PhD Defense by Nathan Chiappa
Biomedical Engineering Ph.D. Thesis Defense
Date: Thursday, April 25, 2019
Location: Suddath Seminar Room, IBB 1128
Edward Botchwey, PhD
Clinton Joiner, MD, PhD
Wilbur Lam, MD, PhD
Alfred Merrill, PhD
Mark Styscynski, PhD
Eberhard Voit, PhD
Title: Combining Sphingolipidomics and Computational Systems Biology to Dissect the Mechanisms of Altered Red Blood Cell Sphingolipid Metabolism in Sickle Cell Disease
Sickle cell disease is among the most common hematologic diseases, affecting over 4 million people worldwide. Despite knowing the genetic origin of this disease for decades, our ability to treat sickle cell disease is still limited. Thus, an improved understanding of the mechanisms of sickle cell pathology is desperately needed. One aspect of sickle cell disease pathology that has not received much attention is the alteration of the membrane lipid metabolism. Sickle red blood cell membranes are subject to intense physical and oxidative damage from sickle hemoglobin. Thus, it is reasonable to hypothesize that sickle red blood cell lipid metabolism is dysfunctional. One branch of lipid metabolism that may be particularly important in red blood cells is sphingolipid metabolism. Research has shown that sphingolipids regulate numerous processes in red blood cells including cell death, adhesion to endothelial cells, and antioxidant defense of other lipids, all of which are areas relevant to sickle cell pathology. Despite this, little is known about red blood cell sphingolipid metabolism under normal or sickle conditions.
In this thesis, we combine liquid chromatography-tandem mass spectrometry with computational modeling to characterize red blood cell sphingolipid metabolism under normal conditions and in the context of sickle cell disease. First, we investigated whether or not the homeostatic concentrations of sphingolipids in normal and sickle cell red blood cells differ using mass spectrometry. We determined that sickle red blood cells have significantly higher concentrations of many different sphingolipids compared to normal red blood cells. Second, we investigated whether or not reticulocytes, which are more abundant in the sickle red blood cell population, could account for the alterations in red blood cell sphingolipid concentrations observed in sickle cell disease. We isolated reticulocyte-enriched and reticulocyte-depleted sickle red blood cell populations and then measured their sphingolipid concentrations using mass spectrometry. Our analysis showed that sickle reticulocytes have elevated concentrations of some, but not all, sphingolipids compared to sickle erythrocytes. Next, we investigated whether or not the altered plasma environment could explain the alterations in red blood cell sphingolipid concentrations. Our analysis showed that sickle plasma can elevate the concentrations of some, but not all, sphingolipids in red blood cells. Finally, we investigated whether or not changes in specific intracellular metabolic enzymes could explain the alterations in red blood cell sphingolipid concentrations. We combined dynamic time series data with a computational model of red blood cell sphingolipid metabolism to estimate sphingolipid metabolic enzyme activities. Our analysis showed that there are significant changes in the activities of a small number of metabolic enzymes in sickle red blood cells.
The results of this work represent significant contributions to our understanding of basic red blood cell biology and to sickle cell disease pathology. We identified roles for the plasma environment, reticulocytes, and specific intracellular metabolic enzymes in causing significant increases in red blood cell sphingolipid concentrations in sickle cell disease. These can serve as novel therapeutic targets to improve the treatment of sickle cell disease.