published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.

我們研究了極端嗜熱古菌Pyrococcus horikoshii 的嗜熱性酰基磷酸酶acylphosphatase (PhAcP) ，以及與它同源的人類嗜溫性酶(HuCTAcP) 的熱穩定性。我們發現PhAcP的熱穩定性之所以比HuCTAcP高出很多，是由於熔融溫度的焓變值的增加以及變性熱容量的減少。研究蛋白質熱穩定性的其中一個推動力，是運用我們的知識去製造耐高溫的酶，這對工業和生物技術非常重要。通過交換 PhAcP的嗜熱核和 HuCTAcP的嗜溫核以及研究變種的熱穩定性，我們認為蛋白表面是改善熱穩定性工程的首選地區。嗜熱和嗜溫蛋白質之間的主要區別，在於嗜熱蛋白質有更多的表面鹽橋。為了探討表面鹽橋對蛋白熱穩定性的貢獻，我們採用雙突變循環，量化嗜熱蛋白T.celer L30e一表面鹽橋的相互作用能。我們的結果顯示，表面鹽橋對蛋白質穩定性的貢獻是獨立於溫度變化的。此外，表面鹽橋對蛋白質變性熱容量的減少起一定作用。 / We characterized the thermodynamic properties of thermophilic acylphosphatase from Pyrococcus horikoshii (PhAcP) and its mesophilic homologue from human (HuCTAcP) and found that the much higher thermostability of PhAcP was the result of increased enthalpy change at melting temperature and decreased heat capacity change of unfolding. One incentive to study protein thermostability is to apply our knowledge to engineer thermostable enzyme which is of great industrial and biotechnological importance. Through swapping the core of thermophilic PhAcP and mesophilic HuCTAcP and characterizing the thermostability of the resulting variants, we concluded that surface is a preferred region for thermostability engineering. The key difference between thermophilic and mesophilic proteins lies in the surface on which thermophilic proteins have more salt-bridges. To investigate the contribution of surface salt-bridge to protein thermostability, we employed double-mutant cycle to quantify the pair-wise interaction energy of a surface salt-bridge in thermophilic T.celer L30e. Our results showed that surface salt-bridge had a temperature independent contribution to the protein stability and plays a role in the reduction of the heat capacity change of unfolding. / Detailed summary in vernacular field only. / Yu, Tsz Ha. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 89-93). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Content --- p.iv / List of Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.ix / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- Definition of protein stability --- p.1 / Chapter 1.2 --- Contribution to thermostability from the protein core --- p.2 / Chapter 1.2.1 --- Definition of hydrophobic effect --- p.2 / Chapter 1.2.2 --- Why the hydrophobic effect has been recognized as the major driving force for protein folding? --- p.2 / Chapter 1.3 --- Contribution to thermostability from the protein surface --- p.6 / Chapter 1.3.1 --- Electrostatic interactions --- p.7 / Chapter 1.3.2 --- Exposed hydrogen bonds and helix propensity --- p.10 / Chapter 1.3.3 --- Surface loop --- p.11 / Chapter 1.4 --- Protein stability curve --- p.13 / Chapter 1.5 --- The incentive to study protein thermostability --- p.17 / Chapter Chapter 2: --- Materials and Methods --- p.18 / Chapter 2.1 --- Generation of DNA clones --- p.18 / Chapter 2.2 --- Plasmid transformation to competent E. coli strain --- p.18 / Chapter 2.3 --- Expression of recombinant proteins --- p.19 / Chapter 2.3.1 --- T. celer L30e --- p.19 / Chapter 2.3.2 --- Acylphosphatase --- p.20 / Chapter 2.4 --- Protein extraction from E. coli by sonication --- p.20 / Chapter 2.5 --- Protein purification --- p.20 / Chapter 2.5.1 --- T. celer L30e --- p.20 / Chapter 2.5.2 --- Acylphosphatase --- p.22 / Chapter 2.6 --- Circular dichroism experiment --- p.22 / Chapter 2.6.1 --- Thermal denaturation --- p.22 / Chapter 2.6.2 --- Denaturant-induced denaturation --- p.23 / Chapter 2.7 --- Differential scanning calorimetry --- p.24 / Chapter 2.8 --- Enzymatic assay of AcPs using benzoyl phosphate as substrate --- p.25 / Chapter 2.9 --- Crystallization and crystal structure refinement --- p.26 / Chapter Chapter 3: --- Thermodynamic characterization of thermophilic acylphosphatase from Pyrococcus horikoshii and its mesophilic homologue from human --- p.27 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- PhAcP has a higher thermostability than HuCTAcP --- p.31 / Chapter 3.2.2 --- PhAcP has an upshifted and broadened PSC compared with the PSC of HuCTAcP --- p.33 / Chapter 3.2.3 --- PhAcP has a highly enhanced ΔH[subscript m] and slightly reduced ΔC[subscript p]. --- p.37 / Chapter 3.3 --- Discussion --- p.41 / Chapter 3.3.1 --- Thermophilic AcPs harness enhanced ΔH[subscript m] and reduced ΔC[subscript p] to attain a higher thermostability. --- p.41 / Chapter 3.3.2 --- Possible structural differences between PhAcP and HuCTAcP that lead to the higher thermostability of PhAcP. --- p.42 / Chapter Chapter 4: --- Protein surface is a preferred region for thermostability