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- Genre: Academic theses
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
Surface plasmon resonance (SPR) has emerged as a popular technique for elucidating subtle signals from biological events in a label-free, high throughput environment. The efficacy of conventional SPR sensors, whose signals are mass-sensitive, diminishes rapidly with the size of the observed target molecules. The following work advances the current SPR sensor paradigm for the purpose of small molecule detection. The detection limits of two orthogonal components of SPR measurement are targeted: speed and sensitivity. In the context of this report, speed refers to the dynamic range of measured kinetic rate constants, while sensitivity refers to the target molecule mass limitation of conventional SPR measurement. A simple device for high-speed microfluidic delivery of liquid samples to a sensor surface is presented to address the temporal limitations of conventional SPR measurement. The time scale of buffer/sample switching is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (kd) up to 130 s-1. The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement. Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. The SPR response to an applied ac potential is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.
ContributorsMacGriff, Christopher Assiff (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / LaBaer, Joshua (Committee member) / Chae, Junseok (Committee member) / Arizona State University (Publisher)
Created2013
Description
This dissertation investigates the condition of skeletal muscle insulin resistance using bioinformatics and computational biology approaches. Drawing from several studies and numerous data sources, I have attempted to uncover molecular mechanisms at multiple levels. From the detailed atomistic simulations of a single protein, to datamining approaches applied at the systems biology level, I provide new targets to explore for the research community. Furthermore I present a new online web resource that unifies various bioinformatics databases to enable discovery of relevant features in 3D protein structures.
ContributorsMielke, Clinton (Author) / Mandarino, Lawrence (Committee member) / LaBaer, Joshua (Committee member) / Magee, D. Mitchell (Committee member) / Dinu, Valentin (Committee member) / Willis, Wayne (Committee member) / Arizona State University (Publisher)
Created2013
Description
Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology.
ContributorsFlores, Julia Anne (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2012
Description
Ebola hemorrhagic fever (EHF) is a severe and often fatal disease in human and nonhuman primates, caused by the Ebola virus. Approximately 30 years after the first epidemic, there is no vaccine or therapeutic medication approved to counter the Ebola virus. In this dissertation, a geminiviral replicon system was used to produce Ebola immune complex (EIC) in plant leaves and tested it as an Ebola vaccine. The EIC was produced in Nicotiana benthamiana leaves by fusing Ebola virus glycoprotein (GP1) to the C-terminus of heavy chain of 6D8 monoclonal antibody (mAb), which is specific to the 6D8 epitope of GP1, and co-expressing the fusion with the light chain of 6D8 mAb. EIC was purified by ammonium sulfate precipitation and protein A or protein G affinity chromatography. EIC was shown to be immunogenic in mice, but the level of antibody against Ebola virus was not sufficient to protect the mice from lethal the Ebola challenge. Hence, different adjuvants were tested in order to improve the immunogenicity of the EIC. Among several adjuvants that we used, Poly(I:C), which is a synthetic analog of double-stranded ribonucleic acid that can interact with a Toll-like receptor 3, strongly increased the efficacy of our Ebola vaccine. The mice immunized with EIC co-administered with Poly(I:C) produced high levels of neutralizing anti-Ebola IgG, and 80% of the mice were protected from the lethal Ebola virus challenge. Moreover, the EIC induced a predominant T-helper type 1 (Th1) response, whereas Poly(I:C) co-delivered with the EIC stimulated a mixed Th1/Th2 response. This result suggests that the protection against lethal Ebola challenge requires both Th1 and Th2 responses. In conclusion, this study demonstrated that the plant-produced EIC co-delivered with Poly(I:C) induced strong and protective immune responses to the Ebola virus in mice. These results support plant-produced EIC as a good vaccine candidate against the Ebola virus. It should be pursued further in primate studies, and eventually in clinical trials.
