Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 82-101). Also issued on the Internet.

Microglia Purinergic Receptor-Mediated Neuroinflammation In Alzheimer’s Disease Neurodegeneration involves a complicated cascade of homeostatic dysfunction that converges on neuron loss and cognitive decline, involving complex immune, metabolic, and cell cell crosstalk pathways. The complicated interplay and heterogeneous nature of these factors in the brain make therapeutic development challenging. Recent advances have placed the immune system as an important driver of neurodegeneration both mechanistically and genetically. Microglia are the professional phagocytes that inhabit the brain and direct these inflammatory pathways, which can have reparative or destructive outcomes on the brain parenchyma. While various genetic risk factors for neurodegeneration reside in microglia, how these trigger and facilitate disease requires further investigation. In the present dissertation, I investigate inflammatory activation in microglia upon various damage or pathology-associated stimuli by utilizing a primary human monocyte-derived microglia-like cell (MDMi) model from a diverse donor cohort, which allows for the examination of genetically driven differences. I find that MDMi stimulated through ATP-mediated P2RX7 activation display reduced phagocytic function for amyloid beta uptake, and this pathway is also influenced by individual donors’ SPI1 genotype which has been associated with Alzheimer’s disease in previous computational studies. These experiments demonstrate functional outcomes related to AD genetics in immune cells. Previous computational studies have identified cognitive-decline associated gene modules expressed in human brain tissues from late-stage AD. I conducted in vitro follow up experiments to interrogate these genetic findings which is crucial for validating RNA sequencing data in a biological model. To interrogate differential MDMi inflammatory pathways, I treated cells with the toxic immunostimulatory molecule lipopolysaccharide (LPS), or its non-toxic derivative monophosphoryl lipid A (MPLA) which has positive immune properties currently utilized in vaccine adjuvants. My results indicated that individual gene expression in this module does not shift in a uniform manner upon LPS or MPLA challenge, suggesting more nuanced in vitro interrogation is required to identify conditions propagating this end stage disease phenotype. Microglia serve as the primary immune cells of the brain but also interact closely with astrocytes, large glial cells that facilitate neuronal homeostasis and are central players in AD due to their high apolipoprotein (APOE) production. Given the newly appreciated role of cellular crosstalk in neurological disease pathogenesis, I sought to optimize a protocol for isolation of primary mouse astrocytes for coculture with MDMi and investigation of non-direct cell contact interactions through astrocyte supernatants. Described in this dissertation is my optimized protocol for purified mouse astrocyte isolation from mice expressing humanized APOE2, APOE3, or APOE4. By developing this model, I was able to discern differential changes to MDMi gene expression in the presence of APOE2, 3, or 4 astrocyte supernatants. Verification of these tools allows further exploration of APOE genotype on glial crosstalk and downstream AD pathology. Overall, this work uncovers important mechanisms of human microglia activation through AD genetics and extracellular P2RX7 receptor behavior. By interrogating these scientific questions in a human microglia model derived from donors of various genetic and age backgrounds, we can assess how real biological variation modulates canonical inflammatory pathways. This adds powerful clinical relevance as AD and other neurodegenerative conditions can present a very heterogenous phenotype pathologically and therefore may require the nuance of more personalized medicine therapeutically.

