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Yu, Chun Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 115-124). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / LIST OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xx / LIST OF ABBREVIATIONS --- p.xxi / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.20 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.22 / Chapter 2.1 --- Major storage proteins in rice --- p.22 / Chapter 2.1.1 --- Structure and composition of glutelin --- p.22 / Chapter 2.1.2 --- Structure and composition of prolamin --- p.22 / Chapter 2.2 --- Biosynthesis pathway --- p.23 / Chapter 2.2.1 --- "The Biosynthesis, processing & compartmentalization of glutelin" --- p.23 / Chapter 2.2.1.1 --- Endoplasmic reticulum as the site of protein folding and compartmentalization --- p.23 / Chapter 2.2.1.2 --- COP-coated vesicles for protien trafficking between ER and Golgi --- p.25 / Chapter 2.2.1.3 --- Glutelin trafficking beyond ER --- p.26 / Chapter 2.2.1.3.1 --- Golgi as the site of post-translational modification of glutelin / Chapter 2.2.1.3.1.1 --- """Sorting for entry"" and ""sorting by retention"" models: mechanism of dense vesicle formation" --- p.26 / Chapter 2.2.1.3.1.2 --- "“Classical ligand-receptor"" and ""aggregation-mediated"" as the model describing protein sorting in Golgi" --- p.27 / Chapter 2.2.1.3.2 --- Pathway bypassing Golgi apparatus --- p.30 / Chapter 2.2.1.4 --- Prevacuolar compartment and protein body --- p.30 / Chapter 2.2.2 --- "The Biosynthesis, processing and compartmentalization of prolamin" --- p.31 / Chapter 2.3 --- Protein processing enzymes --- p.31 / Chapter 2.3.1 --- Luminal chaperone binding protein (BiP) --- p.31 / Chapter 2.3.2 --- Protein disulfide isomerase (PDI) --- p.33 / Chapter 2.4 --- ER quality control： unfolded protein response --- p.34 / Chapter 2.4.1 --- The importance of quality control in ER --- p.34 / Chapter 2.4.2 --- The target of ER quality control: misfolded protein --- p.35 / Chapter 2.4.3 --- Unfolded protein response --- p.36 / Chapter 2.4.3.1 --- IRE1 --- p.37 / Chapter 2.4.3.2 --- PERK --- p.37 / Chapter 2.4.3.3 --- ATF6 --- p.38 / Chapter 2.4.3.4 --- BiP as the master regulator of three transducers --- p.38 / Chapter 2.5 --- The cause of chalkiness --- p.41 / Chapter 2.5.1 --- "The relationship between ER stress, unfolded protein response and chalkiness" --- p.42 / Chapter 2.6 --- Organelle separation： sucrose density gradient centrifugation --- p.43 / Chapter 2.6.1 --- General introduction --- p.43 / Chapter 2.6.2 --- Plant organelle separation --- p.43 / Chapter 2.6.3 --- Organelle marker enzyme as a mean to elucidate the homogeneity of isolated organelle fraction --- p.44 / Chapter 2.7 --- Rice grain quality improvement by genetic engineering --- p.45 / Chapter 2.7.1 --- Increase in lysine content of rice endosperm --- p.45 / Chapter 2.7.2 --- Physiological and phenotypic changes in GT and LRP-fusion lines --- p.46 / Chapter 2.8 --- Hypotheses and objectives --- p.48 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.49 / Chapter 3.1 --- Materials --- p.49 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.49 / Chapter 3.1.2 --- Instruments --- p.49 / Chapter 3.1.3 --- Plant materials --- p.49 / Chapter 3.1.3.1 --- Glutelin-enriched line (GT) --- p.50 / Chapter 3.1.3.2 --- Gtl-LRP-fusion line (LRP fusion) --- p.50 / Chapter 3.2 --- RNA extraction and northern-blot analysis --- p.50 / Chapter 3.2.1 --- Seed harvesting and RNA extraction --- p.50 / Chapter 3.2.2 --- Northern-blot analysis --- p.51 / Chapter 3.3 --- SDS-PAGE and western-blot analysis --- p.52 / Chapter 3.3.1 --- Seed harvesting and protein extraction --- p.52 / Chapter 3.3.2 --- SDS-PAGE and western-blot analysis s --- p.52 / Chapter 3.4 --- Purification of cellular organelles by SDG centrifugation --- p.53 / Chapter 3.4.1 --- Purification of ER by SDG centrifugation --- p.53 / Chapter 3.4.2 --- Purification of protein body by SDG centrifugation --- p.54 / Chapter 