Hao, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 98-115). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / 摘要 --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / List of Primers --- p.xii / Publications arising from this study --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.2 --- APP and its functions --- p.4 / Chapter 1.2.1 --- APP processing --- p.7 / Chapter 1.3 --- APPc-interacting proteins --- p.10 / Chapter 1.3.1 --- FE65 --- p.10 / Chapter 1.3.2 --- Xllα and Xl1β --- p.12 / Chapter 1.3.3 --- JIP-1 --- p.13 / Chapter 1.3.4 --- Dabl and Dab2 --- p.15 / Chapter 1.3.5 --- SNX17 --- p.15 / Chapter 1.3.6 --- Numb --- p.15 / Chapter 1.3.7 --- AIDA-1 --- p.16 / Chapter 1.4 --- Objectives of the project --- p.18 / Chapter 1.4.1 --- Engulfment adaptor protein 1 (GULP1) --- p.19 / Chapter 1.4.2 --- Specific aims of my study --- p.20 / Chapter Chapter 2 --- General Methodology --- p.22 / Chapter 2.1 --- Bacterial culture --- p.22 / Chapter 2.2 --- Mini-preparation/Midi-preparation of plasmid DNA --- p.22 / Chapter 2.3 --- Spectrophotometric analysis of DNA --- p.22 / Chapter 2.4 --- Agarose gel electrophoresis for DNA --- p.23 / Chapter 2.5 --- Preparation of competent E. coli --- p.23 / Chapter 2.6 --- Transformation of competent E. coli --- p.24 / Chapter 2.7 --- Molecular cloning --- p.24 / Chapter 2.7.1 --- Preparation of the cloning vector and insert --- p.25 / Chapter 2.7.2 --- Isolation of DNA from agarose gel --- p.25 / Chapter 2.7.3 --- DNA ligation and transformation --- p.25 / Chapter 2.7.4 --- Rapid screening for ligated plasmid --- p.26 / Chapter 2.8 --- Site-directed mutagenesis --- p.26 / Chapter 2.9 --- Cell culture and transfection --- p.27 / Chapter 2.10 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) --- p.28 / Chapter 2.11 --- Western blotting --- p.29 / Chapter Chapter 3 --- Investigation of the GULP1-APP interaction and the effect of GULP1 on APP processing --- p.31 / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.34 / Chapter 3.2.1 --- DNA constructs --- p.34 / Chapter 3.2.2 --- Antibodies --- p.34 / Chapter 3.2.3 --- GST pull-down assays --- p.35 / Chapter 3.2.4 --- Rat tissues preparation --- p.36 / Chapter 3.2.5 --- Immunostaining --- p.36 / Chapter 3.2.6 --- "siRNA knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.37 / Chapter 3.2.7 --- Luciferase assays --- p.37 / Chapter 3.2.9 --- Tricine-SDS/PAGE analysis for APP CTFs --- p.38 / Chapter 3.2.9 --- Aβ enzyme-linked immunosorbent assay (ELISA) --- p.39 / Chapter 3.2.10 --- Statistical analysis --- p.40 / Chapter 3.3 --- Results --- p.40 / Chapter 3.3.1 --- GULP1 F145V mutant abandons the GULP1-APP interaction --- p.40 / Chapter 3.3.2 --- GULP1 and APP colocalize in neurons --- p.45 / Chapter 3.3.3 --- "siRNA mediated knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.48 / Chapter 3.3.4 --- GULP1 enhances the cleavage of APP in APP-GAL4 cleavage system --- p.49 / Chapter 3.3.5 --- GULP1 alters APP processing by increasing the secretion of APP CTFs --- p.52 / Chapter 3.3.6 --- GULP1 stimulates Aβ secretion --- p.55 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Identification and characterization of GULPl phosphorylation sites --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Materials and Methods --- p.60 / Chapter 4.2.1 --- DNA constructs --- p.61 / Chapter 4.2.2 --- Antibodies --- p.61 / Chapter 4.2.3 --- Expression and purification of GST fusion proteins --- p.61 / Chapter 4.2.4 --- In vitro phosphorylation of GULP1 by cdk5/p35 --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- GULP1 Ser223 can be phosphorylated by cdk5/p35 in vivo --- p.62 / Chapter 4.3.2 --- The phosphorylation ofGULPl Thr35 completely abolished the GULP1-APP interaction --- p.67 / Chapter 4.4 --- Discussion --- p.70 / Chapter Chapter 5 --- Crystallization of the PTB domains of GULPl and GULP1t35d…… --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.72 / Chapter 5.2.1 --- DNA constructs --- p.72 / Chapter 5.2.2 --- Small-scale protein expression and purification --- p.73 / Chapter 5.2.3 --- Large-scale protein expression and purification --- p.73 / Chapter 5.2.4 --- Dynamic light scattering measurement --- p.76 / Chapter 5.2.5 --- Crystallization screening GULP1-PTB --- p.76 / Chapter 5.2.6 --- Optimization of GULP1-PTB crystals by grid screen --- p.76 / Chapter 5.2.7 --- Optimization of GULPl -PTB crystals by additive screen and detergent screen --- p.79 / Chapter 5.3 --- Results --- p.79 / Chapter 5.3.1 --- Large-scale expression and purification of GULP 1-PTB --- p.79 / Chapter 5.3.2 --- Small-scale expression and purification of GULP1T35d-PTB --- p.86 / Chapter 5.3.3 --- Crystallization screening and optimization --- p.88 / Chapter 5.4 --- Discussion --- p.91 / Chapter Chapter 6 --- Conclusion and future perspective --- p.94 / Chapter 6.1 --- Conclusion --- p.94 / Chapter 6.2 --- Future perspective --- p.95 / References --- p.98