engineering --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Results --- p.51 / Chapter 4.2.1 --- Construction of the chimera with Thermophilic Surface and Mesophilic Core (T[subscript surf]M[subscript core]), and the chimera with Mesophilic Surface and Thermophilic Core (M[subscript surf]T[subscript core]). --- p.51 / Chapter 4.2.2 --- The crystal structures of the chimera T[subscript surf]M[subscript core] and M[subscriptsurf]T[subscript core] reveal that anticipated interactions are engineered. --- p.54 / Chapter 4.2.3 --- Characterization of the thermodynamic stabilities of the chimeras at different temperatures --- p.56 / Chapter 4.3 --- Discussion --- p.59 / Chapter 4.3.1 --- Engineering a thermophilic surface onto a mesophilic protein enhances thermostability --- p.59 / Chapter 4.3.2 --- Concluding remarks --- p.64 / Chapter 4.4 --- Supplementary Tables --- p.64 / Chapter Chapter 5: --- Stabilizing surface salt-bridge enhances protein thermostability by upshifting the protein stability curve --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.2 --- Results --- p.70 / Chapter 5.2.1 --- Design of variants --- p.70 / Chapter 5.2.2 --- Determination of the pair-wise interaction energy of K46 and E62 by double-mutant cycles --- p.72 / Chapter 5.2.3 --- Surface salt-bridge K46/E62 is stabilizing and its interaction energy is insensitive to temperature changes --- p.75 / Chapter 5.2.4 --- Stabilizing salt-bridge K46/E62 reduces ΔC[subscript p] and upshifts protein stability curve --- p.77 / Chapter 5.3 --- Discussion --- p.80 / Chapter 5.3.1 --- Stabilization effect brought by surface salt-bridge is insensitive to temperature change --- p.80 / Chapter 5.3.2 --- The pair-wise interaction energy of K46-E62 determined by DMC reflects their electrostatic interaction --- p.80 / Chapter 5.3.3 --- Surface salt-bridge contributes to the reduction of ΔC[subscript p] in thermophilic proteins --- p.81 / Chapter 5.3.4 --- Reduced ΔC[subscript p] upshifts and broadens the PSC resulting a higher T[subscript m] --- p.83 / Chapter 5.4 --- Supplementary Figures and Tables --- p.85 / Chapter Appendix --- List of Publications --- p.88 / References --- p.89

DNA arrays, capable of detecting specific DNA sequences from a sample have become widely used. They rely on DNA heterogeneous hybridization, which is the binding between a single strand of DNA immobilized on a surface (probe) and its complementary strand present in the bulk (target). In order to improve the hybridization time in DNA arrays, it is crucial to understand the kinetics of DNA hybridization. The study of the Damkohler number that compares the DNA supply by diffusion to the DNA consumption by reaction (hybridization) shows that in many cases we can expect DNA hybridization to be a diffusion limited process. This is verified by a finite element study, where a whole microfluidic chamber (bulk and reacting surface) is simulated. In these cases, the formation of a depletion zone above the sensing zone is observed. The reaction rate is much lower than in the ideal case where the reaction would be reaction rate limited. A better DNA transport could be a solution to overcome the diffusion barrier. Therefore, the influence of convection on DNA hybridization was studied. Finite element simulation shows that even a small DNA velocity (10 ƒ�m/s) can greatly enhance the overall reaction rate and help preventing the formation of a depletion zone. These observations are valid when one kind of probe reacts with one kind of target. In reality, non specific hybridization can happen between a probe and a non complementary target. We show that in some cases, non specific hybridization can slow down the kinetics and reduce the fraction of specifically hybridized probes at equilibrium. The fraction of non specific hybrids can reach a maximum before decreasing and reaching equilibrium, suggesting that a longer hybridization time would lead to a better specificity. The addition of convective transport does not affect the equilibrium, but allows to reach it faster and with a better ratio between specific and non specific hybrids during the process. Therefore, convective transport of DNA appears to be beneficial. Another possibility is to act on the DNA itself to focus it near the sensing zone. Our study of the different electrokinetic forces leads us to derive the expression of the dielectrophoretic force in a field resulting from the combination of a DC field and an AC field. This could be a novel way to act on polarizable particles like DNA.