ContributorsPhoolcharoen, Waranyoo (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Thesis advisor) / Arntzen, Charles J. (Committee member) / Change, Yung (Committee member) / Ma, Julian (Committee member) / Arizona State University (Publisher)
Created2010
Description
Vaccines are one of the most effective ways of combating infectious diseases and developing vaccine platforms that can be used to produce vaccines can greatly assist in combating global public health threats. This dissertation focuses on the development and pre-clinical testing of vaccine platforms that are highly immunogenic, easily modifiable, economically viable to produce, and stable. These criteria are met by the recombinant immune complex (RIC) universal vaccine platform when produced in plants. The RIC platform is modeled after naturally occurring immune complexes that form when an antibody, a component of the immune system that recognizes protein structures or sequences, binds to its specific antigen, a molecule that causes an immune response. In the RIC platform, a well-characterized antibody is linked via its heavy chain, to an antigen tagged with the antibody-specific epitope. The RIC antibody binds to the epitope tags on other RIC molecules and forms highly immunogenic complexes. My research has primarily focused on the optimization of the RIC platform. First, I altered the RIC platform to enable an N-terminal antigenic fusion instead of the previous C-terminal fusion strategy. This allowed the platform to be used with antigens that require an accessible N-terminus. A mouse immunization study with a model antigen showed that the fusion location, either N-terminal or C-terminal, did not impact the immune response. Next, I studied a synergistic response that was seen upon co-delivery of RIC with virus-like particles (VLP) and showed that the synergistic response could be produced with either N-terminal or C-terminal RIC co-delivered with VLP. Since RICs are inherently insoluble due to their ability to form complexes, I also examined ways to increase RIC solubility by characterizing a panel of modified RICs and antibody-fusions. The outcome was the identification of a modified RIC that had increased solubility while retaining high immunogenicity. Finally, I modified the RIC platform to contain multiple antigenic insertion sites and explored the use of bioinformatic tools to guide the design of a broadly protective vaccine.
ContributorsPardhe, Mary (Author) / Mason, Hugh S (Thesis advisor) / Chen, Qiang (Committee member) / Mor, Tsafrir (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2021
Description
The TP53 tumor suppressor gene is the most frequently mutated gene in human cancers. In the highly aggressive triple negative breast cancer (TNBC), TP53 is mutated in 80% of cases. TNBC lacks viable drug targets, resulting in a low prognosis (12.2% 5 year survivability rate). As such, the discovery of druggable targets in TNBC would be beneficial. Mutated p53 protein typically occurs as a missense mutation and often endows cancer cells with gain of function (GOF) properties by dysregulating metabolic pathways. One of these frequently dysregulated pathways is the Hippo/Yes-associated protein-1 (YAP1)/WW Domain Containing Transcription Regulator 1 (TAZ) tumor suppressor pathway. This study therefore analyzed the involvement of the Hippo/YAP1/TAZ pathway in p53-mediated breast cancer cell invasion. From an RNA-seq screen in MCF10A cell lines harboring different TP53 missense mutations, each with a differing invasive phenotype, components of the Hippo pathway were found to correlate with cell invasion. To this end, the active and inactive forms of YAP1 and TAZ were studied. Phosphorylated (inactive) YAP1 and TAZ are retained in the cytoplasm and eventually degraded. Unphosphorylated (active) YAP1 and TAZ translocate to the nucleus to activate TEAD-family transcription factors, inducing cell survival and proliferation genes leading to increased cell invasion. Using quantitative western blot analysis, it was found that inactive TAZ expression was lower in the most invasive cell lines and higher in the least invasive cell lines (p = 0.003). Moreover, the ratio of inactive TAZ protein to total TAZ protein was also shown to be predominantly lower in the invasive cell lines compared to the non-invasive lines (p = 0.04). Finally, active TAZ expression was primarily higher in p53-mutant invasive cell lines and lower in non-invasive p53 mutant cells. Additionally, although YAP1 and TAZ are thought to be functionally redundant, the pattern seen in TAZ was not seen in the YAP1 protein. Taken together, the results demonstrated here suggest that TAZ holds a more dominant role in governing TNBC cell invasion compared to YAP1 and further highlights TAZ as a potential therapeutic target in TNBC.