3.4.3 --- Protein body isolation by pepsin treatment --- p.54 / Chapter 3.5 --- Electron-microscopic observation --- p.55 / Chapter 3.5.1 --- Sample preparation for immuno-localization analysis --- p.55 / Chapter 3.5.1.1 --- Sample preparation --- p.55 / Chapter 3.5.1.2 --- Immunocytochemical observation --- p.55 / Chapter 3.5.2 --- Sample preparation for structural analysis --- p.56 / Chapter 3.6 --- Antibodies --- p.56 / Chapter 3.6.1 --- KLH conjugation of synthetic peptide --- p.57 / Chapter 3.6.2 --- Immunization of rabbits --- p.57 / Chapter 3.6.3 --- Antibody purification by affinity column --- p.57 / Chapter 3.6.3.1 --- Preparation of column for coupling --- p.57 / Chapter 3.6.3.2 --- Affinity purification of antibody by prepared column --- p.58 / Chapter 3.6.4 --- Testing of antibody specificity --- p.58 / Chapter CHAPTER 4. --- RESULTS --- p.60 / Chapter 4.1 --- Pro-glutelin accumulation in GT and LRP fusion transgenic lines --- p.60 / Chapter 4.2 --- General morphology and glutelin localization in rice seed --- p.61 / Chapter 4.3 --- "Studies on glutelin, BiP and pdi expression profiles of GT, LRP fusion lines and wild type rice" --- p.63 / Chapter 4.3.1 --- Comparison of the protein and RNA profiles of BiP between wild type and FH transgenic rice lines --- p.64 / Chapter 4.3.2 --- Comparison of the protein and RNA profiles of PDI between wild type and FH transgenic rice lines --- p.66 / Chapter 4.3.3 --- "Comparison of the RNA and protein profiles of BiP between wild type, GH and GL transgenic rice lines" --- p.68 / Chapter 4.3.4 --- "Comparison of the RNA and protein expression profiles of PDI between wild type, GH and GL transgenic lines" --- p.70 / Chapter 4.3.5 --- Summary of RNA and protein level comparison of different transgenic lines with wild type --- p.72 / Chapter 4.4 --- Electron microscopic studies of morphological changes in GLUTELIN OVER-EXPRESSED AND GT1-LRP-FUSION TRANSGENIC LINES AND WILD type rice --- p.73 / Chapter 4.5 --- Isolation of ER-enriched fractions by sucrose density gradient centrifugation --- p.76 / Chapter 4.5.1 --- Cross-contamination assessment by organelle specific marker proteins --- p.77 / Chapter 4.5.2 --- Identification of ER enriched fractions of different transgenic lines --- p.78 / Chapter 4.5.3 --- Studies on ER enriched fraction --- p.85 / Chapter 4.6 --- Isolation and studies on PB enriched fractions of different transgenic lines --- p.91 / Chapter 4.7 --- TEM studies on immuno-localization of ER chaperones (BlP and pdI) in immature rice seeds of different transgenic lines --- p.94 / Chapter CHAPTER 5. --- DISCUSSIONS --- p.101 / Chapter 5.1 --- Distortion of glutelin processing and translocation pathway --- p.101 / Chapter 5.1.1 --- The relationship between proglutelin localization and novel protein body in Gt1-LRP-fusion lines --- p.101 / Chapter 5.1.2 --- The presence of BiP and PDI in novel protein body in Gt1-LR-fusion lines --- p.103 / Chapter 5.1.2.1 --- Glutelin translocation pathway bypassing Golgi --- p.105 / Chapter 5.1.2.2 --- Glutelin translocation pathway through Golgi --- p.105 / Chapter 5.1.2.3 --- Gt1-LRP-fusion protein and proglutelin are trapped in ER --- p.107 / Chapter 5.2 --- "The relationship between novel protein body formation, ER stress, unfolded protein response and chalkiness" --- p.108 / Chapter 5.2.1 --- Relationship between novel protein body formation and unfolded protein response --- p.108 / Chapter 5.2.2 --- Repressing the expression of other storage proteins: consequence of unfold protein response or protein nutrients regulation --- p.109 / Chapter 5.2.3 --- Relationship between novel protein body formation and chalkiness --- p.110 / Chapter 5.3 --- The causes of ER dilation --- p.110 / Chapter 5.4 --- The relationship between different physiological changes in transgenic glutelin lines --- p.111 / Chapter 5.5 --- Future perspectives --- p.112 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.114 / REFERENCES --- p.115 / APPENDIX --- p.125