While the genetic control of ureteric bud (UB) morphogenesis has been extensively studied, the cellular basis of this process remains unclear. The renal organoid system is a novel technique in which embryonic kidneys are dissociated into single cells and then reaggregated, where they reassociate to form organotypic structures. This system may be very beneficial for investigating the cellular basis of ureteric bud development. Here, we first used a fluorescent UB marker, Hoxb7:myrVenus, and time-lapse microscopy to characterize the cellular and tissue-level events during self-organization and UB morphogenesis of E12.5 or E14.5 renal organoids. Briefly, we found that UB structures self-assembled by aggregation of individual cells that sent out long cell processes. The cellular aggregates grew and elongated into epithelial tubes that displayed characteristic ampullae, bifurcated, and appropriately expressed UB tip markers analogous to their in vivo counterparts. We also found that cap mesenchymal cells are attracted to newly formed epithelial structures early in renal organoid development, and were later found in cell clusters surrounding new branches. RET is a trans-membrane tyrosine kinase receptor (RTK), expressed in ureteric bud cells, whose expression is gradually restricted to the tips of the growing ureteric tree. We demonstrate that the renal organoid system can be used, as an alternative to the generation of in vivo chimeric embryos, to study Ret-dependent cell rearrangements previously shown to establish and maintain the UB tip progenitor domain. Chimeric renal organoids that juxtaposed wild-type cells with Sprouty1–/– mutant cells (higher Ret-signaling) or with Ret51/cre (lower Ret-signaling) mutant cells recapitulated the cell sorting pattern observed in similar in vivo chimeras. The cells with higher Ret-signaling preferentially sorted to, and were maintained in, the forming and growing tips of these mosaic ureteric bud structures, out-competing cells with lower Ret-signaling. We then used the mosaic organoid system to ask if fibroblast growth factor receptor 2 (Fgfr2), another RTK expressed in the ureteric bud and important for its development, also mediates individual cell rearrangements that generate and maintain the UB tips. UB cells null for Fgfr2 were largely unable to compete with wild-type cells for occupancy of the UB tips in chimeric renal organoids. Using the innovative MASTR (Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination) technique in vivo, mosaic homozygous deletion of Fgfr2 in newly formed ureteric buds also revealed that mutant cells were slightly deficient in their ability to contribute to Fgfr2 heterozygous UB tips. This demonstrates a novel, cell-autonomous role of Fgfr2 in ureteric bud development. Matrix metalloproteinase 14 (MMP14) is a membrane-bound protein known to participate in a wide variety of cell functions including degradation of the extracellular matrix (ECM), cell signaling, and cell-autonomous cell migration. It is expressed in the UB and was discovered to act downstream of Ret-signaling. Although needed in the ureteric epithelium for ECM degradation and proper UB morphogenesis, its specific function in the UB has not been thoroughly investigated. In generating in vivo chimeras, we discovered that Mmp14 null cells could contribute to wild-type ureteric bud tips at E12.5 and E14.5, demonstrating that, despite its documented role in UB branching, Mmp14 does not have a cell-autonomous role in the cell rearrangements observed during UB morphogenesis.

The aim of this thesis is to evaluate the role of the extracellular environment in regulating breast cancer cell morphological and invasive characteristics. In vitro experiments of breast cancer cell lines in three dimensional matrices, which afford control over variables of interest while maintaining physiological relevance, were utilized for this purpose. We evaluated the sensitivity of cell morphology to the dimensionality, biochemistry, and mechanical properties of the extracellular environment as well as the reciprocal effects cells display when remodeling the extracellular environment during invasion. Chapter 1 introduces background material on breast cancer development, classification systems, and in vitro methods of research. Chapter 2 describes protocols for cell care and experiments used in these studies. In chapter 3, we explore the role of fibrillar collagen I environments in breast cancer cell invasion. This was motivated by previous research that has associated high breast tissue density with breast cancer risk and poor prognosis as well as tissue stiffness with cancer cell aggressiveness. Breast cancer cells were found to regain an invasive phenotype in sterically constrained environments when the extracellular matrix included a fibrillar component. In chapter 4, the relationship between cell morphology and invasive behavior in various dimensional contexts was assessed. Anecdotal evidence has shown stellate morphology may be associated with epithelial to mesenchymal transition and invasive capacity in cancer cells. Differences in the dimensionality and biochemistry of the environment resulted in changes to cell aggregate morphology. Although morphology did not predict invasive capacity as measured by spheroid invasion in collagen I, invasion was found to correlate with cancer-related gene expression profiling, suggesting the ability of cancer cells to utilize more than one mode of invasion. Chapter 5 explores to what degree the presence of invasive cells can give rise to invasive behavior from noninvasive cells. Segregation of cell subtypes during co-culture spheroid formation was found to be altered in the presence of BME. When implanted into collagen gels, invasive cell lines that generate structural changes to the extracellular matrix on their own were able to confer invasive behavior to otherwise noninvasive cell lines in some cases. Chapter 6 summarizes these findings and suggests further studies. Appendix 1 lists useful abbreviations. In Appendices 2 and 3, codes for ImageJ and Matlab-based analyses are recorded. Through this work, we see how cell morphology and invasive capacity are influenced by the extracellular environment. Cells that can interact with components of the extracellular matrix through matrix-specific integrins show a range of capacities for remodeling the extracellular environment, which in turn plays a role in invasive capacity. We anticipate that enhanced understanding of the role of the extracellular environment in regulating cell morphology and invasive behavior will lead to advances in the study of cell locomotion as well as in cancer research, diagnosis, and treatment.