ContributorsGrief, Dustin (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen (Committee member) / Nikkhah, Mehdi (Committee member) / Arizona State University (Publisher)
Created2022
Description
Glioblastoma (GBM), the most common and aggressive primary brain tumor affecting adults, is characterized by an aberrant yet druggable epigenetic landscape. The Histone Deacetylases (HDACs), a major family of epigenetic regulators, favor transcriptional repression by mediating chromatin compaction and are frequently overexpressed in human cancers, including GBM. Hence, over the last decade there has been considerable interest in using HDAC inhibitors (HDACi) for the treatment of malignant primary brain tumors. However, to date most HDACi tested in clinical trials have failed to provide significant therapeutic benefit to patients with GBM. This is because current HDACi have poor or unknown pharmacokinetic profiles, lack selectivity towards the different HDAC isoforms, and have narrow therapeutic windows. Isoform selectivity for HDACi is important given that broad inhibition of all HDACs results in widespread toxicity across different organs. Moreover, the functional roles of individual HDAC isoforms in GBM are still not well understood. Here, I demonstrate that HDAC1 expression increases with brain tumor grade and is correlated with decreased survival in GBM. I find that HDAC1 is the essential HDAC isoform in glioma stem cells and its loss is not compensated for by its paralogue HDAC2 or other members of the HDAC family. Loss of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner and leads to significant suppression of tumor growth in vivo. While no HDAC isoform-selective inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. I show that quisinostat exhibits potent growth inhibition in multiple patient-derived glioma stem cells. Using a pharmacokinetics- and pharmacodynamics-driven approach, I demonstrate that quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM and significantly extends survival both alone and in combination with radiotherapy. The work presented in this thesis thereby unveils the non-redundant functions of HDAC1 in therapy- resistant glioma stem cells and identifies a brain-penetrant HDACi with higher selectivity towards HDAC1 as a potent radiosensitizer in preclinical models of GBM. Together, these results provide a rationale for developing quisinostat as a potential adjuvant therapy for the treatment of GBM.
ContributorsLo Cascio, Costanza (Author) / LaBaer, Joshua (Thesis advisor) / Mehta, Shwetal (Committee member) / Mirzadeh, Zaman (Committee member) / Mangone, Marco (Committee member) / Paek, Andrew (Committee member) / Arizona State University (Publisher)
Created2022
Description
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) that emerged from a zoonotic host at the end of 2019 and caused a public health crisis. In this collection of studies, Nicotiana benthamiana plants are used to set the foundation for producing monoclonal antibodies (mAbs) with homogeneous glycosylation to neutralize SARS-CoV-2 and potentially address the immunopathology often observed with severe COVID-19. Specifically, a mAb against the human interleukin (IL)-6 receptor (sarilumab) was generated and evaluated in vitro for its potential to reduce IL-6 signaling that has been shown to be associated with more severe cases of COVID-19. Furthermore, multiple mAbs that bind to the receptor-binding domain (RBD) of SARS-CoV-2 and efficiently neutralize the virus were developed using plant-based expression. Several of these mAbs are from different classes of RBD-binding mAbs that have distinct binding sites from one another. Several mAbs from different classes showed synergy in neutralizing the ancestral strain of SARS-CoV-2 and a smaller subset showed synergy when tested against the highly mutated Omicron (B.1.1.529) variant. Of interest, a novel RBD-binding mAb, termed 11D7, that was raised against the ancestral strain and derived from a hybridoma, appears to have an epitope on the RBD that contributes more synergy to a mAb combination that efficiently neutralizes the B.1.1.529 variant of SARS-CoV-2. This epitope was partially mapped by competitive binding and shows that it overlaps with another known antibody that binds a cryptic, distal epitope, away from the receptor binding site, giving insight into the potential mechanism by which 11D7 neutralizes SARS-CoV-2, as well as potentially allowing it to resist SARS-CoV-2 immune evasion more efficiently. Furthermore, this mAb carries a highly homogeneous glycan pattern when expressed in N. benthamiana, that may contribute to enhanced effector function and provides a tool to elucidate the precise role of crystallizable fragment (Fc)-mediated protection in SARS-CoV-2 infection. Ultimately, these studies provide evidence of the utility of plant-made mAbs to be used as cocktail members, giving clarity to the use of less potent mAbs as valuable cocktail components which will spur further investigations into how mAbs with unique epitopes work together to efficiently neutralize SARS-CoV-2.
ContributorsJugler, Collin (Author) / Chen, Qiang (Thesis advisor) / Lake, Douglas (Committee member) / Steele, Kelly (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2022
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023