Matching Items (13)
Description
Since the molecular biology revolution in the 1980s, ease of gene editing had led to the resurgence of Oncolytic Virotherapy. Countless viruses have been engineered yet only three are approved for clinical use worldwide, with only one being approved by the U.S Food and Drug Administration (FDA). Vaccinia virus (VACV) has a large genome, contains many immune evasion genes and has been thoroughly studied, making it a popular candidate for an oncolytic platform. VACV mutants with deletions in the E3 immune evasion protein have been shown to have oncolytic efficacy but the mechanism of tumor selectivity has not been fully elucidated. These mutants have been shown to be regulated by the necroptosis pathway, a pathway that has been shown to be deficient in certain cancers. Using a pan-cancer screening method that combines dye exclusion assays, western blot analysis, and viral growth curve, the role of necroptosis in regulating VACV replication and oncolytic efficacy in cancer was further characterized. Results demonstrate a preliminary correlation between necroptosis, viral replication, and oncolytic efficacy. This correlation is clearest in breast cancer and melanomas yet may apply to other cancer subgroups. This data was also used to guide the development of a receptor-interacting protein kinase 3 (RIP3) matched pair mouse model in the E0771 mouse breast cancer line which can be used to further study the role of necroptosis and oncolytic efficacy in vivo. Understanding the contribution necroptosis plays in oncolytic efficacy can guide to design enhance the design of clinical trials to test VACV E3L mutants and may lead to better efficacy in humans and an improvement in clinical oncology.
ContributorsKasimsetty, Aradhana (Author) / Jacobs, Bertram L (Thesis advisor) / McFadden, Douglas G (Committee member) / Borad, Mitesh (Committee member) / Arizona State University (Publisher)
Created2020
Description
Environmental stressors can perturb cellular homeostasis. Cells activate an integrated stress response that will alleviate the effects of the ongoing stress. Stress-activated protein kinases function to phosphorylate the eukaryotic translation initiation factor, eIF2α, which results in inhibition of translation of house-keeping genes. Following these events, formation of cytoplasmic messenger ribonucleoprotein complexes, known as stress granules, will take place. Stress granules typically have a pro-survival function. These studies demonstrate that assembly of stress granules can also lead to necroptosis. Necroptosis is a caspase-independent, receptor-interacting protein kinase 3 (RIPK3)-dependent cell death pathway executed by mixed lineage kinase domain-like (MLKL) protein. Cellular stress is induced using arsenite (oxidative stress) or by infection with vaccinia virus (VACV) E3 protein Z-DNA-binding domain mutant, VACV-E3LΔ83N. In both cases, RIPK3-dependent death was observed in interferon (IFN)-primed L929 cells. This death led to phosphorylation and trimerization of MLKL, indicative of necroptosis. Necroptosis induced by oxidative stress and VACV-E3LΔ83N infection was dependent on the host Z-form nucleic acid sensor, DNA-dependent activator of IFN-regulatory factors (DAI), as it was inhibited in DAI-deficient L929 cells. Under both cellular stresses, DAI associated with RIPK3 and formed high-molecular-weight complexes, consistent with formation of the necrosomes. DAI localized into stress granules during necroptosis induced by arsenite and the mutant virus, and the necrosomes formed only in presence of stress granule assembly. The significance of stress granules for cellular stress-induced necroptosis was demonstrated using knock-out (KO) cell lines unable to form granules: T cell-restricted intracellular antigen 1 (TIA-1) KO MEF cells and Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1/2) KO U2OS cells. Necroptosis was inhibited in absence of stress granule formation as no cell death or activation of MLKL was observed in the knock-out cell lines following arsenite treatment or VACV-E3LΔ83N infection. Furthermore, wild-type VACV was able to inhibit stress granule assembly, which coincided with the virus ability to inhibit necroptosis. These studies have led to a model of Z-form nucleic acids being involved in activation of the stress granule-mediated necroptosis following induction by environmental stressors. These results have significance for understanding the etiology of human diseases and the antiviral innate immunity.
ContributorsSzczerba, Mateusz Bartlomiej (Author) / Jacobs, Bertram L (Thesis advisor) / Langland, Jeffrey (Committee member) / Lake, Douglas (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2021
Description
In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules has significant implications for all aspects of research spanning the basic understanding of biomolecules to the development of new therapeutics. Presented here are four projects that highlight the ability to select for and characterize functional biomolecules through in vitro selection.
Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents.
Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells.
Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis.
Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers.
Chapter one outlines the development of a new characterization tool for in vitro selected binding peptides. The approach enables rapid screening of peptide candidates in small sample volumes using cell-free translated peptides. This strategy has the potential to accelerate the pace of peptide characterization and help advance the development of peptide-based affinity reagents.
Chapter two details an in vitro selection strategy for searching entire genomes for RNA sequences that enhance cap-independent initiation of translation. A pool of sequences derived from the human genome was enriched for members that function to enhance the translation of a downstream coding region. Thousands of translation enhancing elements from the human genome are identified and the function of a subset is validated in vitro and in cells.
Chapter three discusses the characterization of a translation enhancing element that promotes rapid and high transgene expression in mammalian cells. Using this ribonucleic acid sequence, a series of full length human proteins is expressed in a matter of only hours. This advance provides a versatile platform for protein synthesis and is espcially useful in situations where prokaryotic and cell-free systems fail to produce protein or when post-translationally modified protein is essential for biological analysis.
Chapter four outlines a new selection strategy for the identification of novel polymerases using emulsion droplet microfluidics technology. With the aid of a fluorescence-based activity assay, libraries of polymerase variants are assayed in picoliter sized droplets to select for variants with improved function. Using this strategy a variant of the 9°N DNA polymerase is identified that displays an enhanced ability to synthesize threose nucleic acid polymers.
ContributorsLarsen, Andrew Carl (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram L (Committee member) / Karr, Timothy L. (Committee member) / Arizona State University (Publisher)
Created2015