Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

The focus of this thesis is to visually show the significance and relationship between the use of natural materials, and geometric patterns used in Māori weaving. The patterns will reflect indigenous episteme of artistic and tacit knowledge. These patterns are significant to the Māori worldview of kaitiakitanga (stewardship of knowledge), which is cognisant in the ontology of Māori weaving. These patterns are significant forms of Māori cultural symbols that reflect elements of nature, evolution of time and space. The focus is to show how natural materials can be utilised in an art form that embraces bicultural activity, as a reference to customary and new age methods of thinking and practice. This leads to self-enquiry and our own responsibilities, only to ask ourselves; What are the guiding principles within art and design, that upholds the core values of Mātauranga Māori? (Māori epistemological thinking). The concept of this thesis is to define the cultural significance of kaitiakitanga (stewardship), through the preservation of Mātauranga Māori and practice as weavers and artists. This concept challenges our own understanding of what we know and what we don’t know about the relationships between people, place, environment and use. The methods and processes used for this work will be based on customary practices and methods, using native materials, endemic to New Zealand. These materials will be harvested at different time periods. The methodologies used in this project, is a product of intrinsic knowledge and testing new boundaries, through researching more specific detail about varieties of harakeke (New Zealand flax) cultivars, testing the flexibility, functionality and durability of materials. This will challenge the test, of making sure that the methods used will be significantly practiced throughout the processes involved in the making of artistic pieces of work, in accordance to tikanga (protocols). The use of native materials enhances cultural values of kaitiakitanga as a metaphor, which asserts sustainability of Māori epistemological notions of practice and meaning.This also applies to the visual language of Māori. The concept of visual language embraces metaphoric meanings and understanding, which relates to our co-existence with the earth, animals and the elements. All these elements of nature are contained within symbolic traditional patterns. Some of these patterns have derived from phenomena of thought structure, historical events and our co-existence through our connectedness to the land, waters, oceans, sky and universe. How can Māori forms of art be embraced and imbued, in modern society, that signifies place, belonging and cultural enhancement?

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

<p>Thrombospondin-1 (TSP-1) is a multifunctional extracellular matrix glycoprotein that is released from platelets α-granule to regulate angiogenesis process. TSP-1 is well-known as an inhibitory factor of angiogenesis that binds to angiogenesis stimulating factors, for example fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), to inhibit angiogenesis. We have cloned TSP-1 domains separately to allow studying of their function and effect on proliferation of human umbilical vein endothelial cells (HUVECs). We used an <em>Escherichia coli</em> expressionsvektor including poly histidin-tags and lac-promoter for induction of the seven successfully cloned domains by IPTG and arabinose. Our result shows that we have very low expression and induction of our protein in the <em>E.coli</em> by IPTG and arabinose, which is most likely due to complications associated with expressing a human protein in a prokaryotic system.</p>

<p>Cancer is a common disease and due to problems with resistance against cancer drugs and the limited benefit from chemotherapy in many diagnoses, there is a need to develop new cancer drugs. In this thesis new methods to screen for cancer drugs and to evaluate their mechanism of action are discussed. </p><p>In Paper I, it was found that by studying the gene expression of a cell line panel and combining the data with sensitivity data of a number of cytotoxic drugs, it was possible to cluster compounds according to mechanism of action as well as identifying genes associated with chemosensitivity.</p><p>In Paper II, studies of compounds with selective activity in drug-resistant cell lines revealed the glucocorticoids as a group of interesting compounds. The glucocorticoid receptor was overexpressed in 8226/Dox40 and the difference in sensitivity was abolished when the cells were treated with a glucocorticoid receptor antagonist.</p><p>In Paper III, an image-based screening method for new proteasome inhibitors was successfully developed and the compounds disulfiram, PDTC and NSC 95397 were identified as inhibitors of the proteasome.</p><p>In Paper IV, disulfiram and PDTC were shown to induce cytotoxic activity, to inhibit the activation of the transcription factor NFkappaB and to inhibit the degradation of proteins normally degraded by the proteasome.</p><p>In Paper V, NSC 95397 was shown to be cytotoxic to all cells in the resistance-based cell line panel as well as to patient samples from a variety of cancer diagnoses. Connectivity Map was successfully used as a tool to propose a new mechanism of action of NSC 95397. The gene expression induced by NSC 95397-treatment was similar to that induced by several proteasome inhibitors not present in the Connectivity Map.</p>

The overall objective of this research was to define the cellular basis underlying heritable subfertility in roosters homozygous for the rose comb allele (R/R). Fertilization in the hen is preceded by the ascension of motile sperm through the vagina and sperm sequestration within sperm storage tubules (SST). The objective of the first set of experiments was to determine if reduced sperm sequestration could account for subfertility. Sperm sequestration differed between genotypes following intravaginal insemination (p<0.0001). However, sperm sequestration did not differ between genotypes when sperm were incubated with SST in vitro (p>0.05). Therefore, subfertility was attributed to reduced sperm transport within the vagina. To test this hypothesis, an assay was developed to evaluate fowl sperm motility in vitro. Based upon this assay, ejaculates from subfertile males contained smaller subpopulations of highly motile sperm than the ejaculates from controls (p<0.001). The objective of the next set of experiments was to characterize the motility of individual sperm and to identify a mechanism that could account for the genotypic difference in sperm cell motility. Computer-assisted sperm motion analysis evaluation revealed that ejaculates from controls contained 91% motile sperm whereas ejaculates from subfertile males contained 62% motile sperm (p<0.001). The ATP concentration in sperm from subfertile males was 63% less than that of sperm from controls (p<0.001). A link between sperm ATP concentration and immotility was investigated. First, sperm metabolism was evaluated using motility as an endpoint. The genotypic difference in sperm motility persisted when ATP synthesis was limited to glycolysis (p<0.001). Consequently, mitochondrial respiration could not account for the genotypic difference in sperm motility. In contrast, sperm uptake of [1,2-��H] 2-deoxy-D-glucose did differ between genotypes (p<0.001). The activity of key glycolytic enzymes, creatine kinase, and dynein ATPase did not differ between genotypes (p>0.05). Therefore reduced sperm motility did not appear to be due to ATP synthesis, allocation of high energy phosphate bonds along the axoneme, or ATP consumption (p>0.05). In conclusion, subfertility of roosters homozygous for the rose comb allele was attributed to decreased spermatozoal glucose transport. / Graduation date: 1997

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells. / Singapore-MIT Alliance (SMA)

In vivo analyses of the Von Willebrand Factor (VWF) promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells. This fragment is active in all embryonic vessels of transgenic mice but in adult mice its activity is restricted to brain vascular endothelial cells, while endogenous VWF gene is expressed in vasculature of all major organs. In this study we demonstrate that a DNase I hypersensitive (HSS) sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. Our results demonstrated that Nuclear Factor 1 (NF1) and Nuclear transcription Factor Y (NFY) repressors contribute to VWF organ-specific regulation. Mutation of the NF1 binding site resulted in promoter activation in lung and heart, while mutation of the repressor corresponding to a novel binding site for NFY resulted in promoter activation in kidney vasculature. / Experimental Medicine