Author ORCID Identifier

https://orcid.org/0000-0003-3565-4509

Date of Graduation

5-2018

Document Type

Dissertation (PhD)

Program Affiliation

Biochemistry and Molecular Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Yang Xia

Committee Member

Rodney E. Kellems

Committee Member

Darren F. Boehning

Committee Member

Dorothy E. Lewis

Committee Member

Edgar T. Walters

Abstract

Sickle cell disease (SCD) is a dangerous condition caused by a genetic mutation on the human beta-globin gene that contributes to erythrocyte sickling, the hallmark of the disease. Previous metabolomics studies have confirmed that elevated sphingosine kinase 1 (SphK1) mediates sphingosine-1-phosphate (S1P) production to promote erythrocyte sickling. S1P signals via five S1P receptors (S1PR) regulates several pathophysiological functions.

In the first chapter of this dissertation, I explored the role of S1PRs in SCD by utilizing pharmacologic and genetic tools. To determine the role of S1P-S1PRs signaling in SCD, I treated humanized Berkeley sickle mice (Berkeley HBS mice), with FTY720, a US Food and Drug Administration (FDA) approved drug. FTY720 can be phosphorylated by SphK1 thus mimicking S1P to regulate S1PRs signaling. Mechanistically, FTY720 can inhibit S1PR signaling in immune cells by the internalization of the receptor. Although FTY720 did not improve erythrocyte life span or reduce sickling in SCD mice, the complete blood cell analysis showed further reduction of inflammatory cells in the periphery, reduced mRNA and protein levels of pro-inflammatory cytokine interleukin 6 (IL-6), improved multiple tissue and renal function.

In the second chapter of this dissertation, I discuss findings generated from a highly robust, unbiased microarray screen performed in SCD lung. Several upregulated gene pathways were identified in sickle lung, which include heme and iron homeostatic genes and inflammatory genes. Unexpectantly, I also discovered an increased expression of rhythmic genes in SCD lung. Period 2 (Per2), a circadian gene, was increased in SCD. To test whether Per2 mRNA and PER2 protein levels were further induced in sickle lung, I utilized a genetic tool, Per2 Luciferase (Per2Luc) mice, a bioluminescence reporter mouse model to study PER2 circadian expression. I generated Per2Luc mice with SCD or WT phenotype by bone marrow transplant transplantation (BMT) studies. With this genetic tool, I detected PER2 to detect luciferase oscillations in SCD lung explant cultures. Next, I determined whether the loss of Per1/Per2 play a role in SCD progression. To test this, I generated WT and SCD phenotypic Per1/Per2 deficient mice by BMT studies. Interestingly, I observed further multiple organ dysfunction, systemic, and local tissue inflammation in SCD →Per1/Per2 dKO mice compared to SCD→WT mice, which demonstrates that the loss of Per1/Per2 in SCD is detrimental and contributes to these devastating effects.

In the third chapter of my dissertation, I explored the impact of chronic hemolysis mediating elevated heme and iron induction in sickle mice. Elevated heme and iron is toxic to the organs and contributes to multiple organ dysfunction. Unexpectantly, I observed heme and iron deposition in the lung of the Per1/Per2 deficient sickle mice. Moreover, I detected heme oxygenase 1 (HO-1), an enzyme that metabolizes heme, in peripheral macrophages in the lung and discovered increased expression of HO-1 in sickle mice with loss Per1/Per2.

Overall, my work demonstrates the roles of inflammatory and circadian Period genes in SCD. By identifying the roles of these genes in SCD, I demonstrated potential mechanisms involved in SCD progression.

Keywords

sickle cell disease, sphingosine-1-phosphate, S1P receptor 1, circadian Per2 gene, bone marrow transplantion, inflammation, heme and iron metabolism

Available for download on Tuesday, April 30, 2019

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