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- Creators: Barrett, The Honors College
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.
The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1α) and decreased activation of nuclear factor kappa B (NF-κB). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use.
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.
Geology and its tangential studies, collectively known and referred to in this thesis as geosciences, have been paramount to the transformation and advancement of society, fundamentally changing the way we view, interact and live with the surrounding natural and built environment. It is important to recognize the value and importance of this interdisciplinary scientific field while reconciling its ties to imperial and colonizing extractive systems which have led to harmful and invasive endeavors. This intersection among geosciences, (environmental) justice studies, and decolonization is intended to promote inclusive pedagogical models through just and equitable methodologies and frameworks as to prevent further injustices and promote recognition and healing of old wounds. By utilizing decolonial frameworks and highlighting the voices of peoples from colonized and exploited landscapes, this annotated syllabus tackles the issues previously described while proposing solutions involving place-based education and the recentering of land within geoscience pedagogical models. (abstract)
The ASU COVID-19 testing lab process was developed to operate as the primary testing site for all ASU staff, students, and specified external individuals. Tests are collected at various collection sites, including a walk-in site at the SDFC and various drive-up sites on campus; analysis is conducted on ASU campus and results are distributed virtually to all patients via the Health Services patient portal. The following is a literature review on past implementations of various process improvement techniques and how they can be applied to the ABCTL testing process to achieve laboratory goals. (abstract)
First, a simple detection paradigm based on reflectance interferometry is developed. This method is simple, low cost and can be easily applied for protein array detection.
Second, a label-free charge sensitive optical detection (CSOD) technique is developed for detecting of both large and small molecules. The technique is based on that most molecules relevant to biomedical research and applications are charged or partially charged. An optical fiber is dipped into the well of a microplate. It detects the surface charge of the fiber, which does not decrease with the size (mass) of the molecule, making it particularly attractive for studying small molecules.
Third, a method for mechanically amplification detection of molecular interactions (MADMI) is developed. It provides quantitative analysis of small molecules interaction with membrane proteins in intact cells. The interactions are monitored by detecting a mechanical deformation in the membrane induced by the molecular interactions. With this novel method small molecules and membrane proteins interaction in the intact cells can be detected. This new paradigm provides mechanical amplification of small interaction signals, allowing us to measure the binding kinetics of both large and small molecules with membrane proteins, and to analyze heterogeneous nature of the binding kinetics between different cells, and different regions of a single cell.
Last, by tracking the cell membrane edge deformation, binding caused downstream event – granule secretory has been measured. This method focuses on the plasma membrane change when granules fuse with the cell. The fusion of granules increases the plasma membrane area and thus the cell edge expands. The expansion is localized at the vesicle release location. Granule size was calculated based on measured edge expansion. The membrane deformation due to the granule release is real-time monitored by this method.