Finally, the gene tree of the true crabs, Brachyura, confirms that the basal "Podotremata" is paraphyletic, with the Raninoidea and Cyclodorippoidea more closely related to Eubrachyura than to the other podotremes. Within the monophyletic Eubrachyura, the analysis supports the reciprical monophyly of the two subsections, Heterotremata and Thoracotremata. All of the Old World freshwater crabs cluster together, representing an early diverged lineage in the Heterotremata. / From the inferred phylogeny, we have obtained new insights on the evolution of decapods. First, the spiny lobster from the family Palinuridae is found to be paraphyletic with the polyphyletic Synaxidae nested within it. The Stridentes forms a monophyletic assemblage, indicating that the stridulating sound producing organ evolved only once in the spiny lobsters. Moreover, the spiny lobsters originated in the shallower water rocky reefs of the Southern Hemisphere and then invaded deep sea habitats and diversified. / In sum, I demonstrate the utility of the nuclear protein-coding gene markers in decapod phylogeny and they are informative across a wide range of taxonomic levels. I propose that nuclear protein-coding genes should constitute core markers for future phylogenetic studies of decapods, especially for higher systematics. / Second, we show that hermit crabs have a single origin, but surprisingly, that almost all other major clades and body forms within the Anomura, are derived from within the hermit crabs. The crab-like form and squat lobster form have each evolved at least twice from separate symmetrical hermit crab ancestors. These remarkable cases of multiple parallelism suggest considerable phenotypic flexibility within the hermit crab ground plan, with a general tendency towards carcinization. Rather than having a separate origin from other major clades, hermit crabs have given rise to most other major anomuran body types. / The high diversity of decapods has attracted the interest of carcinologists but there is no consensus on decapod phylogeny in spite of the endeavors using both morphological and molecular approaches. New sources of information are necessary to elucidate the phylogenetic relationships among decapods. In the present study, I attempted to develop and apply the nuclear protein-coding gene markers on decapod phylogeny. Using only two protein-coding genes, we have successfully resolved most of the infraordinal relationships with good statistical support, indicating the superior efficiency of these markers compared to nuclear ribosomal RNA and mitochondrial genes commonly used in phylogenetic reconstruction of decapods. Apparently these two types of markers suffer from the problems of alignment ambiguities and rapid saturation, respectively. Subsequently, I tried to apply the nuclear protein-coding genes in revealing interfamilial and intergeneric evolutionary history in three selected decapod groups, the spiny lobster (family Palinuridae), the infraorder Anomura and the true crabs of the infraorder Brachyura to further evaluate the utility of these markers and reconstruct the evolutionary history the groups. Trees with robust support can be obtained using sequences of three to five genes for the infraorders and families tested including the most speciose Brachyura. The genes are shown to be informative in elucidating interspecific phylogeny as well. / Tsang, Ling Ming. / Adviser: Ka Hou Chu. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 127-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Pearl oysters of the genus Pinctada include some economically important species. The taxonomy of some of the species is problematic. Phylogenetic relationship of the species in the genus is also poorly studied. In the present study, phylogenetic relationships of P. chemnitzi, P. fucata, P. margaritifera, P. maxima, P. nigra, P. radiata (from China), P. fucata martensii (from Japan), P. albina and P. imbricata (from Australia) were studied with Pteria penguin as an outgroup, and genetic variation of Chinese P. fucata, Japanese P. fucata martensii and Australian P. imbricata populations were investigated (1) to address the taxonomic confusion and phylogeny of pearl oysters, (2) to understand the genetic connections between the Chinese P. fucata, Japanese P. fucata martensii and Australian P. imbricata in west Pacific and (3) to provide information for the genetic improvement program initiated in China. / Since P. fucata, P. fucata martensii and P. imbricata are synonymous, to study the genetic differentiation and