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Investigating the Role of the Las and Rhl Quorum Sensing Systems in the Pathogenesis of Pseudomonas aeruginosa

Description

Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a

Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a chronic infection. The resultant phenotypes have been associated with chronic infection and can provide important information to track the patient’s individualized disease progression. This study examines the link between the accumulation of QS genetic mutations and phenotypic expression in P. aeruginosa laboratory strains and clinical isolates. We utilized several plate-based and colorimetric assays to quantify the production of pyocyanin, rhamnolipids, and protease from paired clinical early- and late-stage chronic infection isolates across 16 patients. Exoproduct production of each isolate was compared to the mean production of pooled isolates to classify high producing (QS-sufficient) and low producing (QS-deficient) isolates. We found that over time P. aeruginosa isolates exhibit a reduction in QS-related phenotypes during chronic infections. Future research of the QS regulatory networks will identify whether reversion of genotype will result in corresponding phenotypic changes in QS-deficient chronic infection isolates.

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2020-05

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Assessment of the Vertical Stratification of Microbial Community Structure in Permafrost Peatlands

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Peatlands are a type of wetlands where the rate of accumulation of organic matter exceed the rate of decomposition and have accumulated more than 30 cm of peat (Joosten and Clark, 2002). Peatlands store approximately 30% of all terrestrial carbon

Peatlands are a type of wetlands where the rate of accumulation of organic matter exceed the rate of decomposition and have accumulated more than 30 cm of peat (Joosten and Clark, 2002). Peatlands store approximately 30% of all terrestrial carbon as recalcitrant peat, partially decomposed plant and microbial biomass, while simultaneously producing almost 40% of the globally emitted methane (Schmidt et al., 2016), making peatlands an important component of the carbon budgets. Published research indicates that the efficiency of carbon usage among microbial communities can determine the soil-carbon response to rising temperatures (Allison et al. 2010). By determining carbon consumption in peatland soils, total community respiration response, and community structure change with additions, models of carbon use efficiency in permafrost peatlands will be well-informed and have a better understanding of how the peatlands will respond to, and utilize, increased availability of carbon compounds due to the melting permafrost. To do this, we will sequence Lutose deep core samples to observe baseline microbial community structure at different depths and different age-gradients, construct substrate incubations of glucose and propionate and observe community respiration response via a gas chromatography flame ionization detector, track the glucose and propionate additions with high-performance liquid chromatography (HPLC), and sequence the samples once more to determine if there was a deviation from the initial community structure obtained prior to the incubations. We found that our initial sequencing data was supported by previous work (Lin et al., 2014), however we were unable to sequence samples post-incubation due to time constraints. In this sequencing analysis we found that the strongest variable that made samples biologically similar was the age-gradient site in which they were extracted. We found that the group with glucose additions produced the most carbon dioxide compared with the other treatments, but was not the treatment that dominated the production of methane. Finally, in the HPLC samples that were analyzed, we found that glucose is likely forming the most by-product accumulation from mass balance calculations, while propionate is likely forming the least. Future experimentation should focus on the shortcomings of this experiment. Further analysis of 16S rRNA sequencing data from after the incubations should be analyzed to determine the change in microbial community structure throughout the experiment. Furthermore, HPLC analysis for the several samples need to be done and followed up with mass balance to determine where the added glucose and propionate are being allocated within the soil. Once these pieces of the puzzle are put into place, our original question of how the microbial community structure changes at different depths and age-gradients within permafrost peatlands will be conclusively answered.

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2018-05

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Codon Optimization of Human TRAIL Gene for Maximal Expression in a Self-Destructing Salmonella Strain for Efficient Colorectal Cancer Treatment

Description

Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which

Colorectal cancer is the third most common type of cancer that affects both men and women and the second leading cause of death in cancer related deaths[1, 2]. The most common form of treatment is chemotherapy followed by radiation, which is insufficient to cure stage four cancers[3]. Salmonella enteric has long been shown to have inherent tumor targeting properties and have been able to penetrate and exist in all aspects of the tumor environment, something that chemotherapy is unable to achieve. This lab has developed a genetically modified Salmonella typhimurium (GMS) which is able to deliver DNA vaccines or synthesized proteins directly to tumor sites. These GMS strains have been used to deliver human TNF-related apoptosis inducing ligand (TRAIL) protein directly to tumor sites, but expression level was limited. It is the hope of the experiment that codon optimization of TRAIL to S. typhimurium preferred codons will lead to increased TRAIL expression in the GMS. For preliminary studies, BALB/c mice were subcutaneously challenged with CT-26 murine colorectal cancer cells and treated with an intra-tumor injection with either PBS, strain GMS + PCMV FasL (P2), or strain GMS + Pmus FasL). APC/CDX2 mutant mice were also induced to develop human colon polyps and treated with either PBS, strain GMS + vector (P1), P2, or P3. The BALB/c mouse showed statistically significant levels of decreased tumor size in groups treated with P2 or P3. The APC/CDX2 mouse study showed statistically significant levels of decreased colon polyp numbers in groups treated with P3, as expected, but was not significantly significant for groups treated with P1 and P2. In addition, TRAIL was codon optimized for robust synthesis in Salmonella. The construct will be characterized and evaluated in vitro and in vivo. Hopefully, the therapeutic effect of codon optimized TRAIL will be maximal while almost completely minimizing any unintended side effects.