This thesis explores the relationship between journalism as a specific type of socio-cultural practice and the production of meaning in public discussion. Through a case study of newspaper coverage of the issue of genetic engineering in New Zealand (2001-2002), it specifically examines journalists' newsgathering methods, their use of sources and their story-telling frames, and analyses how the news media uses the norm of objectivity to shape public debate on contentious issues. The study argues that elements and structures of journalistic practice both determine a newspaper's ability to address events and issues in a meaningful way and define a newspaper's potential to create a space for public debate. Drawing on field theory, discourse studies and the sociology of journalism, the thesis develops a new operational framework for investigation of journalistic practice by looking at the ideal of objectivity as a method of news gathering, an account of representing reality, and an attitude towards the reality so constructed. This framework is applied in the case study of newspaper coverage of the GE issue where four components of journalistic practice are analysed: journalistic form, transparency of newsgathering, sources and frame. Using content analysis, discourse analysis, interviews and a survey, the thesis explores the relationship between journalistic norms around these elements of the practice and the discursive potential of the news text to represent, interpret and construct reality. The findings of this study highlight the tension between outmoded forms of practice and the complexity of issues in the public domain. The analysis reveals how the norm of objectivity, originally developed as a shield for the defence of the autonomy of the profession to mediate reality, became, in the case of media coverage of genetic engineering, an obstacle in extending journalism's potential to contribute to public debate. As a method, objectivity failed to provide a set of transparent protocols for the representation of the issue in the public arena; as an account, it reflected the impossibility of separating 'facts' from 'views' and positions of detachment from those of partisanship; and as an attitude, objectivity was endangered by the increasing power of economic imperatives in the production of news. Following this analysis, the thesis explores the influence of journalistic norms on public debate by looking at journalism as a text, as a discursive practice and as a field of cultural production. The GE issue, constructed in the New Zealand press as a key component of the 'knowledge economy', drew attention to the dynamics between the economic imperatives and professional standards of the journalistic field. The objectivity norm was reduced in news reports to reporting 'what people say' rather than what the issue or argument meant, which led to a simplification of the genetic engineering issue in the public domain. The study concludes with the call for a re-examination of the journalistic field in light of the press's incapacity to challenge the status quo and map the social world for its readers.