genetic variation of such widely distributed populations is helpful in understanding their genetic connections. For this purpose, five populations, three from China (Daya Bay, Sanya Bay and Beibu Bay), one from Japan (Mie Prefecture) and one from Australia (Port Stephens) were studied using AFLP technique. Three primer pairs generated 184 loci among which 91.8-97.3% is polymorphic. An overall genetic among populations and an average of 0.37 within populations (ranging from 0.35 in Japanese population to 0.39 in Beibu Bay population) were observed. Genetic differentiation among the five populations is low but significant as indicated by pairwise GST (0.0079-0.0404). AMOVA further shows that differentiation is significant among the five populations but is not significant at a broader geographical scale, among the three groups of Chinese. Japanese and Australian populations or among the two groups of Australian and north Pacific populations. The low level of genetic differentiation indicated that P. fucata populations in the west Pacific are genetically linked. Among the five populations, the Australian one is more differentiated from the others, based on both pairwise AMOVA and GST analyses, and is genetically isolated by distance as indicated by Mantel test. However, genetic differences among the three Chinese populations are not correlated with the geographic distances, suggesting that Hainan Island and Leizhou Peninsula may act as barriers blocking gene flow. / The above three wild Chinese populations in southern China were compared with the three adjacent cultured populations using AFLP markers. Three pairs of primers generated 184 loci among 179 individuals in populations from Beibu Bay, Daya Bay and Sanya Bay. A high level of genetic diversity, ranging from 0.363 in a wild population in Sanya Bay to 0.388 in a wild population in Beibu Bay, was observed within both wild and cultured populations, indicating an absence of strong bottleneck effects in the history of cultured P. fucata populations. Yet cultured populations in Sanya Bay and Beibu Bay had more fixed loci than the corresponding wild populations. Genetic differentiation in most pairwise comparisons of populations was significant. AMOVA indicated that genetic variation among populations were very low (1.77%) though significant, while more than 98% variation resided among individuals within population. These findings provide no evidence to show that hatchery practice of pearl oyster in China to date has significantly affected the genetic diversity of the cultured populations, and suggest that all populations are competent for selection. Yet the significant genetic differentiation among populations implies that any translocation of individuals for genetic improvement program should be managed with caution for the preservation of genetic diversity in natural populations. / The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were compared among the above nine taxa, based on sequences determined by the present study and those available from Genl3ank. The phylogenetic analysis indicates that the pearl oysters studied constitute three clades: clade I with the small oysters P. fucata, P. fucata martensii and P. imbricata, clade II with P. albina, P. nigra, P. chemnitzi and P. radiata, and clade III and clade III with the big pearl oysters P. margaritifera and P. maxima forming the basal clade. Clade II is made up two subclades: clade IIA consisting of P. albina and P. nigra and clade IIB consisting of P. chemnitzi and P. radiata. The topology of the phylogenetic tree and substitution pattern of ITS sequences suggest that P. margaritifera and P. maxima are primitive species and P. chemnitzi is a recent species. The genetic divergences between clades ranged from 28% to 76.5%, and between subclades, 8.7-10.2%. In clade I, the interspecific genetic divergences ranged from 0.6% to 1.4%, and overlapped with interspecific divergences (0.6-1.1%), indicating that P. fucata, P. fucata martensii and P. imbricata may be conspecific. Based on amplified fragment length polymorphism (AFLP) markers and ITS sequences from more individuals, analyses of the populations of these three taxa also support the conclusion that Chinese P. fucata, Japanese P. fucata martensii and Australian P. imbricata are the same species, with P. fucata being the correct name. The genetic divergence between P. albina and P. nigra was also very low (1.2%), suggesting that they may represent two subspecies that can only be distinguished by shell color. The genetic divergences between P. maxima and P. margaritifera, and between clade IIA and clade IIB ranged from 8.3% to 10.2%, suggesting that they are closely related, respectively. The ITS1 sequence of P. radiata from GenBank is almost identical to that of P. chemnitzi determined in the present study, suggesting that the specimen used for the P. radiata sequence was possibly misidentified. / Yu Dahui. / "August 2005." / Adviser: Ka Hou Chu. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6125. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 100-124). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

