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The current project is to develop a transient expression system for Chinese Hamster Ovary (CHO) cells based on autonomous replication and retention of plasmid DNA. The expression system, named Epi-CHO comprises (1) a recombinant CHO-K1 cell line encoding the Polyoma (Py) virus large T-Antigen (PyLT-Ag), and (2) a DNA expression vector, pPy/EBV encoding the Py Origin (PyOri) for autonomous replication and encoding the Epstein-Barr virus (EBV), Nuclear Antigen-1 (EBNA-1) and EBV Origin of replication (OriP) for plasmid retention. The CHO-K1 cell line expressing PyLT-Ag, named CHO-T was adapted to suspension growth in serum-free media (EXCELL-302) to facilitate large scale transient transfection and recombinant (r) protein production. PyLT-Ag-expressed in CHO-T supported replication of PyOri-containing plasmids and enhanced growth and r- protein production. A scalable cationic lipid based transfection was optimised for CHO-T cells using LipofectAMINE-2000??. Destabilised Enhanced Green Fluorescence Protein (D2EGFP) and Human Growth Hormone (HGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of CHO-T cells with the vector pPy/EBV encoding D2EGFP showed prolonged and enhanced EGFP expression, and transfection with pPy/EBV encoding HGH resulted in a final concentration of 75 mg/L of HGH in culture supernatant 11 days following transfection.

The traditional view of the genome is based on the dogma that genetic information flows from DNA to RNA to protein. Genes have essentially been synonymous with proteins, with RNA viewed primarily as an intermediate template for protein translation. Intriguingly, only ~2% of the human genome encodes proteins, and the number of protein-coding genes (~20,000) is similar between humans and the simple nematode worm.I have been involved with the analysis of a large-scale transcriptome study, intiated by the RIKEN Genomic Sciences Centre in Japan. As part of this work, it was discovered that the genome carries instructions for tens of thousands of “non protein-coding RNAs” (ncRNAs)(please refer to Appendix A to view the original articles that appeared in Science). The significance of these ncRNAs remains a matter of intense interest and debate. Some have argued that these ncRNAs are simply transcriptional noise, while others have suggested that they comprise a critical regulatory system, which directs the complex patterns of gene expression that underlie differentiation and development.To investigate this further, I have conducted a series of studies that explore the expression and evolution of ncRNAs in mammals. Firstly, I established a comprehensive, on-line database of ncRNAs. This collection provides information on more than twenty thousand ncRNAs, and has proven a valuable resource for ncRNA studies. Secondly, I have systematically analyzed the conservation of known functional ncRNA subsets. I found that small ncRNAs (microRNAs and snoRNAs) were well-conserved similar to protein-coding sequences, whereas longer functional ncRNAs were not. These results indicate that long ncRNAs are evolving more rapidly than other functional genomic elements, and suggest that many of the recently-discovered ncRNAs – most of which are long and of unknown significance – might still be functional, despite having poor sequence conservation. Thirdly, I have shown that many ncRNAs are derived from genuine transcripts, whose expression appears regulated in a biologically-relevant manner. Fourthly, I helped develop a computational strategy to identify extremely large ncRNAs and discovered >60 novel candidates, several of which were characterized experimentally. Prior to this work, only a handful of extremely large ncRNAs had been previously described, and these play critical roles in processes such as genomic imprinting and X chromosome inactivation. This study represented the first systematic discovery of extremely large ncRNAs. Finally, I designed custom microarrays and profiled ncRNA expression across the development of CD8+ T cells. CD8+ T cells serve an important role in immunity by killing virus-infected and tumour cells, and transit through a series of functionally-distinct developmental stages. I found that ~200 novel ncRNAs are dynamically expressed during CD8+ T cell development.Taken together, my findings indicate that ncRNAs are a major, regulated output of the mammalian genome, and are consistent with the notion that ncRNAs represent an important, previously-unrecognised biological control system.

No description available.

