Matching Items (136)
Description
The pathogenesis of type 1 diabetes (T1D) is still not fully understood in the scientific community. Evidence has shown that viral infections are one of the important environmental factors associated with the disease development. Seven of the top T1D related viruses were selected to study the prevalence of viral humoral response in T1D patients using our innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). In this study, each viral gene was individually captured using various PCR based techniques, cloned into a protein expression vector, and assembled as the first version of T1D viral protein array. Humoral responses of IgG, IgA, and IgM were examined. Although each class of immunoglobulin generated a wide-range of reactivity, responses to various viral proteins from different proteins were observed. In summary, we captured most of the T1D related viral genes, established viral protein expression on the protein array, and displayed the serum response on the viral protein array. The successful progress will help to fulfill the long term goal of testing the viral infection hypothesis in T1D development.
ContributorsDavis, Amy Darlene (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Desi, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
AMPylation is a post-translation modification that has an important role in the survival of many bacterial pathogens by affecting the host cell's molecular signaling. In the course of studying this intercellular manipulation, there has only been modest progression in the identification of the enzymes with AMPylation capabilities (AMPylators) and their respective targets. The reason for these minimal developments is the inability to analyze a large subset of these proteins. Therefore, to increase the efficiency of the identification and characterization of the proteins, Yu et al developed a high-throughput non-radioactive discovery platform using Human Nucleic Acid Programmable Protein Arrays (NAPPA) and a validation platform using bead-based assays. The large-scale unbiased screening of potential substrates for two bacterial AMPylators containing Fic domain, VopS and IbpAFic2, had been performed and dozens of novel substrates were identified and confirmed. With the efficiency of this method, the platform was extended to the identification of novel substrates for a Legionella virulence factor, SidM, containing a different adenylyl transferase domain. The screening was performed using NAPPA arrays comprising of 10,000 human proteins, the active AMPylator SidM, and its inactive D110/112A mutant as a negative control. Many potential substrates of SidM were found, including Rab GTPases and non-GTPase proteins. Several of which have been confirmed with the bead-based AMPylation assays.
ContributorsGraves, Morgan C. (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Yu, Xiaobo (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Dental caries also known as tooth decay is a bacterial infection that causes demineralization and destruction of enamel dentin and cementum in the tooth. This bacterium, Streprococcus mutans, feeds on the carbohydrates in the mouth and produces lactic acids that result in dental caries. This thesis discusses the use of plants to produce antibodies, Guy 13 and anti-GTFB to treat this dental disease. We believe these plant-derived antibodies will be effective to treat dental caries and economical to produce.
ContributorsSayegh, Luvenia Crystal (Author) / Chen, Qiang (Thesis director) / Garg, Vikas (Committee member) / Barrett, The Honors College (Contributor) / School of Letters and Sciences (Contributor)
Created2014-12
Description
The purpose of this project was to identify proteins associated with the migration and invasion of non-transformed MCF10A mammary epithelial cells with ectopically expressed missense mutations in p53. Because of the prevalence of TP53 missense mutations in basal-like and triple-negative breast cancer tumors, understanding the effect of TP53 mutations on the phenotypic expression of human mammary epithelial cells may offer new therapeutic targets for those currently lacking in treatment options. As such, MCF10A mammary epithelial cells ectopically overexpressing structural mutations (G245S, H179R, R175H, Y163C, Y220C, and Y234C) and DNA-binding mutations (R248Q, R248W, R273C, and R273H) in the DNA-binding domain were selected for use in this project. Overexpression of p53 in the mutant cell lines was confirmed by western blot and q-PCR analysis targeting the V5 epitope tag present in the pLenti4 vector used to transduce TP53 into the mutant cell lines. Characterization of the invasion and migration phenotypes resulting from the overexpression of p53 in the mutant cell lines was achieved using transwell invasion and migration assays with Boyden chambers. Statistical analysis showed that three cell lines—DNA-contact mutants R248W and R273C and structural mutant Y220C—were consistently more migratory and invasive and demonstrated a relationship between the migration and invasion properties of the mutant cell lines. Two families of proteins were then explored: those involved in the Epithelial-Mesenchymal Transition (EMT) and matrix metalloproteinases (MMPs). Results of q-PCR and immunofluorescence analysis of epithelial marker E-cadherin and mesenchymal proteins Slug and Vimentin did not show a clear relationship between mRNA and protein expression levels with the migration and invasiveness phenotypes observed in the transwell studies. Results of western blotting, q-PCR, and zymography of MMP-2 and MMP-9 also did not show any consistent results indicating a definite relationship between MMPs and the overall invasiveness of the cells. Finally, two drugs were tested as possible treatments inhibiting invasiveness: ebselen and SBI-183. These drugs were tested on only the most invasive of the MCF10A p53 mutant cell lines (R248W, R273C, and Y220C). Results of invasion assay following 30 μM treatment with ebselen and SBI-183 showed that ebselen does not inhibit invasiveness; SBI-183, however, did inhibit invasiveness in all three cell lines tested. As such, SBI-183 will be an important compound to study in the future as a treatment that could potentially serve to benefit triple-negative or basal-like breast cancer patients who currently lack therapeutic treatment options.
