Chan, Wai Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-141). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Content --- p.6 / List of Abbreviations and symbols --- p.10 / Chapter Chapter 1 --- Introduction --- p.13 / Chapter 1.1 --- The Severity of Influenza A Virus --- p.14 / Chapter 1.2 --- Introduction to Influenza A Virus --- p.15 / Chapter 1.3 --- What is Nucleoprotein? --- p.17 / Chapter 1.4 --- Multifunctional role of Nucleoprotein --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Other Viral Components --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Cellular Components --- p.22 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Chemical reagents --- p.25 / Chapter 2.1.2 --- Buffers --- p.28 / Chapter 2.1.2.1 --- Preparation of Buffers --- p.28 / Chapter 2.1.2.2 --- Buffer for Common Use --- p.28 / Chapter 2.1.3 --- Plasmids and Strains --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Molecular Cloning --- p.32 / Chapter 2.2.2 --- "Expression of the Recombinant NP-WT/ mutants and importin α1, 3and 5 in E.coli" --- p.46 / Chapter 2.2.3 --- Purification of the NP WT/ variants --- p.50 / Chapter 2.2.4 --- "Purification of importin αl, 3 and 5" --- p.53 / Chapter 2.2.5 --- In vitro interaction study between NP and importin α --- p.56 / Chapter 2.2.6 --- In vivo interaction study between NP and importin α --- p.59 / Chapter 2.2.7 --- In vivo analysis to study NP-NP homo-oligomerization --- p.64 / Chapter 2.2.8 --- In vitro static light scattering analysis to determine NP oligomeric state --- p.68 / Chapter 2.2.9 --- Control experiments to verify NP-polymerases and NP-RNA interaction --- p.69 / Chapter Chapter 3 --- Functional analysis of influenza H5N1 nucleoprotein tail loop for oligomerization and ribonucleoprotein activities / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Results --- p.75 / Chapter 3.2.1 --- Tail loop insertion is maintained by intra- and inter-molecular interactions --- p.75 / Chapter 3.2.2 --- NP mutants display defective transcription-replication activity --- p.77 / Chapter 3.2.3 --- Expression and purification of defective NP variants --- p.80 / Chapter 3.2.4 --- Defective NP variants interact with RNA and the polymerase complex --- p.85 / Chapter 3.2.5 --- Defective NP variants possess abnormal oligomeric states in vitro --- p.91 / Chapter 3.2.6 --- NP variants with impaired RNP activity cannot form homo-oligomers in vivo --- p.95 / Chapter 3.3 --- Discussion --- p.98 / Chapter Chapter 4 --- Biophysical characterization of the interaction between influenza nucleoprotein and importin α / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Results --- p.108 / Chapter 4.2.1 --- Expression and Purification of NP-WT/ NLS variants and Importin a --- p.108 / Chapter 4.2.2 --- Pull down Assay --- p.113 / Chapter 4.2.3 --- Light scattering analysis (NP-lmportin α5) --- p.114 / Chapter 4.2.4 --- Binding assay of NP NLS variants with importin α5 --- p.115 / Chapter 4.2.5 --- BIAcore 3000 Surface Plasmon Resonance (NP-lmportin α5) --- p.118 / Chapter 4.2.6 --- QRT-PCR (NP-lmportin a5) --- p.123 / Chapter 4.3 --- Discussion --- p.125 / References --- p.134

The nucleus of epidermal keratinocytes (KCs) is a complex and highly compartmentalized organelle, whose structure is markedly changed during terminal differentiation and transition of the genome from a transcriptionally active state seen in the basal and spinous epidermal cells to a fully inactive state in the keratinized cells of the cornified layer. Here, using multicolor confocal microscopy, followed by computational image analysis and mathematical modeling, we demonstrate that in normal mouse footpad epidermis, transition of KCs from basal epidermal layer to the granular layer is accompanied by marked differences in nuclear architecture and microenvironment including the following: (i) decrease in the nuclear volume; (ii) decrease in expression of the markers of transcriptionally active chromatin; (iii) internalization and decrease in the number of nucleoli; (iv) increase in the number of pericentromeric heterochromatic clusters; and (v) increase in the frequency of associations between the pericentromeric clusters, chromosomal territory 3, and nucleoli. These data suggest a role for nucleoli and pericentromeric heterochromatin clusters as organizers of nuclear microenvironment required for proper execution of gene expression programs in differentiating KCs, and provide important background information for further analyses of alterations in the topological genome organization seen in pathological skin conditions, including disorders of epidermal differentiation and epidermal tumors.