ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
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- All Subjects: Biology
- All Subjects: Protein microarrays
- Creators: LaBaer, Joshua
Description
ABSTRACT
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation.
In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work.
In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro.
ContributorsMorales Betanzos, Carlos (Author) / LaBaer, Joshua (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA).
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
ContributorsBian, Xiaofang (Author) / LaBaer, Joshua (Thesis advisor) / Mandarino, Lawrence (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
Description
Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR pathway is still unexplored. Unfortunately, NanoBRET, as with all other high throughput methods, cannot determine binding kinetics or affinities. To address this shortcoming, I developed a hybrid platform that characterizes > 400 PPIs quantitatively and simultaneously in < 1 hour by combining the high throughput and flexible nature of nucleic programmable protein arrays (NAPPA) with the quantitative abilities of surface plasmon resonance imaging (SPRi). NAPPA-SPRi was then used to study the kinetics and affinities of > 12,000 PPIs in the BCR signaling pathway, revealing unique kinetic mechanisms that are employed by proteins, phosphorylation and activation states to regulate PPIs. In one example, activation of the GTPase RAC1 with nonhydrolyzable GTP-γS minimally affected its binding affinities with phosphorylated proteins but increased, on average, its on- and off-rates by 4 orders of magnitude for one-third of its interactions. In contrast, this phenomenon occurred with virtually all unphosphorylated proteins. The majority of the interactions (85%) were novel, sharing 40% of the same interactions as NanoBRET as well as detecting 55% more interactions than NanoBRET. In addition, I further validated four novel interactions identified by NAPPA-SPRi using SDS-PAGE migration and Western blot analyses. In one case, we have the first evidence of a direct enzyme-substrate interaction between two well-known proto-oncogenes that are abnormally regulated in > 30% of cancers, PI3K and MYC. Herein, PI3K is demonstrated to phosphorylate MYC at serine 62, a phosphosite that increases the stability of MYC. This study provides valuable insight into how PPIs, phosphorylation, and GTPase activation regulate the BCR signal transduction pathway. In addition, these methods could be applied toward understanding other signaling pathways, pathogen-host interactions, and the effect of protein mutations on protein interactions.
ContributorsPetritis, Brianne Ogata (Author) / LaBaer, Joshua (Thesis advisor) / Lake, Douglas (Committee member) / Wang, Shaopeng (Committee member) / Arizona State University (Publisher)
Created2018
Description
Emerging pathogens present several challenges to medical diagnostics. Primarily, the exponential spread of a novel pathogen through naïve populations require a rapid and overwhelming diagnostic response at the site of outbreak. While point-of-care (PoC) platforms have been developed for detection of antigens, serologic responses, and pathogenic genomes, only nucleic acid diagnostics currently have the potential to be developed and manufactured within weeks of an outbreak owing to the speed of next-generation sequencing and custom DNA synthesis. Among nucleic acid diagnostics, isothermal amplification strategies are uniquely suited for PoC implementation due to their simple instrumentation and lack of thermocycling requirement. Unfortunately, isothermal strategies are currently prone to spurious nonspecific amplification, hindering their specificity and necessitating extensive empirical design pipelines that are both time and resource intensive. In this work, isothermal amplification strategies are extensively compared for their feasibility of implementation in outbreak response scenarios. One such technology, Loop-mediated Amplification (LAMP), is identified as having high-potential for rapid development and PoC deployment. Various approaches to abrogating nonspecific amplification are described including a novel in silico design tool based on coarse-grained simulation of interactions between thermophilic DNA polymerase and DNA strands in isothermal reaction conditions. Nonspecific amplification is shown to be due to stabilization of primer secondary structures by high concentrations of Bst DNA polymerase and a mechanism of micro-complement-mediated cross-priming is demonstrated as causal via nanopore sequencing of nonspecific reaction products. The resulting computational model predicts primer set background in 64% of 67 test assays and its usefulness is illustrated further by determining problematic primers in a West Nile Virus-specific LAMP primer set and optimizing primer 3’ nucleotides to eliminate micro-complements within the reaction, resulting in inhibition of background accumulation. Finally, the emergence of Orthopox monkeypox (MPXV) as a recurring threat is discussed and SimCycle is utilized to develop a novel technique for clade-specific discrimination of MPXV based on bridging viral genomic rearrangements (Bridging LAMP). Bridging LAMP is implemented in a 4-plex microfluidic format and demonstrates 100% sensitivity in detection of 100 copies of viral lysates and 45 crude MPXV-positive patient samples collected during the 2022 Clade IIb outbreak.
ContributorsKnappenberger, Mark Daniel (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Roberson, Robert (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2023
Description
According to the World Health Organization, cancer is one of the leading causes of death around the world. Although early diagnostics using biomarkers and improved treatments with targeted therapy have reduced the rate of cancer related mortalities, there remain many unknowns regarding the contributions of the tumor microenvironment to cancer progression and therapeutic resistance. The tumor microenvironment plays a significant role by manipulating the progression of cancer cells through biochemical and biophysical signals from the surrounding stromal cells along with the extracellular matrix. As such, there is a critical need to understand how the tumor microenvironment influences the molecular mechanisms underlying cancer metastasis to facilitate the discovery of better therapies. This thesis described the development of microfluidic technologies to study the interplay of cancer cells with their surrounding microenvironment. The microfluidic model was used to assess how exposure to chemoattractant, epidermal growth factor (EGF), impacted 3D breast cancer cell invasion and enhanced cell motility speed was noted in the presence of EGF validating physiological cell behavior. Additionally, breast cancer and patient-derived cancer-associated fibroblast (CAF) cells were co-cultured to study cell-cell crosstalk and how it affected cancer invasion. GPNMB was identified as a novel gene of interest and it was shown that CAFs enhanced breast cancer invasion by up-regulating the expression of GPNMB on breast cancer cells resulting in increased migration speed. Lastly, this thesis described the design, biological validation, and use of this microfluidic platform as a new in vitro 3D organotypic model to study mechanisms of glioma stem cell (GSC) invasion in the context of a vascular niche. It was confirmed that CXCL12-CXCR4 signaling is involved in promoting GSC invasion in a 3D vascular microenvironment, while also demonstrating the effectiveness of the microfluidic as a drug screening assay. Taken together, the broader impacts of the microfluidic model developed in this dissertation include, a possible alternative platform to animal testing that is focused on mimicking human physiology, a potential ex vivo platform using patient-derived cells for studying the interplay of cancer cells with its surrounding microenvironment, and development of future therapeutic strategies tailored toward disrupting key molecular pathways involved in regulatory mechanisms of cancer invasion.
ContributorsTruong, Danh, Ph.D (Author) / Nikkhah, Mehdi (Thesis advisor) / LaBaer, Joshua (Committee member) / Smith, Barbara (Committee member) / Mouneimne, Ghassan (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Cancer autoantibody biomarker discovery and validation using nucleic acid programmable protein array
Description
Currently in the US, many patients with cancer do not benefit from the population-based screening, due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and get diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies is a promising class of molecules that have potential to serve as early detection biomarkers for cancers. This Ph.D thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancer, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformation on protein array. This method adopted a novel protein tag technology, called HaloTag, to covalently immobilize proteins on glass slide surface. The covalent attachment allowed these proteins to endure harsh treatment without getting dissociated from slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to significantly increase signal to noise ratio of protein array based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases / 145 controls / 70 non-basal breast cancer) by ELISA, a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. Similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed-tomography positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA level in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights on the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.
ContributorsWang, Jie (Author) / LaBaer, Joshua (Thesis advisor) / Anderson, Karen S (Committee member) / Lake, Douglas F (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2015
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
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