conditions.

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