Lau, Ka Hoi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-151). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENT --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.iv / ABSTRACT IN CHINESE --- p.vii / TABLE OF CONTENTS --- p.x / Chapter CHAPTER I - --- INTRODUCTION / Chapter 1.1 --- Regulation of human airway surface liquid --- p.1 / Chapter 1.2 --- Cysteinyl leukotrienes in asthma --- p.2 / Chapter 1.3 --- Cysteinyl leukotriene receptor in epithelial cells --- p.5 / Chapter 1.4 --- Particular interest on CysLT1 receptor --- p.7 / Chapter 1.5 --- Cysteinyl leukotrienes receptor antagonists --- p.10 / Chapter 1.6 --- Purinergic receptors in epithelial cells --- p.11 / Chapter 1.7 --- P2Y receptors in epithelial cells --- p.13 / Chapter 1.8 --- Signalling pathways of P2Y receptors by nucleotide stimulation --- p.15 / Chapter 1.9 --- The importance of P2Y6 receptor on inflammation --- p.17 / Chapter 1.10 --- Relation between CysLT1 receptor and P2Y receptor --- p.18 / Chapter 1.11 --- The properties of 16HBE14o- cell line --- p.21 / Chapter 1.12 --- Objectives of the present project --- p.22 / Chapter CHAPTER II - --- MATERIALS AND METHODS / Chapter 2.1 --- Solutions and chemicals --- p.23 / Chapter 2.2 --- Cell culture --- p.25 / Chapter 2.3 --- Measurement of intracellular calcium concentration ([Ca2+ ]i) with fluorescent imaging / Chapter 2.3.1 --- Preparation of 16HBE14o- cells for fluorescent imaging --- p.26 / Chapter 2.3.2 --- Measurement of [Ca2+]j with fluorescent imaging --- p.28 / Chapter 2.4 --- Measurement of short-circuit current (Isc) and transepithelial resistance with Ussing chamber / Chapter 2.4.1 --- Preparation of 16HBE14o- cells for Isc and transepithelial resistance measurement --- p.31 / Chapter 2.4.2 --- Measurement of Isc and transepithelial resistance with Ussing chamber --- p.33 / Chapter 2.5 --- Immunoblot analysis for CysLT1 and P2Y6 receptors --- p.35 / Chapter 2.6 --- Measurement of protein kinase A activity --- p.36 / Chapter 2.7 --- Data analysis --- p.37 / Chapter CHAPTER III - --- RESULTS / Chapter 3.1 --- Expressions of CysLTi and P2Y6 receptor in 16HBE14o- cell monolayers --- p.38 / Chapter 3.2 --- "Differential inhibitory effects of montelukast, pranlukast and zafirlukast to UDP on Isc and [Ca2+]i in 16HBE14o- cells" / Chapter 3.2.1 --- Effect of apical or basolateral application of UDP on Isc and [Ca2+]i --- p.41 / Chapter 3.2.2 --- Effect of montelukast to the application of UDP on Isc and [Ca2+]i --- p.48 / Chapter 3.2.3 --- Effect of pranlukast to the application of UDP on Isc and [Ca2+ ]i --- p.57 / Chapter 3.2.4 --- Effect of zafirlukast to the application of UDP on Isc and [Ca2+]j --- p.63 / Chapter 3.2.5 --- "Summary of the effects of montelukast, pranlukast, zafirlukast to UDP application on Isc and [Ca2+]i" --- p.69 / Chapter 3.3 --- Cellular mechanism(s) underlying the effect of montelukast to apical UDP application on 16HBE14o-cells / Chapter 3.3.1 --- Effect of various blockers inhibiting Ca2 226}Bؤdependent pathway on UDP-induced [Ca2+]i in the presence or absence of montelukast --- p.70 / Chapter 3.3.2 --- "Effects of montelukast, pranlukast and zafirlukast to PKA or Epac on Isc induced by apical UDP" --- p.86 / Chapter 3.4 --- "Effects of montelukast, pranlukast and zafirlukast on other P2Y receptor agonists on 16HBE14o- cells" / Chapter 3.4.1 --- "Effects of montelukast, pranlukast and zafirlukast on 2-methio-ADP-induced Isc and [Ca2+]i responses on 16HBE14o- cellsl" --- p.14 / Chapter 3.4.2 --- "Effects of montelukast, pranlukast and zafirlukast on UTP-induced Isc and [Ca2+]i responses on 16HBE14o- cells" --- p.116 / Chapter CHAPTER IV - --- DISCUSSION / Chapter 4.1 --- Differential effects of CysLT1 antagonists to P2Y6 agonist on Isc and [Ca2+]i in 16HBE14o-cells --- p.120 / Chapter 4.2 --- Possible cellular mechanism(s) underlying the effects of CysLT1 antagonists on UDP-induced [Ca2+]j increase in 16HBE14o- cells --- p.125 / Chapter 4.3 --- Possible cellular mechanism(s) underlying the effects of CysLT1 antagonists on UDP-induced Isc in 16HBE14o- cells --- p.129 / Chapter 4.4 --- Effects of CysLT1antagonists on other P2Y receptor subtypes in 16HBE14o- cells --- p.132 / Chapter 4.5 --- Summary: Possible interaction between CysLT1 antagonists and P2Y6 receptor --- p.135 / Chapter 4.6 --- Clinical implications and perspectives --- p.138 / Chapter CHAPTER V - --- REFERENCES --- p.139