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2019-05

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Enhancing Escherichia coli Fermentative Performance with Lactobacillus plantarum WCFS1 Genes

Description

Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of

Renewable bioproduction through fermentation of microbial species such as E. coli shows much promise in comparison to conventional fossil fuel based chemical production. Although Escherichia coli is a workhorse for bioproduction, there are inherent limitations associated with the use of this organism which negatively affect bioproduction. One example is E. coli fermentative growth being less robust compared to some microbes such as Lactobacilli under anaerobic and microaerobic fermentation conditions. Identification and characterization of its fermentative growth constraints will help in making E. coli a better fermentation host. In this thesis, I demonstrate that Lactobacillus plantarum WCFS1 has desirable fermentative capabilities that may be transferrable to E. coli through genetic engineering to alleviate growth restraints. This has led to the hypothesis that these L. plantarum DNA sequences are transferrable through a genomic library. A background of comparative genomics and complementary literature review has demonstrated that E. coli growth may be hindered by stress from many toxin-antitoxin systems. L. plantarum WCFS1 optimizes amino acid catabolism over glycolysis to generate high ATP levels from reducing agents and proton motive force, and Lactobacilli are resistant to acidic environments and encodes a wide variety of acid transporters that could help E. coli fermentative growth. Since a great variety of L. plantarum genes may contribute to its fermentative capabilities, a gDNA library containing L. plantarum WCFS1 genes has been successfully constructed for testing in E. coli bioproducers to search for specific genes that may enhance E. coli fermentative performance and elucidate the molecular basis of Lactobacillus fermentative success.

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2019-05

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Quantifying Intragenomic Variability in the 18S Gene of Trichonympha from Zootermopsis

Description

The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and

The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.

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2020-05

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Inhibition of PKR phosphorylation by Vaccinia Virus' E3 Protein

Description

Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can

Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.

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2017-05

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Coronavirus Envelope Protein Transmembrane Domain: Impact of Positive Charges on Virus-like Particle Assembly

Description

Coronaviruses are a significant group of viruses that cause enteric and respiratory infections in a variety of animals, including humans. Outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS) in the past 15 years has increased

Coronaviruses are a significant group of viruses that cause enteric and respiratory infections in a variety of animals, including humans. Outbreaks of Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS) in the past 15 years has increased research into coronaviruses to gain an understanding of their structure and function so one day therapies and vaccines may be produced. These viruses have four main structural proteins: the spike, nucleocapsid, envelope, and membrane proteins. The envelope (E) protein is an integral membrane protein in the viral envelope that acts as a viroporin for transport of cations and plays an important role in pathogenesis and viral assembly. E contains a hydrophobic transmembrane domain with polar residues that is conserved across coronavirus species and may be significant to its function. This experiment looks at the possible role of one polar residue in assembly, the 15th residue glutamine, in the Mouse Hepatitis Virus (MHV) E protein. The glutamine 15 residue was mutated into positively charged residues lysine or arginine. Plasmids with these mutations were co-expressed with the membrane protein (M) gene to produce virus-like particles (VLPs). VLPs are produced when E and M are co-expressed together and model assembly of the coronavirus envelope, but they are not infectious as they do not contain the viral genome. Observing their production with the mutated E protein gives insight into the role the glutamine residue plays in assembly. The experiment showed that a changing glutamine 15 to positive charges does not appear to significantly affect the assembly of the VLPs, indicating that this specific residue may not have a large impact on viral assembly.

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2017-05

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Development and evaluation of selective and differential media for polymicrobial communities from the International Space Station potable water system

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The International Space Station (ISS) utilizes recycled water for consumption, cleaning and air humidity control. The Environmental Control and Life Support Systems (ECLSS) have been rigorously tested at the NASA Johnson Space Center. Despite the advanced engineering of the water

