Matching Items (141)
Description
Disturbances in the protein interactome often play a large role in cancer progression. Investigation of protein-protein interactions (PPI) can increase our understanding of cancer pathways and will disclose unknown targets involved in cancer disease biology. Although numerous methods are available to study protein interactions, most platforms suffer from drawbacks including high false positive rates, low throughput, and lack of quantification. Moreover, most methods are not compatible for use in a clinical setting. To address these limitations, we have developed a multiplexed, in-solution protein microarray (MISPA) platform with broad applications in proteomics. MISPA can be used to quantitatively profile PPIs and as a robust technology for early detection of cancers. This method utilizes unique DNA barcoding of individual proteins coupled with next generation sequencing to quantitatively assess interactions via barcode enrichment. We have tested the feasibility of this technology in the detection of patient immune responses to oropharyngeal carcinomas and in the discovery of novel PPIs in the B-cell receptor (BCR) pathway. To achieve this goal, 96 human papillomavirus (HPV) antigen genes were cloned into pJFT7-cHalo (99% success) and pJFT7-n3xFlag-Halo (100% success) expression vectors. These libraries were expressed via a cell-free in vitro transcription-translation system with 93% and 96% success, respectively. A small-scale study of patient serum interactions with barcoded HPV16 antigens was performed and a HPV proteome-wide study will follow using additional patient samples. In addition, 15 query proteins were cloned into pJFT7_nGST expression vectors, expressed, and purified with 93% success to probe a library of 100 BCR pathway proteins and detect novel PPIs.
ContributorsRinaldi, Capria Lakshmi (Author) / LaBaer, Joshua (Thesis director) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
ABSTRACT Peptide microarrays may prove to be a powerful tool for proteomics research and clinical diagnosis applications. Fodor et al. and Maurer et al. have shown proof-of-concept methods of light- and electrochemically-directed peptide microarray fabrication on glass and semiconductor microchips respectively. In this work, peptide microarray fabrication based on the abovementioned techniques were optimized. In addition, MALDI mass spectrometry based peptide synthesis characterization on semiconductor microchips was developed and novel applications of a CombiMatrix (CBMX) platform for electrochemically controlled synthesis were explored. We have investigated performance of 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) derivatives as photo-labile protecting group. Specifically, influence of substituents on 4 and 5 positions of phenyl ring of NPPOC group on the rate of photolysis and the yield of the amine was investigated. The results indicated that substituents capable of forming a π-network with the nitro group enhanced the rate of photolysis and yield. Once such properly substituted NPPOC groups were used, the rate of photolysis/yield depended on the nature of protected amino group indicating that a different chemical step during the photo-cleavage process became the rate limiting step. We also focused on electrochemically-directed parallel synthesis of high-density peptide microarrays using the CBMX technology referred to above which uses electrochemically generated acids to perform patterned chemistry. Several issues related to peptide synthesis on the CBMX platform were studied and optimized, with emphasis placed on the reactions of electro-generated acids during the deprotection step of peptide synthesis. We have developed a MALDI mass spectrometry based method to determine the chemical composition of microarray synthesis, directly on the feature. This method utilizes non-diffusional chemical cleavage from the surface, thereby making the chemical characterization of high-density microarray features simple, accurate, and amenable to high-throughput. CBMX Corp. has developed a microarray reader which is based on electro-chemical detection of redox chemical species. Several parameters of the instrument were studied and optimized and novel redox applications of peptide microarrays on CBMX platform were also investigated using the instrument. These include (i) a search of metal binding catalytic peptides to reduce overpotential associated with water oxidation reaction and (ii) an immobilization of peptide microarrays using electro-polymerized polypyrrole.
