Matching Items (19)
Description
Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.
ContributorsVenkatagopalan, Pavithra (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram L (Committee member) / Roberson, Robert W. (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Cancer is a heterogeneous disease with discrete oncogenic mechanisms. P53 mutation is the most common oncogenic mutation in many cancers including breast cancer. This dissertation focuses on fundamental genetic alterations enforced by p53 mutation as an indirect target. p53 mutation upregulates the mevalonate pathway genes altering cholesterol biosynthesis and prenylation. Prenylation, a lipid modification, is required for small GTPases signaling cascades. Project 1 demonstrates that prenylation inhibition can specifically target cells harboring p53 mutation resulting in reduced tumor proliferation and migration. Mutating p53 is associated with Ras and RhoA activation and statin prevents this activity by inhibiting prenylation. Ras-related pathway genes were selected from the transcriptomic analysis for evaluating correlation to statin sensitivity. A gene signature of seventeen genes and TP53 genotype (referred to as MPR signature) is generated to predict response to statins. MPR signature is validated through two datasets of drug screening in cell lines. As advancements in targeted gene modification are rising, the CRISPR-Cas9 technology has emerged as a new cancer therapeutic strategy. One of the important risk factors in gene therapy is the immune recognition of the exogenous therapeutic tool, resulting in obstruction of treatment and possibly serious health consequences. Project 2 describes a method development that can potentially improve the safety and efficacy of gene-targeting proteins. A cohort of 155 healthy individuals was screened for pre-existing B cell and T cell immune response to the S. pyogenes Cas9 protein. We detected antibodies against Cas9 in more than 10% of the healthy population and identified two immunodominant T cell epitopes of this protein. A de-immunized Cas9 that maintains the wild-type functionality was engineered by mutating the identified T cell epitopes. The gene signature and method described here have the potential to improve strategies for genome-driven tumor targeting.
ContributorsRoshdi Ferdosi, Shayesteh (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Thesis advisor) / Woodbury, Neel (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2017
Description
CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
ContributorsKrishna, Sri (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Jacobs, Bertram L (Committee member) / Lake, Douglas F (Committee member) / Arizona State University (Publisher)
Created2017
Description
Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.
ContributorsEwaisha, Radwa Mohamed Emadeldin Mahmoud (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas F (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2018
Description
Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings.
This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV.
The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use.
The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost.
The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.
This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV.
The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use.
The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost.
The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.
ContributorsObahiagbon, Uwadiae (Author) / Blain Christen, Jennifer M (Thesis advisor) / Anderson, Karen S (Committee member) / Goryll, Michael (Committee member) / Smith, Barbara S. (Committee member) / Arizona State University (Publisher)
Created2018
Description
Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.
ContributorsHarahap, Indira (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda G (Thesis advisor) / Lake, Douglas (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2015
Description
The immune system plays a dual role during neoplastic progression. It can suppress tumor growth by eliminating cancer cells, and also promote neoplastic expansion by either selecting for tumor cells that are fitter to survive in an immunocompetent host or by establishing the right conditions within the tumor microenvironment. First, I present a model to study the dynamics of subclonal evolution of cancer. I model selection through time as an epistatic process. That is, the fitness change in a given cell is not simply additive, but depends on previous mutations. Simulation studies indicate that tumors are composed of myriads of small subclones at the time of diagnosis. Because some of these rare subclones harbor pre-existing treatment-resistant mutations, they present a major challenge to precision medicine. Second, I study the question of self and non-self discrimination by the immune system, which is fundamental in the field in cancer immunology. By performing a quantitative analysis of the biochemical properties of thousands of MHC class I peptides, I find that hydrophobicity of T cell receptors contact residues is a hallmark of immunogenic epitopes. Based on these findings, I further develop a computational model to predict immunogenic epitopes which facilitate the development of T cell vaccines against pathogen and tumor antigens. Lastly, I study the effect of early detection in the context of Ebola. I develope a simple mathematical model calibrated to the transmission dynamics of Ebola virus in West Africa. My findings suggest that a strategy that focuses on early diagnosis of high-risk individuals, caregivers, and health-care workers at the pre-symptomatic stage, when combined with public health measures to improve the speed and efficacy of isolation of infectious individuals, can lead to rapid reductions in Ebola transmission.
ContributorsChowell-Puente, Diego (Author) / Castillo-Chavez, Carlos (Thesis advisor) / Anderson, Karen S (Thesis advisor) / Maley, Carlo C (Committee member) / Wilson Sayres, Melissa A (Committee member) / Blattman, Joseph N (Committee member) / Arizona State University (Publisher)
Created2016
Description
Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles.