Wong, Miu Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 140-152). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENT --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.iv / ABSTRACT IN CHINESE --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xviii / Chapter CHAPTER I - --- INTRODUCTION / Chapter 1.1 --- Regulation of human airway surface liquid --- p.1 / Chapter 1.2 --- Sodium reabsorption and chloride secretion in airway epithelium --- p.3 / Chapter 1.3 --- Purinergic receptors --- p.7 / Chapter 1.4 --- P2Y receptors in epithelial cells --- p.11 / Chapter 1.5 --- Autocrine or paracrine regulation of ion transport in epithelial cells --- p.13 / Chapter 1.6 --- Signaling pathways underlying the regulation of ion transport by P2Y receptors stimulation --- p.16 / Chapter 1.7 --- The therapeutic potential of P2Y receptors in treating cystic fibrosis --- p.18 / Chapter 1.8 --- Particular interest on P2Y6 receptor as potential target for treatment of cystic fibrosis --- p.21 / Chapter 1.9 --- Properties of 16HBE14o- cell line --- p.23 / Chapter 1.10 --- Objectives of the present experiments --- p.25 / Chapter CHAPTER II - --- MATERIALS AND METHODS / Chapter 2.1 --- Solutions and Chemicals --- p.26 / Chapter 2.2 --- Cell culture --- p.28 / Chapter 2.3 --- Simultaneous measurement of short-circuit current (Isc) and intracellular calcium concentration ([Ca2+ ])i --- p.29 / Chapter 2.3.1 --- Preparation of 16HBE14o- cells for simultaneous measurement of Isc and [Ca2+]i --- p.29 / Chapter 2.3.2 --- Measurement of Isc and transepithelial resistance with Ussing chamber --- p.32 / Chapter 2.3.3 --- Simultaneous measurement of Isc and [Ca2+]i --- p.35 / Chapter 2.4 --- Measurement of protein kinase A activity --- p.38 / Chapter 2.5 --- Data analysis --- p.39 / Chapter CHAPTER III - --- RESULTS / Chapter 3.1 --- Apical and basolateral application of P2Y agonists induced Isc and [Ca2+］i responses in 16HBE14o- cells --- p.40 / Chapter 3.1.1 --- Effect of apical and basolateral application of ATP on Isc and [Ca2+̐]ư --- p.40 / Chapter 3.1.2 --- Effect of apical and basolateral application of UTP on Isc and [Ca2+̐]ư --- p.45 / Chapter 3.1.3 --- Effect of apical and basolateral application of UDP on Isc and [Ca2+̐]ư --- p.50 / Chapter 3.1.4 --- "Summary of the effects of apical and basolateral application of ATP, UTP and UDP on Isc and [Ca2+̐]ư" --- p.55 / Chapter 3.2 --- Ionic mechanisms underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.56 / Chapter 3.2.1 --- Differential effect of apical and basolateral UDP on Isc --- p.56 / Chapter 3.2.2 --- Effect of various apical CI－ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.59 / Chapter 3.2.3 --- Effect of various basolateral K+ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.83 / Chapter 3.3 --- Involvement of other signaling molecules or pathways in regulation of the chloride secreting response evoked by apical and basolateral UDP --- p.108 / Chapter 3.3.1 --- Effect of apical and basolateral UDP on PKA activity --- p.109 / Chapter 3.3.2 --- Effect of PKC inhibitors on Isc response induced by apical and basolateral application of UDP --- p.111 / Chapter CHAPTER IV - --- DISCUSSION / Chapter 4.1 --- Simultaneous measurement of Isc and [Ca2+ ̐]ư upon apical and basolateral application of P2Y agonists in 16HBE14o- cells --- p.125 / Chapter 4.2 --- Ionic mechanism underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.128 / Chapter 4.2.1 --- Possible ionic mechanism for chloride secretion mediated by apical P2Y6 receptors --- p.131 / Chapter 4.2.2 --- Possible ionic mechanism for chloride secretion mediated by basolateral P2Y6 receptors --- p.133 / Chapter 4.3 --- Involvement of other possible signaling molecules or pathway underlying the action of apical and basolateral UDP --- p.135 / Chapter 4.4 --- Summary --- p.138 / Chapter CHAPTER V - --- REFERENCES --- p.140 / Publications --- p.153