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

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes. / Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

Variantes genéticas trombogênicas podem aumentar o risco de eventos adversos em pacientes com coronariopatia crônica. Estudos prévios demonstraram que o Haplótipo H2 do gene do receptor P2Y12 apresenta uma maior agregação plaquetária e está associado com a presença de isquemia arterial periférica. A metaloprotease ADAMTS13 é responsável pela clivagem do fator de von Willebrand e recentemente foi associada com doença isquêmica coronariana. O objetivo deste trabalho foi avaliar o efeito das variantes genéticas funcionais trombogênicas dos Haplótipos H1 e H2 do receptor plaquetário P2Y12 e dos polimorfismos C1342G (Q448E), C1852G (P618A) e C2699T (A900V) da metaloprotease ADAMTS13 em 611 pacientes com doença arterial coronariana multiarterial com função ventricular preservada, acompanhados por um período de 05 anos no ensaio clínico do projeto MASS II (Medical, Angioplasty, or Surgery Study II) em relação aos eventos morte, infarto agudo do miocárdio, angina refratária necessitando um novo procedimento e acidente vascular cerebral. Neste estudo, a avaliação dos Haplótipos H1 e H2 nos pacientes do MASS II não encontrou diferença entre estes haplótipos e os eventos estudados. A análise dos polimorfismos da ADAMTS13 não encontrou associação entre os polimorfismos e os eventos estudados, exceto para a variante genética T2699 (Val900) que está associada com o evento morte (OR: 1,67 95%IC: 1-2,78, p= 0,049) e morte por causa cardiovascular (OR: 2,23 95%IC: 1,2-3,94, p=0,004) e apresenta uma diminuição na sobrevida livre de morte por causa cardíaca para os portadores do genótipo TT relacionado à este polimorfismo. A análise dos haplótipos e das combinações alélicas destes polimorfismos não apresentou associação com eventos ou com a sobrevida livre dos eventos nestes pacientes. / Thrombotic genetic variants could improve the risk of adverse events related to coronary arterial disease (CAD). P2Y12 platelet receptor H2 haplotype showed higher aggregation index and a positive association was described between such genetic variant and peripheral artery disease. DAMTS13 is a metaloprotease responsible to von Willebrand factor cleavage recently found correlated to CAD. We tested the genetic variants P2Y12 receptor H1 and H2 haplotypes and ADAMTS13 polymorphisms C1342G (Q448E), C1852G (P618A) and C2699T (A900V) in a group of 611 patients enrolled in the Medical, Angioplasty, or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function in a follow up period of 05 years. The incidence of the end points of death and death from cardiac causes, myocardial infarction, refractory angina requiring revascularization and cerebrovascular accident was determined for P2Y12 H1 and H2 haplotypes and ADAMTS polymorphisms. In our study, we did not disclose any association between H1 or H2 haplotype groups regarding the incidence of any of the studied cardiovascular end-points. The association of ADAMTS13 genotypes and cardiovascular events did not showed any association between C1342G (Q448E), C1852G (P618A) variants and cardiovascular end points. Our date provide a strong association between T2699 variant and increased risk to death (OR: 1,67 CI: 1-2,78, p= 0,049) and cardiac death (OR: 2,23 CI: 1,2-3,94, p=0,004) in a population with CAD. The allelic combinations and haplotypes obtained from ADAMTS13 polymorphisms were not associated to cardiac end points and survival differences between MASS II patients.

Indiana University-Purdue University Indianapolis (IUPUI) / The first part of the study describes the development of a microCT based engineering model to study orthodontic responses. The second part investigated the relationship between orthodontic stimulus, root resorption and bone modeling. It was hypothesized that stress magnitudes are insufficient to portray the mechanical environment and explain the clinical response; directions also play a role. An idealized tooth model was constructed for finite element analysis. The principal stress magnitudes and directions were calculated in tipping and translation. It was concluded that within the same region of root, PDL and bone, there can be compression in one structure, tension in another. At a given point in a structure, compression and tension can coexist in different directions. Magnitudes of compression or tension are typically different in different directions. Previously published data presenting only stress magnitude plots can be confusing, perhaps impossible to understand and/or correlate with biological responses. To avoid ambiguities, a reference to a principal stress should include its predominant direction. Combined stress magnitude/direction results suggest that the PDL is the initiator of mechanotransduction. The third part of this project tested the role of the P2X7 receptor in the dentoalveolar morphology of C57B/6 mice. P2X7R KO (knockout) mice were compared to C57B/6 WT to identify differences in a maxillary molar and bone. Tooth dimensions were measured and 3D bone morphometry was conducted. No statistically significant differences were found between the two mouse types. P2X7R does not have a major effect on alveolar bone or tooth morphology. The final part examines the role of the P2X7 receptor in a controlled biomechanical model. Orthodontic mechanotransduction was compared in wild-type (WT) and P2X7R knock-out (KO) mice. Using Finite Element Analysis, mouse mechanics were scaled to produce typical human stress levels. Relationships between the biological responses and the calculated stresses were statistically tested and compared. There were direct relationships between certain stress magnitudes and root resorption and bone formation. Hyalinization and root and bone resorption were different in WT and KO. Orthodontic responses are related to the principal stress patterns in the PDL and the P2X7 receptor plays a significant role in their mechanotransduction.