Two yeast/E.coli shuttle vector plasmids were studied in 1994, termed pIBIDB and pBP33. According to this study, each plasmid should contain at least one ADH2UAS (upstream activation sequence in the alcohol dehydrogenase 2 gene) insert. In the present study, the constructed plasmids were analysed and transformed into laboratory strain yeast. The aim of this project was to identify the orientation, quantity and quality of the insert in the selected plasmids. Methods such as restriction analysis, polymerase chained reaction (PCR), sequencing, plate assays and enzyme assays were used to identify and evaluate the novel inserts. The data presented in this thesis suggest the inserted ADH2UAS fragment did enhance the production of maltose permease and maltase when the transformants were cultivated in maltose and ethanol-glycerol medium. The results suggested that transformants containing two inserts of ADH2UAS had a greater influence on the transformants than a single insert. But the inserts within the vectors and in transformed laboratory stain yeast appeared unstable. This could be due to the method used for plasmid construction and the storage condition of the transformants / Master of Science (Hons)

Legume-rhizobia symbioses contribute at least 20% of the biosphere's supply of reactive nitrogen. These unique associations rely on the exchange of specific molecular signals between nitrogen-fixing soil bacteria, collectively called rhizobia, and their host plants and, with few exceptions, result in the formation of root nodules, which provide an environment suitable for nitrogen fixation. However, nitrogen fixation is energetically expensive and nodule proliferation, in much the same manner as the proliferation of other meristems in plants, must be controlled in order to attain equilibrium between cell proliferation and differentiation. Nodule proliferation is controlled primarily and systemically by autoregulation of nodulation (AON). A gene central to this process was first isolated by map-based cloning from soybean (Glycine max) and was named G. max Nodule Autoregulation Receptor Kinase (GmNARK) in accordance with its biochemical and physiological functions. Expression patterns of GmNARK have been described by several investigators; however, these reports were based on either non-quantitative methods or a limited number of tissue types. More importantly, the expression domains of GmNARK were completely unknown. The study described in this thesis utilised techniques such as quantitative RT-PCR (QRTPCR), transcription start site mapping, promoter-reporter gene fusion, and promoter deletion, to analyse the expression levels and domains of GmNARK across a variety of tissues as well as identify the promoter elements that are responsible for the basal and tissue-specific expression of GmNARK. In addition, the promoter activity of GmNARK was also compared with that of Lotus japonicus HAR1, the GmNARK orthologue, in both homologous and heterologous transformation systems. Based on QRT-PCR, GmNARK was expressed to varying levels throughout the plant; the transcript was detected at high levels in mature leaves and roots but to a lesser extent in young leaves, shoot tips and nodules. The transcript level was not significantly affected by Bradyrhizobium japonicum during the first week following inoculation. Histochemical analysis of L. japonicus plants carrying either a 1.7 kb GmNARK promoter or 2.0 kb LjHAR1 promoter fused to a beta-glucuronidase reporter gene localised GUS activity to living cells within vascular bundles, especially phloem cells in leaves, stems, roots, and nodules. Phloem-specific expression was also detected in soybean hairy roots carrying these constructs. These results suggested that both cis- and trans-acting elements required for the transcriptional regulation of these orthologous genes are likely to be conserved. In contrast, 1.7 kb of the GmNARK promoter did not drive phloem-specific expression in Arabidopsis thaliana, indicating the absence of the trans-acting elements required for the tissue-specificity of GmNARK in this distantly related species. The comparison of 2.0 kb of promoter sequences of GmNARK, LjHAR1 and Medicago truncatula SUNN, another GmNARK orthologue, using bioinformatics and computational approaches indicated several highly conserved motifs including a putative negative regulatory region (NRR), which was previously reported to repress gene expression in non-phloem cell types. Deletion analysis of the GmNARK promoter, however, ruled out the possibility that this motif, found at -308 bp with respect to the translation start site, was truly functional and located the region controlling phloem-specific expression to DNA sequence between 908 bp and 1.7 kb upstream of the start codon. Two other candidate regions were identified by Multiple EM for Motif Elicitation (MEME). These regions, namely MEME3 and MEME4 showed strong sequence similarity to the corresponding regions of the LjHAR1 promoter. Interestingly, the MEME3 motif was also found in the MtSUNN promoter at a similar location to that of LjHAR1. Potential NRRs in the LjHAR1 and MtSUNN promoters were found in the MEME3 motifs, whereas only a variant form of a NRR in the GmNARK promoter was found in this region. Additionally, an identical semi-palindromic sequence was also observed in the MEME3 motifs of the three orthologous promoters. Based on these findings, the semi-palindromic sequence and the variant form of the NRR are proposed to be positive and negative regulatory elements for the phloem-specific expression of GmNARK, respectively. The computational approaches also identified two potential TATA elements in the GmNARK promoter. Rapid amplification of 5' cDNA ends and promoter deletion analysis have confirmed that they were functional. The two TATA elements in GmNARK promoter appeared to cooperatively direct transcription of GmNARK, but either was adequate for basal transcription. The finding that the expression of AON receptor-like kinase genes is phloem-specific has contributed to a better understanding of AON signalling pathways. The QRT-PCR study and the discovery of cis-acting regulatory regions have also provided crucial information on the transcriptional regulation of GmNARK as well as plant genes in general. Additionally, the promoters of GmNARK and LjHAR1 could potentially be used to drive phloem-specific expression in legume biotechnology research.

<p>To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. </p><p>In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. </p><p>The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. </p><p>In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.</p>

Graduation date: 1999

To investigate mechanisms involved in virus-host interactions, global changes in host gene expression were examined during infection with adenovirus type 2 (Ad2) using cDNA microarray technology. In paper I and II, transcriptional changes in HeLa cells were investigated during the early and late phase of infection, respectively. A limited number of genes, mainly implicated in cell growth and antiviral defence, were found to be differentially expressed in the early phase, whereas modulation of host cell gene expression during the late phase was augmented and mainly focused on growth inhibition and cell architecture. The experimental set-up was then redesigned to follow transcriptional regulatory events in growth synchronised, human primary lung fibroblasts. The immediate response of the host cell within two hours of infection was investigated in paper III, revealing a transient induction of a small number of cellular alert genes. This was followed by an expanded time course presented in paper IV, which included gene expression profiling at eight consecutive time points throughout the infectious cycle. The results indicated that specific sets of cellular genes were targeted at different stages of the infection, and four distinct periods were identified. In summary, the studies presented in this thesis demonstrate that adenovirus interferes with many cellular processes during the progression of infection to optimize the cellular environment for viral replication. These include cell cycle control, cell growth and growth inhibition, as well as DNA, RNA and protein metabolism. However, a transient induction of cellular genes involved in immune response and growth inhibition was observed before the onset of viral gene expression. During the very late stages of infection, the expression of a large number of genes involved in maintaining the cell structure was down-regulated, presumably to facilitate the spread of progeny virus.

Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.

Thesis (Ph. D.)--University of California, San Diego, 2001. / Vita. Includes bibliographical references (leaves 276-291).