SINGLE-CELL ANALYSIS OF LIPID ENZYME ACTIVITY USING AUTOMATED CAPILLARY ELECTROPHORESIS Public Deposited

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  • March 19, 2019
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  • Dickinson, Alexandra
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Lipid enzymes play critical roles in regulating the function and behavior of cells. Dysregulation of lipid enzymes is an important factor in a variety of diseases. For instance, sphingosine kinase is a crucial regulator of cell proliferation, survival, and differentiation. Phospholipase C is another key lipid enzyme that modulates proliferation, differentiation, and cytokinesis. Aberrant activity of both enzymes has been implicated in diseases involving the immune system, including leukemic, inflammatory, and autoimmune diseases. However, very little is known about the activity of these enzymes at the single-cell level, although cellular heterogeneity is a characteristic feature of the immune system and of many immune system diseases. In fact, assays typically used to directly measure lipid enzyme activity require bulk cell lysates comprised of thousands of cells. Therefore, the development of a technology capable of measuring lipid enzyme activity in single cells may be essential in further elucidating the role of lipid enzymes in both normal and aberrant immune cell biology. Capillary electrophoresis (CE) is a powerful tool for single-cell analysis, but use of CE for single-cell applications has been limited by low throughput. Typically, the rate of single-cell CE is limited to 1 - 2 cells per hour. This dissertation will describe the development and characterization of a single-cell CE system capable of performing single-cell analysis at rates as high as 3.5 cells/min. The system was used to characterize sphingosine kinase activity in hundreds of tissue-cultured cells derived from myeloid leukemia. Sphingosine kinase activity was additionally analyzed in primary leukemic cells from patients with different types of leukemia, including the deadliest form of leukemia, acute myeloid leukemia. The automated CE system was further used to characterize the sphingosine kinase pathway in primary natural killer cells from healthy subjects. Finally, a method for blocking lipid adsorption to device surfaces was developed to enable coupling of single-cell sphingosine kinase and phospholipase C activity measurements to immunofluorescence microscopy. In addition to the lipid enzyme activity measurements presented in this work, automated single-cell CE has the potential to analyze a variety of biomolecules and cell types for a broad range of biological applications.
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  • In Copyright
Advisor
  • Armistead, Paul
  • Allbritton, Nancy
  • Jorgenson, James
  • Jaspers, Ilona
  • Lockett, Matthew
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2014
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  • Chapel Hill, NC
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  • This item is restricted from public view for 2 years after publication.
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