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Abstract: Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have four sialidases, but their biological functions are unclear. In this report, we show that incubation of human neutrophils with the human sialidase NEU3, but not NEU1, NEU2 or NEU4, inducess human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed neutrophil markers CD66, CD11B, and CD18 to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

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Abstract: Chemorepulsion is the movement of cells away from a chemical signal and is a fundamental process in developmental biology and immune responses. The ability to manipulate chemorepulsion could lead to new therapeutics for neutrophil driven diseases such as acute respiratory distress syndrome (ARDS). However, little is known about eukaryotic chemorepulsion. Autocrine proliferation repressor protein A (AprA) is a protein that acts as an endogenous chemorepellent continuously secreted by Dictyostelium cells. AprA has structural and functional similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV acts as a chemorepellent for neutrophils by activating protease activated receptor 2 (PAR2). In this dissertation, my work on Dictyostelium showed that some of the key proteins that appear to mediate AprA chemorepulsion include Ras GTPases. I have also identified proteins that are involved in regulating the distribution of active Ras protein during chemorepulsion. To identify novel proteins, I utilized restriction enzyme mediated integration, and found 17 proteins that appear to be required for AprA-induced chemorepulsion. One of these proteins is phosphatidylinositol phosphate kinase A (PIPkinA). I showed that PIPkinA is required for both AprA-induced chemorepulsion and proliferation inhibition, and that PipKinA regulates PIP and PIP3 levels in Dictyostelium. In this dissertation, while working with human neutrophils to study how a chemorepellent could also induces apoptosis of activated neutrophils, I have also explored the effect of human iii sialidases NEU1-4 on neutrophil priming. I found that NEU3, previously shown to be upregulated in fibrosis, primes neutrophils and increases expression of neutrophil markers CD11b, CD18, and CD66a to localize to the cell cortex, and decreases the localization of the unprimed neutrophil markers CD43 and CD62-L at the cell cortex. The inhibition of NEU3 by the NEU3 inhibitor 2- acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these data expand the crucial regulation of Ras GTPases and the role of novel proteins in eukaryotic chemorepulsion. This furthers our understanding of the underlying chemorepulsion mechanism in eukaryotes, and facilitates the development of therapeutics for ARDS and neutrophil-driven diseases.