ContributorsKumar, Pallav (Author) / Woodbury, Neal (Thesis advisor) / Allen, James (Committee member) / Johnston, Stephen (Committee member) / Arizona State University (Publisher)
Created2013
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
Computational models have long been used to describe and predict the outcome of complex immunological processes. The dissertation work described here centers on the construction of multiscale computational immunology models that derives biological insights at the population, systems, and atomistic levels. First, SARS-CoV-2 mortality is investigated through the lens of the predicted robustness of CD8+ T cell responses in 23 different populations. The robustness of CD8+ T cell responses in a given population was modeled by predicting the efficiency of endemic MHC-I protein variants to present peptides derived from SARS-CoV-2 proteins to circulating T cells. To accomplish this task, an algorithm, called EnsembleMHC, was developed to predict viral peptides with a high probability of being recognized by CD T cells. It was discovered that there was significant variation in the efficiency of different MHC-I protein variants to present SARS-CoV-2 derived peptides, and countries enriched with variants with high presentation efficiency had significantly lower mortality rates. Second, a biophysics-based MHC-I peptide prediction algorithm was developed. The MHC-I protein is the most polymorphic protein in the human genome with polymorphisms in the peptide binding causing striking changes in the amino acid compositions, or binding motifs, of peptide species capable of stable binding. A deep learning model, coined HLA-Inception, was trained to predict peptide binding using only biophysical properties, namely electrostatic potential. HLA-Inception was shown to be extremely accurate and efficient at predicting peptide binding motifs and was used to determine the peptide binding motifs of 5,821 MHC-I protein variants. Finally, the impact of stalk glycosylations on NL63 protein dynamics was investigated. Previous data has shown that coronavirus crown glycans play an important role in immune evasion and receptor binding, however, little is known about the role of the stalk glycans. Through the integration of computational biology, experimental data, and physics-based simulations, the stalk glycans were shown to heavily influence the bending angle of spike protein, with a particular emphasis on the glycan at position 1242. Further investigation revealed that removal of the N1242 glycan significantly reduced infectivity, highlighting a new potential therapeutic target. Overall, these investigations and associated innovations in integrative modeling.
ContributorsWilson, Eric Andrew (Author) / Anderson, Karen (Thesis advisor) / Singharoy, Abhishek (Thesis advisor) / Woodbury, Neal (Committee member) / Sulc, Petr (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
Description
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, declared in March 2020 resulted in an unprecedented scientific effort that led to the deployment in less than a year of several vaccines to prevent severe disease, hospitalizations, and death from coronavirus disease 2019 (COVID-19). Most vaccine models focus on the production of neutralizing antibodies against the spike (S) to prevent infection. As the virus evolves, new variants emerge that evade neutralizing antibodies produced by natural infection and vaccination, while memory T cell responses are long-lasting and resilient to most of the changes found in variants of concern (VOC). Several lines of evidence support the study of T cell-mediated immunity in SARS-CoV-2 infections. First, T cell reactivity against SARS-CoV-2 is found in both (cluster of differentiation) CD4+ and CD8+ T cell compartments in asymptomatic, mild, and severe recovered COVID-19 patients. Second, an early and stronger CD8+ T cell response correlates with less severe COVID-19 disease [1-4]. Third, both CD4+ and CD8+ T cells that are reactive to SARS-CoV-2 viral antigens are found in healthy unexposed individuals suggesting that cross-reactive and conserved epitopes may be protective against infection.
The current study is focused on the T cell-mediated response, with special attention to conserved, non-spike-cross-reactive epitopes that may be protective against SARS-CoV-2. The first chapter reviews the importance of epitope prediction in understanding the T cell-mediated responses to a pathogen. The second chapter centers on the validation of SARS-CoV-2 CD8+ T cell predicted peptides to find conserved, immunodominant, and immunoprevalent epitopes that can be incorporated into the next generation of vaccines against severe COVID-19 disease. The third chapter explores pre-existing immunity to SARS-CoV-2 in a pre-pandemic cohort and finds two highly immunogenic epitopes that are conserved among human common cold coronaviruses (HCoVs). To end, the fourth chapter explores the concept of T cell receptor (TCR) cross-reactivity by isolating SARS-CoV-2-reactive TCRs to elucidate the mechanisms of cross-reactivity to SARS-CoV-2 and other human coronaviruses (HCoVs).
ContributorsCarmona, Jacqueline (Author) / Anderson, Karen S (Thesis advisor) / Lake, Douglas (Thesis advisor) / Maley, Carlo (Committee member) / Mangone, Marco (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Evolution is a key feature of undergraduate biology education: the AmericanAssociation for the Advancement of Science (AAAS) has identified evolution as one of
the five core concepts of biology, and it is relevant to a wide array of biology-related
careers. If biology instructors want students to use evolution to address scientific
challenges post-graduation, students need to be able to apply evolutionary principles to
real-life situations, and accept that the theory of evolution is the best scientific
explanation for the unity and diversity of life on Earth. In order to help students progress
on both fronts, biology education researchers need surveys that measure evolution
acceptance and assessments that measure students’ ability to apply evolutionary concepts.
This dissertation improves the measurement of student understanding and acceptance of
evolution by (1) developing a novel Evolutionary Medicine Assessment that measures
students’ ability to apply the core principles of Evolutionary Medicine to a variety of
health-related scenarios, (2) reevaluating existing measures of student evolution
acceptance by using student interviews to assess response process validity, and (3)
correcting the validity issues identified on the most widely-used measure of evolution
acceptance - the Measure of Acceptance of the Theory of Evolution (MATE) - by
developing and validating a revised version of this survey: the MATE 2.0.
