No description available.

This study investigated the position, ultrastructure and life history of glia in the midline of the Drosophila melanogaster Central Nervous System (CNS) by using enhancer traps (AA142, X81, argoswll, pointed1277) and reporter constructs (EEl, slilacZ 1.0, slilacZ 4.5) as glial-specific markers. Previous work has established that glia are necessary for proper formation and morphogenesis of longitudinal and commissural axon tracts, and axon ensheathment (Jacobs, 1993; Klambt et al., 1991; Jacobs and Goodman, 1989). By the end of embryogenesis there are three midline glial (MG) cells remaining in each segment (Sonnenfeld and jacobs, in press) which this study verifies. In the third instar larval MG cells proliferate to 24 cells per segment as followed with the E. coli lacZ expressing pointed1277 strain. These E. coli lacZ expressing pointed1277 MG cells begin dividing 57 hours after hatching as seen with 5 -bromodeoxyuridine and hydroxyurea treatment. Some MG genes cease midline expression before MG proliferation (seen with EEl, X81), others (seen with AA142) continue to be expressed until the beginning of MG proliferation. Only the argoswll, slilacZ 1.0, slilacZ 4.5, and pointed1277 expression strains continue E. coli lacZ expression to the end of the larval stages. In the first larval stage a few perineuropilar glia begin to express the E. coli lacZ gene and increase to 400 cells per CNS in the third ins tar as seen in the pointed1277 marker strain. pointed1277 EM micrographs show that E. coli lacZ labeled cells have a glial-like ultrastructure. There - was no co-localization of the E. coli lacZ expression in pointed12 77 and an anti-RK2 (repo) antibody in third instar larvae. In pointed1277 pupae the MG cell E. coli lacZ expression stops after 48- 72 hours and the prerineuropilar signal stops after 24 hours. In newly hatched pointed1277 adults perineuropilar E. coli lacZ expression is present with a cluster of 12 cells in the center of the neuropil. To summarize, after embryogenesis, in the pointed1277 marker strain, the MG cells begin dividing after 57 hours and the E. coli lacZ gene expression ends after the second day of the pupal stage. In the first instar, perineuropilar glia begin to label for the E. coli lacZ product and this expression ends by one day into the pupal stage, with re-appearance in the adult CNS. / Thesis / Master of Science (MSc)

No description available.

No description available.

Ko Chun-wai, Hardy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 107-132). / Acknowledgement --- p.3 / Declaration of originality --- p.4 / Summary of thesis --- p.5-6 / Chapter Chapter 1 --- Introduction and aims of study --- p.7-26 / Chapter Chapter 2 --- Materials and methods --- p.27-39 / Chapter Chapter 3 --- Results of study --- p.40-77 / Chapter Chapter 4 --- Discussion --- p.78-103 / Chapter Chapter 5 --- Conclusion of study --- p.104-106 / References --- p.107-132