ContributorsZhang, Kathie Q (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
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
Coronavirus disease 2019 (COVID-19), an illness caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
coronavirus 2 (SARS-CoV-2), has been responsible for significant social and economic
disruption, prompting an urgent search for therapeutic solutions. The spike protein of the virus
has been examined as an immunogenic target because of its role in viral binding and fusion
necessary for infection of host cells. Previous studies have identified a recombinant protein
(denoted as S1) that has been shown to potentially induce a neutralizing antibody response by
mimicking the structure of the SARS-CoV-2 spike protein. We have produced the S1 in plants
using agroinfiltration, a plant transformation technique whereby plasmid-containing
Agrobacterium tumefaciens is injected into Nicotiana benthamiana plants, resulting in transfer of
the desired gene from bacteria to plant cells. S1 was expressed to high levels within 5 days of
infiltration, and Western blot analysis showed recognition of the S1 by an anti-S1 antibody.
ELISA results exhibited increased binding activity to anti-S1 with increasing concentrations of
S1, indicating their specific interaction. This ongoing study will demonstrate the potential of a
plant-produced S1 as a vaccine, therapeutic, and diagnostic tool against COVID-19 that is not
only effective, but also cost-efficient and scalable in comparison to conventional mammalian cell
culture production methods.
ContributorsNguyen, Katherine (Author) / Chen, Qiang (Thesis director) / Ghirlanda, Giovanna (Committee member) / Jugler, Collin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12
Description
The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).
Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
ContributorsGong, Zhen (Author) / Martin Garcia, Jose Manuel (Author) / Daskalova, Sasha (Author) / Craciunescu, Felicia (Author) / Song, Lusheng (Author) / Dorner, Katerina (Author) / Hansen, Debra (Author) / Yang, Jay-How (Author) / LaBaer, Joshua (Author) / Hogue, Brenda (Author) / Mor, Tsafrir (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Innovations in Medicine (Contributor) / Personalized Diagnostics (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-08-21
Description
Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides (Ilp1 and 2) and manganese superoxide dismutase (mnSOD). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior (llp1 and 2) and genetic background (Ilp2), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD, suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown.
ContributorsIhle, Kate (Author) / Fondrk, M. Kim (Author) / Page, Robert (Author) / Amdam, Gro (Author) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-01-01
Description
Plant viral vectors have previously been used to produce high expression levels of antibodies and other proteins of interest. By utilizing a transformed Agrobacterium with the vector containing the protein of interest for infiltration, viral vectors can easily reach the plant cells making it an effective form of transient protein expression. For this project two different plant viral vectors were compared; the geminiviral vector derived from Bean yellow dwarf virus (BeYDV) and the MagnICON vector system derived from Tobacco Mosaic Virus(TMV) and Potato Virus X(PVX). E16, an antibody against West Nile virus, has previously been expressed using both systems but expression levels between the systems were not directly compared. Agrobacterium tumefaciens EHA105 cells were transformed with both systems and expression levels of E16 were quantified using ELISAs. Results showed very low expression levels of E16 using the geminiviral vector indicating a need for further investigation into the clone used as previous studies reported much higher expression levels with the system.
ContributorsMurphy, Skylar (Author) / Chen, Qiang (Thesis director) / Jugler, Collin (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05