<p> The proper organization of receptors and signal transduction protein complexes of epithelial and neuronal cells is crucial in tissue formation, cellular differentiation and proper overall development and function. Scaffolding proteins are major components involved in protein targeting and protein complex assembly. MAGUK.s, a family of scaffolding proteins with multiple binding domains such as PDZ, SH3 and GUK, are important regulators of cellular polarity by recruiting and assembling signal and cytoskeletal components into large complexes. Cell polarity is established and maintained by the proper formation and placement of cellular junctions, which separate the plasma membrane into two distinct domains: apical and basolateral. Epithelial polarity determinants from the Bazooka, Crumbs and Scribble complexes establish the boundaries between the apical and basolateral membrane domains and situate the adherens junctions (AJ) at the interface between the two domains. In neuronal cells, the organization and polarization of the presynaptic and the postsynaptic membranes is organized by the CASKIVELIIMINTl/Xllalpha complex. Both CASK and VELI also play a role in epithelial cells. </p> <p> Two novel proteins, originally discovered by Far Western overlay assay in Mus musculus, have been identified as additional binding partners of VELI: PALS I and PALS2. Both proteins are MAGUK.s and are thought to compete with CASK for binding VELI via L27 domain dimerization. PALSl, a major component of the Crumbs complex, is essential for the formation of AJ and the establishment of cellular polarity. PALS2 has been shown to co localize with E-cadherin below tight junctions and directly associate with nectin-like molecule-2 (Necl-2) at extra junctional regions, however its function remains unknown. </p> <p> Using Drosophila melanogaster as a model organism, we have identified the potential Drosophila ortholog of P ALS2, termed dP ALS2, and found that it is conserved across other species. We have done extensive sequence analysis of dP ALS2 at the nucleotide and amino acid level and determined the RNA transcript distribution and protein localization. </p> <p> dP ALS2 expression begins around stage 13 in embryonic tissues in a transversestriped pattern in the epithelia and continues in this striped pattern until the end of stage 17. dP ALS2 is expressed in adult tissues but undetectable in larval tissues. Based on homology and the expression pattern, dP ALS2 may play a role in cell adhesion or cell polarity, similar to the mammalian orthologs. However the striped expression pattern of dPALS2 is similar to segment polarity proteins thus implying dPALS2 may play a role in segment polarity. </p> / Thesis / Master of Science (MSc)

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No description available.

No description available.

The renin-angiotensin system (RAS) is a major physiological regulator of body fluid volume, electrolyte balance and blood pressure. The principal effector peptides for this system are the angiotensins, particularly angiotensin II (Ang II), whose biological cations are mediated by specific angiotensin receptors. The use of highly specific nonpeptide angiotensin antagonists has allowed identification and characterisation of two major Ang II receptor subtypes, designated AT1 and AT2. The aims of this thesis were to investigate AT1 and AT2 receptor expression at both mRNA and protein level in freshly isolated and primary cultures of rat proximal tubular (PT) cells, and to determine the effect of culture upon Ang II receptor subtype expression. The possible role of Ang II upon receptor expression was also investigated. This study demonstrated that only the AT1 receptor is expressed in freshly isolated rat PT cells, with no evidence for AT2 receptor expression. Although continuously present throughout the culture period, a significant decrease in AT1 receptor expression was observed with time. Conversely AT2 receptor expression was absent in freshly isolated cells but was observed after 24 hours in culture with expression then remaining stable throughout culture. Clearly Ang II receptor expression is not stable during culture. Primary cultures of rat PT cells exhibit a change in receptor expression similar to those observed in vivo following tissue damage and repair, with an increase in AT2 receptor expression possibly mediated by locally released Ang II. Initial studies however, involving incubation of rat PT cells with exogenous Ang II, have demonstrated no effect upon AT1 receptor mRNA expression.

