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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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
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
Description
The intracellular motility seen in the cytoplasm of angiosperm plant pollen tubes is known as reverse fountain cytoplasmic streaming (i.e., cyclosis). This effect occurs when organelles move anterograde along the cortex of the cell and retrograde down the center of the cell. The result is a displacement of cytoplasmic volume causing a cyclic motion of organelles and bulk liquid. Visually, the organelles appear to be traveling in a backwards fountain hence the name. The use of light microscopy bioimaging in this study has documented reverse fountain cytoplasmic streaming for the first time in fungal hyphae of Rhizopus oryzae and other members in the order Mucorales (Mucoromycota). This is a unique characteristic of the mucoralean fungi, with other fungal phyla (e.g., Ascomycota, Basidiomycota) exhibiting unidirectional cytoplasmic behavior that lacks rhythmic streaming (i.e., sleeve-like streaming). The mechanism of reverse fountain cytoplasmic streaming in filamentous fungi is currently unknown. However, in angiosperm plant pollen tubes it’s correlated with the arrangement and activity of the actin cytoskeleton. Thus, the current work assumes that filamentous actin and associated proteins are directly involved with the cytoplasmic behavior in Mucorales hyphae. From an evolutionary perspective, fungi in the Mucorales may have developed reverse fountain cytoplasmic streaming as a method to transport various organelles over long and short distances. In addition, the mechanism is likely to facilitate driving of polarized hyphal growth.
ContributorsShange, Phakade Mdima (Author) / Roberson, Robert W. (Thesis advisor) / Gile, Gillian (Committee member) / Baluch, Debra (Committee member) / Arizona State University (Publisher)
Created2020
Description
Lipolysis or hydrolysis of triglyceride (TG) stored within intracellular lipid droplets (LD), is vital to maintaining metabolic homeostasis in mammals. Regulation of lipolysis and subsequent utilization of liberated fatty acids impacts cellular and organismal functions including body fat accumulation and thermogenesis. Adipose triglyceride lipase (ATGL) is the intracellular rate-limiting enzyme responsible for catalyzing hydrolysis of TG to diacylglycerol (DAG), the initial step of the lipolytic reaction. G0/G1 switch gene-2 (G0S2) and hypoxia-inducible gene-2 (HIG2) are selective inhibitors of ATGL. G0S2 facilitates accumulation of TG in the liver and adipose tissue, while HIG2 functions under hypoxic conditions. Sequence analysis and mutagenesis were used to confirm the presence of conserved domains between these proteins, and that these domains are required for efficient binding and inhibition of ATGL. Further analysis revealed a Positive sequence (Pos-Seq)-LD binding motif in G0S2 but not HIG2. The Pos-Seq mediated ATGL-independent localization to LD and was required for achieving maximal inhibition of ATGL activity by G0S2. Identification and mutational analysis of this motif revealed distinct mechanisms for HIG2 and G0S2 LD association. In addition to molecular characterization of known protein inhibitors of lipolysis, an intracellular member of the apolipoprotein L (ApoL) family, ApoL6, was also identified as a LD and mitochondria associated protein expressed in adipose tissue. Brown adipose tissue uses fatty acids as fuel for increasing its energy output as heat during acute responses to cold exposure. A Comprehensive Lab Animal Monitoring System was used to compare heat production at room temperature (RT) and 4oC in transgenic animals overexpressing ApoL6 in brown adipose tissue. Overexpression of ApoL6 delayed utilization of long-chain fatty acids (LCFAs) as a fuel source while promoting an enhanced thermogenic response during initial cold exposure. ApoL6 mediated inhibition of LCFA utilization results from binding of ApoL6 to Mitochondrial Trifunctional Protein (MTP/TFP), which catalyzes mitochondrial β-oxidation. Indirect calorimetry and fasting acute cold exposure experiments suggest the augmented thermogenic profile of ApoL6 transgenic animals is a result of enhanced utilization of medium-chain fatty acids (MCFAs), glucose, and amino acids as fuel sources. Cumulatively these results indicate multiple mechanisms for regulation lipolysis and fatty acid utilization.
ContributorsCampbell, Latoya E (Author) / Lake, Douglas (Thesis advisor) / Liu, Jun (Committee member) / Folmes, Clifford (Committee member) / Sweazea, Karen (Committee member) / Baluch, Debra (Committee member) / Arizona State University (Publisher)
Created2021