Chiu, Yu Hin Teddy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Content --- p.vi / Abbreviations --- p.x / Naming system for mutant proteins --- p.xi / Abbreviation for amino acid --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- What are acidic ribosomal proteins? --- p.1 / Chapter 1.2 --- Why P-proteins are so important? --- p.13 / Chapter 1.3 --- Research objectives --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- List of buffers and media --- p.17 / Chapter 2.1.1 --- Preparation of buffers and media --- p.17 / Chapter 2.1.2 --- Buffers for preparing competent cells --- p.17 / Chapter 2.1.3 --- Media for bacterial culture --- p.17 / Chapter 2.1.4 --- Buffers for nucleic acid electrophoresis --- p.19 / Chapter 2.1.5 --- Buffers for protein electrophoresis --- p.19 / Chapter 2.1.6 --- Buffers for interaction studies using BIAcore 3000 --- p.21 / Chapter 2.2 --- General methods --- p.23 / Chapter 2.2.1 --- Preparation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.2 --- Transformation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.3 --- DNA cloning --- p.24 / Chapter 2.2.3.1 --- DNA cloning by polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.3.2 --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.2.3.3 --- Extraction and purification of DNA from agarose gels --- p.25 / Chapter 2.2.3.4 --- Restriction digestion of DNA --- p.25 / Chapter 2.2.3.5 --- Ligation of digested insert and expression vector --- p.27 / Chapter 2.2.3.6 --- Verification of insert by PCR --- p.27 / Chapter 2.2.3.7 --- Mini-preparation of plasmid DNA --- p.28 / Chapter 2.2.4 --- Polyacrylamide gel electrophoresis (PAGE) of protein --- p.29 / Chapter 2.2.4.1 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.4.2 --- Tricine SDS-polyacrylamide gel electrophoresis --- p.30 / Chapter 2.2.4.3 --- Native polyacrylamide gel electrophoresis --- p.31 / Chapter 2.2.4.4 --- Commassie brilliant blue staining of proteinin polyacrylamide gel --- p.32 / Chapter 2.2.4.5 --- Zinc Imidazole staining of protein in polyacrylamide gel --- p.33 / Chapter 2.2.5 --- Protein concentration determination --- p.33 / Chapter 2.2.6 --- Expression of recombinant proteins --- p.33 / Chapter 2.2.6.1 --- Expression of recombinant proteins using LB --- p.33 / Chapter 2.2.6.2 --- Expression of recombinant proteins using minimal medium --- p.34 / Chapter 2.2.6.3 --- Harvest and lysis of bacterial cell culture --- p.34 / Chapter 2.3 --- Protein purification --- p.36 / Chapter 2.3.1 --- Purification of ribosomal protein P1 and its deletion mutants --- p.36 / Chapter 2.3.1.1 --- Purification of P1 --- p.36 / Chapter 2.3.1.2 --- Purification of P1ΔC25 --- p.36 / Chapter 2.3.1.3 --- Purification of HisMBP-P1ΔC40 and HisMBP-P1ΔC47 --- p.37 / Chapter 2.3.2 --- Purification of ribosomal protein P2 and its deletion mutants --- p.38 / Chapter 2.3.2.1 --- Purification of P2 --- p.38 / Chapter 2.3.2.2 --- Purification of P2ΔC46 and P2ΔC55 --- p.39 / Chapter 2.4 --- "Preparation and purification of protein complexes formed by P1, P2 and their truncation mutants" --- p.40 / Chapter 2.4.1 --- Preparation of complexes by Co-refolding in urea buffer --- p.40 / Chapter 2.4.1.1 --- Preparation of P1 or P1ΔC25 involved complexes --- p.40 / Chapter 2.4.1.2 --- Preparation of P1ΔC40/ P2ΔC46 and P1ΔC47/ P2ΔC46 --- p.41 / Chapter 2.4.2 --- Preparation of complexes by direct mixing --- p.42 / Chapter 2.5 --- Laser light scattering for the determination of molecular weight of protein and their complexes --- p.43 / Chapter 2.5.1 --- Chromatography mode light scattering experiment (SEC/LS) --- p.43 / Chapter 2.6 --- Interaction study of P1 and P2 using BIAcore 3000 surface plasmon resonance (SPR) biosensor --- p.45 / Chapter 2.6.1 --- Immobilization of P2 onto CM5 sensor chips --- p.45 / Chapter 2.6.2 --- Kinetic measurements of P1 and P2 interaction --- p.46 / Chapter Chapter 3 --- Determination of domain boundaries for dimerization of P1/P2 --- p.46 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Preparation of P1，P2 and their truncation mutants --- p.50 / Chapter 3.2.1 --- Construction of P1 and P2 N-terminal domains (NTDs) --- p.50 / Chapter 3.2.2 --- P1 and its truncation mutants were purified in denaturing condition --- p.53 / Chapter 3.2.3 --- "P2, P2AC46 and P2AC55 were purified" --- p.56 / Chapter 3.3 --- Formation of complexes from P1，P2 and their truncation mutants --- p.59 / Chapter 3.3.1 --- "P1, P2 and their truncation mutants interact to yield protein complexes" --- p.49 / Chapter 3.3.2 --- P1AC47/P2AC46 is the smallest N-terminal domain complex --- p.63 / Chapter 3.4 --- Perturbation of P2 NTD upon binding with P1 --- p.65 / Chapter 3.4.1 --- "1H, 15N 一 HSQC spectrum of P2AC46 changed significantly upon binding with P1" --- p.65 / Chapter 3.4.2 --- P1/P2AC46 prepared by co-refolding and direct mixing give the same HSQC spectra --- p.66 / Chapter 3.5 --- Discussion --- p.69 / Chapter Chapter 4 --- Stochiometry of P1/P2 Complex is revealed by Light scattering --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- P1 and P2 interact in 1:1 molar ratio --- p.77 / Chapter 4.2.1 --- Purified P2 exists as homo-dimer in solution --- p.77 / Chapter 4.2.2 --- The stochiometry of P1/P2 complex is 1:1 --- p.78 / Chapter 4.3 --- Stochiometries of P1 and P2 truncation mutant complexes varied from the full-length counterparts --- p.81 / Chapter 4.3.1 --- P2AC46 and P2AC55 exist as homo-dimer in solution --- p.81 / Chapter 4.3.2 --- "P1/P2AC46, P1AC25/P2 and P1AC40/P2AC46 retain the hetero-dimeric stochiometry of 1:1" --- p.82 / Chapter 4.3.3 --- P2AC55 involved complexes show a different stochiometry --- p.83 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- Binding kinetics of P1/P2 complex studied by surface plasmon resonance --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Kinetic parameters of P1 and P2 interaction is revealed by surface plasmon resonance --- p.95 / Chapter 5.2.1 --- P2 was coupled to CM5 sensor chip surface for kinetic studies --- p.95 / Chapter 5.2.2 --- Reduction of basal response after the 1st binding of P1 --- p.96 / Chapter 5.2.3 --- P1 induced a great change in response unit than P2 upon binding with immobilized P2 --- p.99 / Chapter 5.2.4 --- Kinetic parameters of P1 and P2 interaction was studied by introducing P1 to the sensor chip surface --- p.101 / Chapter 5.2.5 --- Dissociation constant derived from 1:1 Langmuir binding isotherm --- p.102 / Chapter 5.2.6 --- Dissociation constant derived from responses at equilibrium (Req) --- p.103 / Chapter 5.3 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion and discussion of the study --- p.112 / References --- p.118 / Appendix --- p.130

No description available.

No description available.

No description available.

No description available.