by Sze, Lung Yam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 106-112). / Abstract --- p.i / Acknowledgements --- p.iv / Contents --- p.v / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Morphology of the Mammalian Somite and Limb --- p.1 / Chapter 1.1.1. --- The Somite --- p.1 / Chapter 1.1.2. --- The Limb --- p.3 / Chapter 1.2. --- Somite-Limb Relationship --- p.5 / Chapter 1.2.1. --- Somite Contribution to the Appendicular Musculature --- p.5 / Chapter 1.2.2. --- Somite Contribution to Limb Morphogenesis --- p.6 / Chapter 1.3. --- Control of Directionality of Somitic Cell Migration in Appendicular Environment --- p.8 / Chapter 1.4. --- Reasons and Objective of the Present Study --- p.10 / Chapter Chapter 2 --- The Origin of the Mammalian Limb Skeletal Muscles / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.2. --- Materials and Methods --- p.19 / Chapter 2.2.1. --- Embryo Collection --- p.19 / Chapter 2.2.2. --- Isolation of Somites --- p.20 / Chapter 2.2.3. --- DiI-labelling of Rat Donor Somites --- p.21 / Chapter 2.2.4. --- Somite Transplantation --- p.21 / Chapter 2.2.5. --- Embryo Culture --- p.22 / Chapter 2.2.6. --- Analysis of Cultured Embryos --- p.22 / Chapter 2.2.7. --- Cryosection --- p.23 / Chapter 2.2.8. --- Limb Explant Cultures --- p.23 / Chapter 2.2.9. --- Immunohistochemistry --- p.24 / Chapter 2.2.10. --- X-gal Staining --- p.25 / Chapter 2.2.11. --- Histology --- p.25 / Chapter 2.3. --- Results --- p.27 / Chapter 2.3.1. --- Gross Morphology of Cultured Embryos --- p.27 / Chapter 2.3.2. --- Distribution of DiI Labelled Somitic Cells in Rat Embryos --- p.27 / Chapter 2.3.3. --- Histogenetic Potential of Labelled Somitic Cells in the Limbs --- p.29 / Chapter 2.3.4. --- Chimeaic Limb Culture --- p.30 / Chapter 2.4. --- Discussion --- p.32 / Chapter 2.4.1. --- Relationship Between the Somites and the Limb Musculature in Rat Embryos --- p.33 / Chapter 2.4.2. --- Myogenic Potential of Somitic Cells in the Mouse Limb Bud --- p.36 / Chapter 2.4.3. --- The Regulatory Potentials of Mammalian Somites --- p.37 / Chapter Chapter 3 --- The Migration of Somitic Cells into the Mammalian Fore- limb Bud / Chapter 3.1. --- Introduction --- p.52 / Chapter 3.2. --- Materials and Methods --- p.57 / Chapter 3.2.1. --- Embryo Collection --- p.57 / Chapter 3.2.2. --- Embryo Culture and Analysis of Cultured Embryos --- p.58 / Chapter 3.2.3. --- Experimental Series I --- p.58 / Chapter A. --- Micro-injection of DiI --- p.58 / Chapter B. --- Explant Cultures of Rat Fore-limb Bud --- p.59 / Chapter C. --- Histology and Immunohistochemistry --- p.60 / Chapter 3.2.4. --- Experimental Series II --- p.61 / Chapter A. --- Preparation of Conditioned and Unconditioned Medium --- p.61 / Chapter B. --- Coating of Nucleopore Membrane with Fibronectin --- p.62 / Chapter C. --- Preparation of Somitic Cells --- p.62 / Chapter D. --- Analysis of Chemotatic Effect --- p.63 / Chapter 3.2.5. --- Experiment Series III --- p.64 / Chapter A. --- Micro-injection of Latex Beads --- p.64 / Chapter B. --- Isolation of Somatopleure and Transplantation --- p.64 / Chapter C. --- "Somite Isolation, Labelling, and Transplantation" --- p.65 / Chapter D. --- Histology --- p.65 / Chapter 3.3. --- Results --- p.66 / Chapter 3.3.1. --- Development of Embryos In vitro --- p.66 / Chapter 3.3.2. --- Experimental Series I --- p.66 / Chapter A. --- Distribution of Somitic Cells in DiI Injected Embryos --- p.66 / Chapter B. --- Histogenetic Potential of Limb Explants Cultured Under the Kidney Capsule --- p.68 / Chapter C. --- Histogenetic Potential of Limb Explants Cultured In vitro --- p.69 / Chapter 3.3.3. --- Experimental Series II --- p.70 / Chapter A. --- Chemotatic Behaviour of Somitic Cells --- p.70 / Chapter 3.3.4. --- Experimental Series III --- p.71 / Chapter A. --- Ability of Latex Beads to Invade the Limb Bud --- p.71 / Chapter B. --- Distribution Pattern of Somatopleural and Somitic Cells --- p.71 / Chapter 3.4. --- Discussion --- p.75 / Chapter 3.4.1. --- Experimental Series I --- p.75 / Chapter A. --- Distribution of Somitic Cells in DiI-Injected Rat Embryos --- p.75 / Chapter B. --- Histogenetic Potential of Rat Fore-limb Bud --- p.77 / Chapter 3.4.2. --- Experimental Series II --- p.80 / Chapter A. --- Chemotatic Behaviour of Somitic Cells --- p.80 / Chapter 3.4.3. --- Experimental Series III --- p.80 / Chapter A. --- Ability of Latex Beads to Invade the Limb Bud --- p.80 / Chapter B. --- Ability of Somatopleure and Somite to Invade Limb Bud --- p.83 / Chapter 3.4.4. --- Conclusion --- p.83 / References --- p.106 / Appendix --- p.113