Date of Award

Summer 8-17-2018

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

Julie A. Gosse

Second Committee Member

John T. Singer

Third Committee Member

Paul J. Millard

Additional Committee Members

Rebecca Van Beneden,

Robert E. Gundersen.

Abstract

Mast cells contribute to numerous physiological processes and diseases including immunological and neurological roles. Mast cells degranulate, releasing potent mediators, following signaling transduction initiated by receptor crosslinking. Previously, we showed that the environmental toxicant arsenic and the antibacterial agent triclosan inhibit mast cell degranulation; thus, we have investigated the mechanisms underlying their inhibitory action. We have discovered that arsenic targets early steps in the mast cell signaling pathway: it inhibits phosphorylation of early tyrosine kinase Syk and of Syk’s direct substrate PI3K. Arsenic’s tyrosine phosphorylation inhibition causes inhibition of calcium influx into the cytosol, a key event necessary for degranulation.

Pharmaceutical agent triclosan also inhibits calcium influx but, in contrast to arsenic, does not inhibit early steps in the pathway. We have discovered that one of triclosan’s targets in the mast cell signaling pathway is phospholipase D (PLD) enzyme: PLD activity is decreased by triclosan. Triclosan does not inhibit activity of key enzyme protein kinase C but does delay its

translocation to the plasma membrane and translocation of its substrate MARCKS to the cytosol. In addition, we have shown that another mechanism of triclosan toxicity, mitochondrial uncoupling, also occurs in vivo. We developed a high-throughput assay to measure bioenergetics of living zebrafish embryos and showed that triclosan exposure decreases ATP production without causing mortality of zebrafish.

Finally, in preparation for comparative studies of toxicant effects on mast cells in vivo using zebrafish and in vitro using the mast cell model RBL-2H3, we aimed to develop a tryptase assay of mast cell degranulation that could be used in both systems. However, we discovered that RBL-2H3 cells express neither mRNA nor protein of several tryptase genes. Overall, the findings in this thesis provide biochemical and cellular explanations for known human health effects of triclosan and arsenic and set the stage for future comparative toxicology studies of mast cell function.

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