The International Space Station (ISS) utilizes recycled water for consumption, cleaning and air humidity control. The Environmental Control and Life Support Systems (ECLSS) have been rigorously tested at the NASA Johnson Space Center. Despite the advanced engineering of the water recovery system, bacterial biofilms have been recovered from this potable water source. Microbial contamination of potable water poses a potential threat to crew members onboard the ISS. Because astronauts have been found to have compromised immune systems, bacterial strains that would not typically be considered a danger must be carefully studied to better understand the mechanisms enabling their survival, including polymicrobial interactions. The need for a more thorough understanding of the effect of spaceflight environment on polymicrobial interactions and potential impact on crew health and vehicle integrity is heightened since 1) several potential pathogens have been isolated from the ISS potable water system, 2) spaceflight has been shown to induce unexpected alterations in microbial responses, and 3) emergent phenotypes are often observed when multiple bacterial species are co- cultured together, as compared to pure cultures of single species. In order to address these concerns, suitable growth media are required that will not only support the isolation of these microbes but also the ability to distinguish between them when grown as mixed cultures. In this study, selective and/or differential media were developed for bacterial isolates collected from the ISS potable water supply. In addition to facilitating discrimination between bacteria, the ideal media for each strain was intended to have a 100% recovery rate compared to traditional R2A media. Antibiotic and reagent susceptibility and resistance tests were conducted for the purpose of developing each individual medium. To study a wide range of targets, 12 antibiotics were selected from seven major classes, including penicillin, cephalosporins, fluoroquinolones, aminoglycosides, glycopeptides/lipoglycopeptides, macrolides/lincosamides/streptogramins, tetracyclines, in addition to seven unclassified antibiotics and three reagents. Once developed, medium efficacy was determined by means of growth curve experiments. The development of these media is a critical step for further research into the mechanisms utilized by these strains to survive the harsh conditions of the ISS water system. Furthermore, with an understanding of the complex nature of these polymicrobial communities, specific contamination targeting and control can be conducted to reduce the risk to crew members. Understanding these microbial species and their susceptibilities has potential application for future NASA human explorations, including those to Mars.

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2018-12

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The Role of Lipolysis in Regulating Plasma Glucose Concentrations in Mourning Doves

Description

Birds have unusually high plasma glucose concentrations compared to mammals of similar size despite their high metabolic rate. While birds use lipids as their main source of energy, it is still unclear how and why they maintain high plasma glucose

Birds have unusually high plasma glucose concentrations compared to mammals of similar size despite their high metabolic rate. While birds use lipids as their main source of energy, it is still unclear how and why they maintain high plasma glucose concentrations. To investigate a potential underlying mechanism, this study looks at the role of lipolysis in glucose homeostasis. The purpose of this study is to examine the effects of decreased glycerol availability (through inhibition of lipolysis) on plasma glucose concentrations in mourning doves. The hypothesis is that decreased availability of glycerol will result in decreased production of glucose through gluconeogenesis leading to reduced plasma glucose concentrations. In the morning of each experiment, mourning doves were collected at the Arizona State University Tempe campus, and randomized into either a control group (0.9% saline) or experimental group (acipimox, 50mg/kg BM). Blood samples were collected prior to treatment, and at 1, 2, and 3 hours post-treatment. At 3 hours, doves were euthanized, and tissue samples were collected for analysis. Acipimox treatment resulted in significant increases in blood glucose concentrations at 1 and 2 hours post- treatment as well as renal triglyceride concentrations at 3 hours post-treatment. Change in plasma free glycerol between 0h and 3h followed an increasing trend for the acipimox treated animals, and a decreasing trend in the saline treated animals. These results do not support the hypothesis that inhibition of lipolysis should decrease blood glycerol and blood glucose levels. Rather, the effects of acipimox in glucose homeostasis appear to differ significantly between birds and mammals suggesting differing mechanisms for glucose homeostasis.

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2015-05

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The Environmental Contribution to Emerging Antibiotic Resistance

Description

Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account for the function of the environment. Over the past 15

Antibiotic resistance in the modern era has reached near-epidemic levels, resulting in much more difficult treatment of previously well-managed pathogens. Previous understandings of how antibiotic resistance emerges failed to account for the function of the environment. Over the past 15 years, new research has provided a link between the environmental and clinical spheres of antibiotic use. This data suggests that environmental bacteria, particularly those found in livestock farming ecosystems, may significantly contribute to the overall flow of antibiotic resistance genes into human populations. The main force behind this is the utilization of antibiotics as growth promoters in animal feed supplements, seeding individual animals and their surroundings with low doses of antibiotics. Notable increases in resistance have been observed within areas that utilize these supplements, as well as in connected but unrelated systems. Waste management strategies are poorly implemented, leading to the dispersal of contaminated runoff into groundwater and riverine environments. Furthermore, existing waste processing is limited in efficacy, often releasing large amounts of unprocessed antibiotics as well as a concentrated population of resistant bacteria. Within these resistant populations, horizontal gene transfer has emerged as a vehicle for the distribution of resistance genes into other populations of bacteria. Due to the prevalence of these transfer events, a new role for the environment as a reservoir and incubator of resistance genes is proposed. Current strategies for managing the spread of antibiotic resistance are woefully inadequate, and the continued emergence of new resistance mechanisms due to negligence highlights the need for global, multidisciplinary solutions. To corral the spread of antibiotic resistance, a system is proposed that utilizes metagenomic monitoring and the enforcement of core global policies to slow the advance of resistance while waiting for novel treatment strategies to bear fruit.

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2016-05