目的： / TGF-β/Smad信号通路在慢性肾脏纤维化疾病中有着重要的作用。大量研究证实Smad3在TGF-β/Smad信号介导的肾脏纤维化过程中发挥着关键的作用，但TGF-β/Smad3这一关键信号通路的分子机制尚不明确。该论文研究假设TGF-β通过Smad3介导的microRNA-21（miR-21）导致肾脏纤维化；特异性的针对miR-21将有助于提供有效的、创新性的方法治疗慢性肾脏纤维化疾病。 / 方法： / 该论文研究利用大鼠肾小管上皮细胞株（TEC）及系膜细胞株(MC)，探讨TGF-β1诱导miR-21表达增高的机制；通过过表达及抑制miR-21在上述细胞株的表达，研究miR-21在TGF-β1的刺激及高糖环境下，对肾脏纤维化的影响。进一步通过采用超声微泡介导基因转入技术，将miR-21 shRNA质粒特异性的诱导入梗阻性肾病小鼠模型（UUO）及糖尿病肾病db/db小鼠模型的肾脏中，体内研究抑制miR-21对纤维化的治疗作用。通过荧光素酶报告分析，检测miR-21的靶基因。 / 结果： / 通过微阵列（microarray）及实时荧光定量PCR（realtime PCR）技术，检测miR-21在TGF-β1及高糖的刺激下的表达水平，结果发现其表达在TEC及MC均明显升高。进一步通过体内体外实验，在TGF-β1及高糖的刺激下，高表达的miR-21和TGF-β/Smad3信号通路的激活有关。体外对miR-21的功能进行研究，结果表明在TGF-β1及高糖的刺激下过表达miR-21促进TEC及MC纤维化的发生，而抑制miR-21的表达有效的降低TEC及MC的纤维化损伤。体内利用梗阻性肾病小鼠模型，通过采用超声微泡介导基因转入技术，将miR-21 shRNA质粒分别于模型前后特异性的诱导入小鼠肾脏，结果发现抑制miR-21的表达能有效地阻止肾脏纤维化的进展，减轻梗阻肾纤维化的程度；利用2型糖尿病肾病db/db小鼠模型，发现抑制miR-21的表达能减轻糖尿病肾病小鼠肾脏的纤维化及炎症程度，并改善糖尿病肾病小鼠的肾脏功能。采用荧光素酶报告分析，结果发现Smad7是miR-21的直接靶基因，miR-21通过直接抑制Smad7的表达从而影响肾脏纤维化和炎症。该论文的研究结果提示miR-21在慢性肾脏纤维化疾病中的治疗作用和前景。 / 结论： / miR-21作为TGF-β/Smad3信号通路的下游因子，在肾脏纤维化的发生发展中起着重要作用。特异性针对miR-21为肾脏纤维化疾病的治疗提供了创新性的有效方法。 / Objectives: / TGF-β/Smad signaling plays a critical role in renal fibrosis in chronic kidney disease (CKD). It is well known that Smad3 is a key mediator of downstream TGF-β/Smad signaling in renal fibrosis, however, the exact mode of TGF-β/Smad3 in renal fibrosis remains unclear. In this thesis, we tested a novel hypothesis that TGF-β may act by regulating the Smad3-dependent microRNA-21（miR-21） to mediate renal fibrosis and that specific targeting miR-21 may represent an effective and novel therapy for chronic kidney disease. / Methods: / The regulatory mechanism of TGF-β1-induced miR-21 expression via the Smad3-dependent pathway was studied in a rat NRK52E tubular epithelial cell (TEC) line and mesangial cell (MC) line. The functional role of miR-21 in renal fibrosis was investigated by overexpressing or down-regulating of miR-21 both in TGF-β1 and high glucose (HG) conditions in TEC and MC. The therapeutic potential role of miR-21 in kidney diseases were examined in unilateral ureteral obstructive (UUO) mouse model and in db/db mice by applying an ultrasound-microbubble-mediated anti-miR-21 gene transfer technique. The target gene of miR-21 was identified by luciferase reporter assays. / Results: / By microarray and realtime PCR, upregulation of miR-21 was observed in tubular epithelial cells (TECs) and mesangial cells (MCs) in response to TGF-β1 and high glucose (HG). Both in vitro and in vivo studies demonstrated that the upregulation of miR-21 expression during renal fibrosis and diabetic conditions was dependent on the activation of TGF-β/Smad3 signaling. The findings that overexpression of miR-21 promoted but knockdown of miR-21 suppressed TGF-β1-induced renal fibrosis and HG-induced diabetic kidney injury demonstrated the functional importance for miR-21 in fibrosis and inflammation in vitro. More importantly, ultrasound-microbubble-mediated gene transfer of a miR-21 knockdown plasmid into the mouse kidney before and after established unilateral ureteral obstructive (UUO) nephropathy was able to prevent and halt the progression of renal fibrosis. Furthermore, we also found that blockade of miR-21 was capable of attenuating diabetic kidney injury including progressive renal fibrosis and inflammation, as well as renal functional injury in a mouse model of type 2 diabetes in db/db mice. The functional role of miR-21 on renal fibrosis and inflammation was through Smad7, which was identified as a direct target gene of miR-21. All these results revealed a therapeutic potential for targeting miR-21 in chronic kidney disease. / Conclusions: / In conclusion, miR-21 is a downstream mediator of TGF-β/Smad3 signaling and plays a critical role in the development of renal fibrosis. Targeting miR-21 may represent a novel and effective therapy to combat renal fibrosis in chronic kidney disease. