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Title:Investigating sphingolipid behavior and function using metabolic labeling
Author(s):Kim, Raehyun
Director of Research:Kraft, Mary L
Doctoral Committee Chair(s):Kraft, Mary L
Doctoral Committee Member(s):Bailey, Ryan C; Leckband, Deborah E; Schroeder, Charles M
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Isotope labeling
Metabolic labeling
Abstract:The past few decades of research have accumulated a body of evidence that membrane lipids are far more than merely the structural components of biological membranes. Instead, membrane lipids play important roles in cellular functions in multiple ways. Sphingolipids are a group of lipids that are involved in various cellular processes that are crucial for cell survival and proliferation. However, our understanding of sphingolipid function is limited due to the complexity of their behaviors and the lack of proper tools to address and decipher this complexity. Chapters 2 and 3 present metabolic labeling with fluorophores and stable isotope tags, respectively, as tools to investigate sphingolipid behaviors. Metabolic labeling enables one to detect and directly observe sphingolipids, and not the activities or levels of the enzymes that metabolize them. Metabolic labeling of cells with fluorescent sphingosines enabled visualization of the sphingosine metabolites in live cells and also showed potential for studies of metabolism and in vitro assays. Use of stable isotope tagged sphingolipid precursors, in conjunction with LC-MS/MS analysis, provided a more comprehensive and complete dataset than traditional radiolabeling, including information about unlabeled as well as labeled species. These tools offer great opportunities to explore sphingolipid behaviors. In Chapter 4, based on the observations that sphingolipids have significant roles in membrane organization and that virus infection requires intense membrane reorganization, the involvement of acid sphingomyelinase or sphingomyelin phosphodiesterase 1 (SMPD1), a sphingolipid metabolizing enzyme, in influenza virus infection and particularly its entry was evaluated using RNAi and a pharmacological inhibitor. Western blotting performed prior to infection showed that a significantly higher level of SMPD1 was present in the medium than in the cells. Lowering SMPD1 levels by RNAi or a functional pharmacologic inhibitor, desipramine, did not cause a statistically meaningful change in influenza virus entry. However, influenza virus infection itself was correlated with upregulated SMPD1 levels at the early phase of infection, opening the possibility that sphingolipids may still play an important role in influenza virus infection. Further investigation of the role of SMPD1 in influenza virus infection is necessary. Lastly, in Chapter 5, the cellular uptake of protein-coated nanoparticles was investigated in an effort to understand how plasma proteins interact with the nanoparticle surface, and to enhance the efficiency of targeted nanoparticle delivery with an in vitro system that mimics the in vivo environment. Formation of the protein corona, the protein layer that adsorbs on the surface of the nanoparticle when it is exposed to a biological fluid, is reported to prevent the desired interactions between the nanoparticles and the target cells. Exploiting the well-established mechanism of opsonin-mediated endocytosis in immune cells, we tested whether the protein corona itself can be used as a targeting moiety. Pre-coating the nanoparticles with γ-globulins provided a simple route to enrich the protein corona with opsonins. However, the increased opsonin levels in the protein corona did not enhance cellular uptake, but instead significantly decreased it. Immunodot blot assay and confocal fluorescence microscopy showed that these nanoparticles were internalized through opsonin-receptor interactions, but the opsonins on the nanoparticle were not accessible. This indicates that other components in the protein corona shielded the opsonins, preventing them from interacting with their target receptor. This study demonstrates that the spatial organization of the targeting moieties is critical, and it must be optimized for more efficient targeted nanoparticle delivery.
Issue Date:2016-07-13
Rights Information:Copyright 2016 Raehyun Kim
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08

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