ContributorsArndt, Ariel L (Author) / Hogue, Brenda G (Thesis advisor) / Jacobs, Bertram (Committee member) / Francisco, Wilson (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2010
Description
The innate immune system serves as an immediate response to pathogenic infection and an informant to the adaptive immune system. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase-L system is a component of the innate immune system induced by interferons (IFNs) and serves to eliminate viral infections. In humans, three enzymatically active OAS proteins exist, OAS1, OAS2, and OAS3. Recent evidence suggests variations in cellular localization of OAS proteins may influence the impact and influence of those proteins on viral replication. However, viral suppression mechanisms involving specific OAS proteins are still unclear for most viruses. Here, I overexpress different isoforms of OAS and determined that though viruses within the same family have similar replication strategies, the extent to which each OAS protein impacts viral replication for Flaviviruses, and Alphaviruses varies. In contrast to the innate immune system, the adaptive immune system provides specific and long-lived immune responses. In the context of cancer, T cells have been shown to play a prominent role in tumor regression. It has previously been demonstrated that administration α-CTLA-4/α-PD-L1 immune checkpoint blockade (ICB) to mice inoculated with a K7M2 metastatic osteosarcoma (mOS) cell line resulted in ~50% survival. Here, I sought to determine biological differences among murine responders and non-responders to ICB for mOS to understand better what factors could increase ICB efficacy. A prospective culprit is a variance in circulating antibodies (Abs). I have shown that sera from mice, before inoculation with mOS or ICB, display distinct differences in Ab repertoire between responders and non-responders, suggesting the presence or absence of particular Abs may influence the outcome of ICB. Recent studies have also shown that malleable environmental factors, such as differences in microbiome composition, can yield subsequent changes in circulating Abs.
Strong associations have been made between host-microbiome interactions and their effects on health. Here, I study potential associations of microbiome-mediated impacts on ICB efficacy for mOS. Additionally, I sought to determine potential changes in T-cellular response to mOS due to modulations in microbiome composition and showed that ICB efficacy can change in conjunction with microbiome composition changes in a murine model.
ContributorsDi Palma, Michelle Pina (Author) / Blattman, Joseph N (Thesis advisor) / Li, Yize (Thesis advisor) / Anderson, Karen S (Committee member) / McFadden, Grant (Committee member) / Arizona State University (Publisher)
Created2023
Description
Resistance to existing anti-cancer drugs poses a key challenge in the field of medical oncology, in that it results in the tumor not responding to treatment using the same medications to which it responded previously, leading to treatment failure. Adaptive therapy utilizes evolutionary principles of competitive suppression, leveraging competition between drug resistant and drug sensitive cells, to keep the population of drug resistant cells under control, thereby extending time to progression (TTP), relative to standard treatment using maximum tolerated dose (MTD). Development of adaptive therapy protocols is challenging, as it involves many parameters, and the number of parameters increase exponentially for each additional drug. Furthermore, the drugs could have a cytotoxic (killing cells directly), or a cytostatic (inhibiting cell division) mechanism of action, which could affect treatment outcome in important ways. I have implemented hybrid agent-based computational models to investigate adaptive therapy, using either a single drug (cytotoxic or cytostatic), or two drugs (cytotoxic or cytostatic), simulating three different adaptive therapy protocols for treatment using a single drug (dose modulation, intermittent, dose-skipping), and seven different treatment protocols for treatment using two drugs: three dose modulation (DM) protocols (DM Cocktail Tandem, DM Ping-Pong Alternate Every Cycle, DM Ping-Pong on Progression), and four fixed-dose (FD) protocols (FD Cocktail Intermittent, FD Ping-Pong Intermittent, FD Cocktail Dose-Skipping, FD Ping-Pong Dose-Skipping). The results indicate a Goldilocks level of drug exposure to be optimum, with both too little and too much drug having adverse effects. Adaptive therapy works best under conditions of strong cellular competition, such as high fitness costs, high replacement rates, or high turnover. Clonal competition is an important determinant of treatment outcome, and as such treatment using two drugs leads to more favorable outcome than treatment using a single drug. Switching drugs every treatment cycle (ping-pong) protocols work particularly well, as well as cocktail dose modulation, particularly when it is feasible to have a highly sensitive measurement of tumor burden. In general, overtreating seems to have adverse survival outcome, and triggering a treatment vacation, or stopping treatment sooner when the tumor is shrinking seems to work well.
ContributorsSaha, Kaushik (Author) / Maley, Carlo C (Thesis advisor) / Forrest, Stephanie (Committee member) / Anderson, Karen S (Committee member) / Cisneros, Luis H (Committee member) / Arizona State University (Publisher)
Created2023