ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
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- All Subjects: Biology
- Creators: Mason, Hugh
- Creators: Krajmalnik-Brown, Rosa
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation.
ContributorsHong, Fan (Author) / Mason, Hugh (Thesis advisor) / Gaxiola, Roberto (Committee member) / Chang, Yung (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2011
Description
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
Description
Photosynthesis converts sunlight to biomass at a global scale. Among the photosynthetic organisms, cyanobacteria provide an excellent model to study how photosynthesis can become a practical platform of large-scale biotechnology. One novel approach involves metabolically engineering the cyanobacterium Synechocystis sp. PCC 6803 to excrete laurate, which is harvested directly.
This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s.
Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI.
How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently.
Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM).
Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity.
This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity.
This work begins by defining a working window of light intensity (LI). Wild-type and laurate-excreting Synechocystis required an LI of at least 5 µE/m2-s to sustain themselves, but are photo-inhibited by LI of 346 to 598 µE/m2-s.
Fixing electrons into valuable organic products, e.g., biomass and excreted laurate, is critical to success. Wild-type Synechocystis channeled 75% to 84% of its fixed electrons to biomass; laurate-excreting Synechocystis fixed 64 to 69% as biomass and 6.6% to 10% as laurate. This means that 16 to 30% of the electrons were diverted to non-valuable soluble products, and the trend was accentuated with higher LI.
How the Ci concentration depended on the pH and the nitrogen source was quantified by the proton condition and experimentally validated. Nitrate increased, ammonium decreased, but ammonium nitrate stabilized alkalinity and Ci. This finding provides a mechanistically sound tool to manage Ci and pH independently.
Independent evaluation pH and Ci on the growth kinetics of Synechocystis showed that pH 8.5 supported the fastest maximum specific growth rate (µmax): 2.4/day and 1.7/day, respectively, for the wild type and modified strains with LI of 202 µE/m2-s. Half-maximum-rate concentrations (KCi) were less than 0.1 mM, meaning that Synechocystis should attain its µmax with a modest Ci concentration (≥1.0 mM).
Biomass grown with day-night cycles had a night endogenous decay rate of 0.05-1.0/day, with decay being faster with higher LI and the beginning of dark periods. Supplying light at a fraction of daylight reduced dark decay rate and improved overall biomass productivity.
This dissertation systematically evaluates and synthesizes fundamental growth factors of cyanobacteria: light, inorganic carbon (Ci), and pH. LI remains the most critical growth condition to promote biomass productivity and desired forms of biomass, while Ci and pH now can be managed to support optimal productivity.
ContributorsNguyen, Binh Thanh (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Westerhoff, Paul (Committee member) / Arizona State University (Publisher)
Created2015
Description
Phytoplankton comprise the base of the marine food web, and, along with heterotrophic protists, they are key players in the biological pump that transports carbon from the surface to the deep ocean. In the world's subtropical oligotrophic gyres, plankton communities exhibit strong seasonality. Winter storms vent deep water into the euphotic zone, triggering a surge in primary productivity in the form of a spring phytoplankton bloom. Although the hydrographic trends of this "boom and bust" cycle have been well studied for decades, community composition and its seasonal and annual variability remains an integral subject of research. It is hypothesized here that proportions of different phytoplankton and protistan taxa vary dramatically between seasons and years, and that picoplankton represent an important component of this community and contributor to carbon in the surface ocean. Monthly samples from the Bermuda Atlantic Time-series Study (BATS) site were analyzed by epifluorescence microscopy, which permits classification by morphology, size, and trophic type. Epifluorescence counts were supplemented with flow cytometric quantification of Synechococcus, Prochlorococcus, and autotrophic pico- and nanoeukaryotes. Results from this study indicate Synechococcus and Prochlorococcus, prymnesiophytes, and hetero- and mixotrophic nano- and dinoflagellates were the major players in the BATS region plankton community. Ciliates, cryptophytes, diatoms, unidentified phototrophs, and other taxa represented rarer groups. Both flow cytometry and epifluorescence microscopy revealed Synechococcus to be most prevalent during the spring bloom. Prymnesiophytes likewise displayed distinct seasonality, with the highest concentrations again being noted during the bloom. Heterotrophic nano- and dinoflagellates, however, were most common in fall and winter. Mixotrophic dinoflagellates, while less abundant than their heterotrophic counterparts, displayed similar seasonality. A key finding of this study was the interannual variability revealed between the two years. While most taxa were more abundant in the first year, prymnesiophytes experienced much greater abundance in the second year bloom. Analyses of integrated carbon revealed further stark contrasts between the two years, both in terms of total carbon and the contributions of different groups. Total integrated carbon varied widely in the first study year but displayed less fluctuation after June 2009, and values were noticeably reduced in the second year.