ContributorsMisheva, Anastasia Taya (Author) / Brownell, Sara (Thesis advisor) / Barnes, Elizabeth (Committee member) / Collins, James (Committee member) / Cooper, Katelyn (Committee member) / Sterner, Beckett (Committee member) / Arizona State University (Publisher)
Created2023
Description
Redox homeostasis is described as the net physiologic balance between inter-convertible oxidized and reduced equivalents within subcellular compartments that remain in a dynamic equilibrium. This equilibrium is impacted by reactive oxygen species (ROS), which are natural by-products of normal cellular activity. Studies have shown that cancer cells have high ROS levels and altered redox homeostasis due to increased basal metabolic activity, mitochondrial dysfunction, peroxisome activity, as well as the enhanced activity of NADPH oxidase, cyclooxygenases, and lipoxygenases. Glioblastoma (GBM) is the most prevalent primary brain tumor in adults with a median survival of 15 months. GBM is characterized by its extreme resistance to therapeutic interventions as well as an elevated metabolic rate that results in the exacerbated production of ROS. Therefore, many agents with either antioxidant or pro-oxidant mechanisms of action have been rigorously employed in preclinical as well as clinical settings for treating GBM by inducing oxidative stress within the tumor. Among those agents are well-known antioxidant vitamin C and small molecular weight SOD mimic BMX-001, both of which are presently in clinical trials on GBM patients. Despite the wealth of investigations, limited data is available on the response of normal brain vs glioblastoma tissue to these therapeutic interventions. Currently, a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of a panel of oxidative stress biomarkers: glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), and cysteine disulfide in human-derived brain tumor and mouse brain samples; this method will be enriched with additional oxidative stress biomarkers homocysteine (Hcy), methionine (Met), and cystathionine (Cyst). Using this enriched method, we propose to evaluate the thiol homeostasis and the redox state of both normal brain and GBM in mice after exposure with redox-active therapeutics. Our results showed that, compared to normal brain (in intact mice), GBM tissue has significantly lower GSH/GSSG and Cys/CySS ratios indicating much higher oxidative stress levels. Contralateral “normal” brain tissue collected from the mice with intracranial GBM were also under significant oxidative stress compared to normal brains collected from the intact mice. Importantly, normal brain tissue in both studies retained the ability to restore redox homeostasis after treatment with a redox-active therapeutic within 24 hours while glioblastoma tissue does not. Ultimately, elucidating the differential redox response of normal vs tumor tissue will allow for the development of more redox-active agents with therapeutic benefit.
ContributorsShaik, Kamal (Author) / LaBaer, Joshua (Thesis director) / Tovmasyan, Artak (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor) / Dean, W.P. Carey School of Business (Contributor)
Created2022-12
Description
DNA methylation (DNAm) is an epigenetic mark with a critical role in regulating gene expression. Altered clinical states, including toxin exposure and viral infections, can cause aberrant DNA methylation in cells, which may persist during cell division. Current methods to study genome-wide methylome profiles of the cells require a long processing time and are expensive. Here, a novel technique called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which is amenable to automation. Up to 15 different samples can be combined into the same run of Mx-MeDIP-Seq, using only 25 ng of DNA per sample. Mx-MeDIP-Seq was used to study DNAm profiles of peripheral blood mononuclear cells (PBMCs) in two biologically distinct RNA viral infections with different modes of transmission, symptoms, and interaction with the host immune system: human immunodeficiency virus1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Analysis of 90 hospitalized patients with SARS-CoV-2 and 57 healthy controls revealed that SARS-CoV-2 infection led to alterations in 920 methylated regions in PBMCs, resulting in a change in transcription that affects host immune response and cell survival. Analysis of publicly available RNA-Sequencing data in COVID-19 correlated with DNAm in several key pathways. These findings provide a mechanistic view toward further understanding of viral infections. Genome-wide DNAm changes post HIV-1-infection from 37 chronically ill patients compared to 17 controls revealed dysregulation of the actin cytoskeleton, which could contribute to the establishment of latency in HIV-1 infections. Longitudinal DNAm analysis identified several potentially protective and harmful genes that could contribute to disease suppression or progression.
ContributorsRidha, Inam (Author) / LaBaer, Joshua (Thesis advisor) / Murugan, Vel (Thesis advisor) / Plaisier, Christopher (Committee member) / Nikkhah, Mehdi (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2022