Applications of chemical biology and mass spectrometry for the analysis of the O-GlcNAcome and adeno-associated viruses
Abstract/Contents
- Abstract
- Proteins are involved in nearly all cellular processes and functions. Though there are some 20000 protein-coding genes in the human genome, the functional space of these proteins is expanded dramatically by post-translational modifications (PTMs). Therefore, understanding the functional significance of any given protein in a biological context necessitates the identification and characterization of the protein PTMs. Glycosylation is one of the most ubiquitous forms of protein PTM, and advances in bioanalytical and chemical technologies have begun to enable the study of glycoconjugates more thoroughly. O-GlcNAcylation has garnered significant attention due to its involvement in co-regulating protein phosphorylation and for its myriad functions in cellular signaling, gene transcription, translation, and development. In Chapter 1 of this dissertation, I present an overview of O-GlcNAcylation, detailing the various roles it plays in biology and the high level of regulation for this PTM. I discuss enzymatic substrate specificity related to O-GlcNAcylation, and I briefly introduce the interplay of this modification with various other PTMs. In Chapter 2, I present a brief overview of different technologies available for the study of the O-GlcNAcome. Additionally, I present preliminary data using many of these methods for the application of understanding the dynamics of protein O-GlcNAcylation during neural development. Chapter 3 of this dissertation focuses on the effects of perturbing normal O-GlcNAc cycling during stem cell differentiation. I discuss chemical inhibition of the enzymes controlling the addition and removal of the modification and introduce a potential workflow for achieving end-stage neuronal cells faster from pluripotent human embryonic stem cells. In Chapter 4, I describe more broadly a variety of orthogonal comparisons between two leading production mechanisms for adeno-associated virus (AAV) gene therapeutics used in the clinic. Using a combination of analytical approaches, including MS-based deep proteomic profiling, two-dimensional isoelectric focusing in combination with enzymatic modification removal, transmission electron microscopy, comparative capsid structural modeling by cryo-EM, human cytokine profiling in response to vector transduction, comparative functional transduction assessments in murine liver and skeletal muscle tissues in vivo, primary human fetal brain slices ex vivo, and a variety of primary and immortalized cell types in vitro, I detail how the therapeutics are chemically, structurally, and biologically distinct.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2018; ©2018 |
| Publication date | 2018; 2018 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Rumachik, Neil G |
|---|---|
| Degree supervisor | Bertozzi, Carolyn R, 1966- |
| Thesis advisor | Bertozzi, Carolyn R, 1966- |
| Thesis advisor | Elias, Joshua |
| Thesis advisor | Pitteri, Sharon |
| Degree committee member | Elias, Joshua |
| Degree committee member | Pitteri, Sharon |
| Associated with | Stanford University, Department of Chemistry. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Neil G. Rumachik. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2018. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2018 by Neil Gregory Rumachik
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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