ContributorsHansen, Amy (Author) / Neuer, Susanne (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Sommerfeld, Milton (Committee member) / Arizona State University (Publisher)
Created2010
Description
The expression of complex proteins was studied in multiple plant systems. Recombinant spider silk, which could be utilized for biomedical applications such as coatings or doped into silk fibers, was successfully expressed in Nicotiana. benthamiana wild type and GnGn glycoengineered transgenic plants and purified from endogenous plant proteins which could be utilized for biomedical applications such as coatings or doped into silk fibers. However, the purification process requires further optimization to result in commercialized production of recombinant spider silk. Green fluorescent protein and Norovirus virus-like particles were expressed in multiple plant systems including alfalfa, beets, lettuce, and spinach, in addition to N. benthamiana, to determine the ability of these plant expression systems to produce vaccine candidates for edible vaccine applications in the agricultural sector as well as low-to-middle income countries. It was determined that alfalfa, beets, and lettuce are potential high production expression systems for edible vaccines however they require further optimization to be commercialized. Lastly, novel virus-like particles and antigen presenting nanoparticles based on the bacteriophage AP205 coat protein and norovirus capsid proteins fused to human papillomavirus L2 protein segments (S and P) were expressed in N. benthamiana and utilized to vaccinate mice against the L2 capsid protein (aa14-38x2 and aa14-122) of Human Papillomavirus 16 to study a potential boosting effect of the Recombinant Immune Complex vaccine platform upon prime-boost dosing with the virus-like particle being the prime and the Recombinant Immune Complex being the boost in this vaccine schema.
ContributorsHunter, Joseph G (Author) / Mason, Hugh (Thesis advisor) / Arizona State University (Publisher)
Created2023
Characterization and Manipulation of Microbiomes From Arid Landfills for Improved Methane Production
Description
Environmentally harmful byproducts from solid waste’s decomposition, including methane (CH4) emissions, are managed through standardized landfill engineering and gas-capture mechanisms. Yet only a limited number of studies have analyzed the development and composition of Bacteria and Archaea involved in CH4 production from landfills. The objectives of this research were to compare microbiomes and bioactivity from CH4-producing communities in contrasting spatial areas of arid landfills and to tests a new technology to biostimulate CH4 production (methanogenesis) from solid waste under dynamic environmental conditions controlled in the laboratory. My hypothesis was that the diversity and abundance of methanogenic Archaea in municipal solid waste (MSW), or its leachate, play an important role on CH4 production partially attributed to the group’s wide hydrogen (H2) consumption capabilities. I tested this hypothesis by conducting complementary field observations and laboratory experiments. I describe niches of methanogenic Archaea in MSW leachate across defined areas within a single landfill, while demonstrating functional H2-dependent activity. To alleviate limited H2 bioavailability encountered in-situ, I present biostimulant feasibility and proof-of-concepts studies through the amendment of zero valent metals (ZVMs). My results demonstrate that older-aged MSW was minimally biostimulated for greater CH4 production relative to a control when exposed to iron (Fe0) or manganese (Mn0), due to highly discernable traits of soluble carbon, nitrogen, and unidentified fluorophores found in water extracts between young and old aged, starting MSW. Acetate and inhibitory H2 partial pressures accumulated in microcosms containing old-aged MSW. In a final experiment, repeated amendments of ZVMs to MSW in a 600 day mesocosm experiment mediated significantly higher CH4 concentrations and yields during the first of three ZVM injections. Fe0 and Mn0 experimental treatments at mesocosm-scale also highlighted accelerated development of seemingly important, but elusive Archaea including Methanobacteriaceae, a methane-producing family that is found in diverse environments. Also, prokaryotic classes including Candidatus Bathyarchaeota, an uncultured group commonly found in carbon-rich ecosystems, and Clostridia; All three taxa I identified as highly predictive in the time-dependent progression of MSW decomposition. Altogether, my experiments demonstrate the importance of H2 bioavailability on CH4 production and the consistent development of Methanobacteriaceae in productive MSW microbiomes.
ContributorsReynolds, Mark Christian (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Wang, Xuan (Committee member) / Kavazanjian, Edward (Committee member) / Arizona State University (Publisher)
Created2022
Description
Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis advisor) / Mason, Hugh (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020