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhong, Xiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 206-221). / Abstract also in Chinese. / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.vi / DECLARATION --- p.xiv / ACKNOWLEDGEMENTS --- p.xv / LISTS OF ABBREVIATION --- p.xvii / LISTS OF FIGURES AND TABLES --- p.xx / PUBLICATIONS --- p.xxvi / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- MicroRNA --- p.1 / Chapter 1.1.1 --- Biogenesis and Function of MicroRNA --- p.2 / Chapter 1.1.2 --- Recognition of MicroRNA Target --- p.5 / Chapter 1.2 --- MicroRNA-21 --- p.6 / Chapter 1.2.1 --- The Role of miR-21 In Fibrosis-related Disease --- p.7 / Chapter 1.2.2 --- The Role of miR-21 In Inflammatory Disease --- p.10 / Chapter 1.2.3 --- The Regulation of miR-21 --- p.12 / Chapter 1.3 --- TGF-β/SMADS SIGNALING IN RENAL FIBROSIS --- p.15 / Chapter 1.3.1 --- TGF-β/Smads Signaling --- p.15 / Chapter 1.3.2 --- The Diverse Role of TGF-β/Smads Signaling In Renal Fibrosis and Inflammation --- p.19 / Chapter 1.3.2.1 --- The Diverse Role of TGF-β1 In Renal Fibrosis and Inflammation --- p.19 / Chapter 1.3.2.2 --- The Diverse Role of Smad2 and Smad3 In Renal Fibrosis --- p.20 / Chapter 1.3.2.3 --- The Inhibitory Role of Smad7 In Renal Fibrosis and Inflammation --- p.22 / Chapter 1.4 --- THE POTENTIAL ROLE OF MIR-21 IN RENAL FIBROSIS --- p.24 / Chapter CHAPTER II --- MATERIALS AND METHODS --- p.26 / Chapter 2.1 --- MATERIALS --- p.26 / Chapter 2.1.1 --- Reagents --- p.26 / Chapter 2.1.1.1 --- Reagents for Cloning --- p.26 / Chapter 2.1.1.2 --- Reagents for Cell Culture --- p.27 / Chapter 2.1.1.3 --- Reagents for Realtime RT-PCR --- p.27 / Chapter (1) --- For miR-21 Assay --- p.27 / Chapter (2) --- For Fibrotic and Inflammatory Index Assay --- p.28 / Chapter 2.1.1.4 --- Reagents for Western Blot --- p.28 / Chapter 2.1.1.5 --- Reagents for In Situ Hybridization (ISH) --- p.29 / Chapter 2.1.1.6 --- Reagents for Immunochemistry Staining --- p.30 / Chapter 2.1.1.7 --- Reagents for Luciferase Activity Assay --- p.30 / Chapter 2.1.1.8 --- Reagents for CHIP Assay --- p.31 / Chapter 2.1.1.9 --- Reagents for Urine Albumin Excretion Measurement --- p.31 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.2.1 --- Buffers for Western Blot --- p.31 / Chapter (1) --- RIPA Lysis Buffer --- p.31 / Chapter (2) --- 4× SDS Loading Sample Buffer --- p.32 / Chapter (3) --- 10% Ammonia Persulfate (10% APS) --- p.33 / Chapter (4) --- 1.5 M Tris Buffer Mix (For 15% Resolving Gel) --- p.33 / Chapter (5) --- 1.5 M Tris Buffer Mix (For 12% Resolving Gel) --- p.33 / Chapter (6) --- 1.5 M Tris Buffer Mix (For 10% Resolving Gel) --- p.33 / Chapter (7) --- 0.5 M Tris Buffer Mix (For 4% Stacking Gel) --- p.34 / Chapter (8) --- 15% Resolving Gel --- p.34 / Chapter (9) --- 12% Resolving Gel --- p.34 / Chapter (10) --- 10% Resolving Gel --- p.35 / Chapter (11) --- 4% Stacking Gel --- p.35 / Chapter (12) --- Tris Buffered Saline (TBS) --- p.35 / Chapter (13) --- TBS-Tween 20 (TBS-T) --- p.36 / Chapter (14) --- SDS-PAGE Electrophoresis Running Buffer --- p.36 / Chapter (15) --- Transfer Buffer without SDS --- p.36 / Chapter (16) --- Transfer Buffer --- p.37 / Chapter (17) --- Blocking Buffer --- p.37 / Chapter (18) --- Antibody Diluent Buffer --- p.37 / Chapter 2.1.2.2 --- Buffers for Immunochemistry Staining --- p.37 / Chapter (1) --- Methyl Carnoy's Fixative --- p.37 / Chapter (2) --- Phosphate Buffered Saline (PBS) --- p.38 / Chapter (3) --- Horseradish Peroxidase (HRP) Inactivation Solution --- p.38 / Chapter (4) --- Microwave-based Antigen-retrieval Solution --- p.38 / Chapter (5) --- Blocking Buffer --- p.39 / Chapter (7) --- Substrate Solution for Fast Blue Staining --- p.39 / Chapter (8) --- Substrate Solution for DAB Staining --- p.39 / Chapter 2.1.2.3 --- Buffers for In Situ Hybridization (ISH) --- p.40 / Chapter (1) --- Fixative Solution --- p.40 / Chapter (2) --- DEPC-treated Water --- p.40 / Chapter (3) --- DEPC-treated PBS --- p.40 / Chapter (4) --- 0.2N HCl --- p.41 / Chapter (5) --- Proteinase K Solution --- p.41 / Chapter (6) --- 5XSSC/50% Deionized Formamide --- p.41 / Chapter (7) --- 5XSSC --- p.41 / Chapter (8) --- 2XSSC --- p.41 / Chapter (9) --- 0.2XSSC --- p.42 / Chapter (10) --- Hybridization Solution --- p.42 / Chapter (11) --- Solution B1 --- p.42 / Chapter (12) --- Solution B2 --- p.42 / Chapter 2.1.3 --- Antibodies --- p.43 / Chapter 2.1.3.1 --- The Primary Antibodies --- p.43 / Chapter 2.1.3.2 --- The Second Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.1.4.1 --- Primers for Realtime RT-PCR --- p.45 / Chapter 2.1.4.2 --- Primers for Luciferase Activity Assay --- p.46 / Chapter 2.1.4.3 --- Primers for CHIP Assay --- p.47 / Chapter 2.1.5 --- Equipments --- p.47 / Chapter 2.1.5.1 --- Equipments for Cloning --- p.47 / Chapter 2.1.5.2 --- Equipments for Cell Culture --- p.47 / Chapter 2.1.5.3 --- Equipments for Realtime RT-PCR --- p.48 / Chapter 2.1.5.4 --- Equipments for Immunochemistry Staining --- p.48 / Chapter 2.1.5.5 --- Equipments for Western Blot --- p.48 / Chapter 2.1.5.6 --- Equipments for Luciferase Activity Assay --- p.49 / Chapter 2.1.5.7 --- Equipments for CHIP Assay --- p.49 / Chapter 2.1.5.8 --- Equipments for Urine Albumin Excretion Measurement --- p.49 / Chapter 2.2 --- METHODS --- p.50 / Chapter 2.2.1 --- Cloning --- p.50 / Chapter 2.2.1.1 --- Cloning Doxcycline-inducible overexpression of MiR-21 and Knockdown of MiR-21 expression plasmids --- p.50 / Chapter 2.2.1.2 --- Cloning Smad7 3’UTR Luciferase Reporter Plasmids --- p.51 / Chapter 2.2.2 --- Cell Cultures --- p.52 / Chapter 2.2.2.1 --- NRK52E Cell Lines and rat Mesengial Cell Lines --- p.52 / Chapter 2.2.2.2 --- Transient Transfection with microRNAs in TECs --- p.52 / Chapter 2.2.2.3 --- Construct Doxcycline-inducible Overexpression of MiR-21 and Knockdown of MiR-21 Stable Cell Lines in NRK52E and MCs --- p.53 / Chapter 2.2.3 --- Animal Models --- p.53 / Chapter 2.2.3.1 --- Unilateral Ureteral Obstruction (UUO) Mouse Model --- p.54 / Chapter 2.2.3.2 --- Diabetes Model --- p.54 / Chapter 2.2.4 --- Ultrasound-Mediated Gene Transfer --- p.55 / Chapter 2.2.5 --- Real Time RT-PCR --- p.56 / Chapter 2.2.5.1 --- Total RNA Isolation --- p.56 / Chapter 2.2.5.2 --- Reverse Transcription --- p.56 / Chapter (1) --- RT For MiR-21 Assay --- p.57 / Chapter (2) --- RT for Fibrotic and Inflammatory Index Assay --- p.57 / Chapter 2.2.5.3 --- Realtime PCR --- p.58 / Chapter (1) --- Realtime PCR For MiR-21 Assay --- p.58 / Chapter (2) --- Realtime PCR for Fibrotic and Inflammatory Index Assay --- p.58 / Chapter 2.2.5.4 --- Analysis of Realtime RT-PCR --- p.59 / Chapter 2.2.6 --- Western Blot --- p.59 / Chapter 2.2.6.1 --- Protein Preparation --- p.59 / Chapter 2.2.6.2 --- Running in SDS-PAGE --- p.60 / Chapter 2.2.6.3 --- Transfer --- p.61 / Chapter 2.2.6.4 --- Blocking --- p.61 / Chapter 2.2.6.5 --- Incubation --- p.62 / Chapter 2.2.6.6 --- Scanning --- p.62 / Chapter 2.2.6.7 --- Stripping --- p.62 / Chapter 2.2.7 --- PAS Staining --- p.63 / Chapter 2.2.7.1 --- Tissue Handling and Fixation --- p.63 / Chapter 2.2.7.2 --- Tissue Embedding and Sectioning --- p.63 / Chapter 2.2.7.3 --- Preparation of Paraffin Tissue Sections for PAS Staining --- p.64 / Chapter 2.2.7.4 --- PAS Staining --- p.64 / Chapter 2.2.7.5 --- Quantitative Analysis of PAS Staining --- p.65 / Chapter 2.2.8 --- Immunochemistry Staining --- p.65 / Chapter 2.2.8.1 --- Tissue Handling and Fixation --- p.65 / Chapter 2.2.8.2 --- Tissue Embedding and Sectioning --- p.65 / Chapter 2.2.8.3 --- Preparation of Paraffin Tissue Sections for Immunostaining --- p.65 / Chapter 2.2.8.4 --- Immunostaining --- p.66 / Chapter (1) --- Antigen-Antibody Reaction --- p.66 / Chapter (2) --- Signal Detection --- p.67 / Chapter 2.2.8.5 --- Quantitative Analysis of Immunohistochemistry --- p.67 / Chapter 2.2.9 --- In Situ Hybridization(ISH) --- p.68 / Chapter 2.2.9.1 --- Tissue Handling and Fixation --- p.68 / Chapter 2.2.9.2 --- Tissue Embedding and Sectioning --- p.68 / Chapter 2.2.9.3 --- Deparaffinization and Dewaxing --- p.68 / Chapter 2.2.9.4 --- Digestion --- p.69 / Chapter 2.2.9.5 --- Pre-Hybridization --- p.69 / Chapter 2.2.9.6 --- Hybridization --- p.69 / Chapter 2.2.9.7 --- Washing --- p.70 / Chapter 2.2.9.8 --- Blocking --- p.70 / Chapter 2.2.9.9 --- Incubation with anti-DIG Reagent --- p.70 / Chapter 2.2.9.10 --- Equilibration --- p.71 / Chapter 2.2.9.11 --- Signaling Detection --- p.71 / Chapter 2.2.10 --- Luciferase Activity Assay --- p.71 / Chapter 2.2.11 --- CHIP Analysis --- p.72 / Chapter 2.2.12 --- Urine Albumin Excretion Measurement --- p.73 / Chapter 2.2.12.1 --- Microalbuminuria Measurement --- p.73 / Chapter 2.2.12.2 --- Creatinine Measurement --- p.74 / Chapter 2.2.13 --- Statistical Analysis --- p.74 / Chapter CHAPTER III --- THE ROLE OF MIR-21 IN TGF-BETA-INDUCED RENAL FIBROSIS IN VITRO --- p.75 / Chapter 3.1 --- INTRODUCTION --- p.75 / Chapter 3.2 --- MATERIAS AND METHODS --- p.77 / Chapter 3.2.1 --- Cell Culture --- p.77 / Chapter 3.2.2 --- Transient Transfection with microRNAs --- p.78 / Chapter 3.2.3 --- Construction of Inducible Stable Cell Lines of miR-21 Overexpression and Knockdown --- p.78 / Chapter 3.2.4 --- Realtime RT-PCR --- p.79 / Chapter 3.2.5 --- Chromatin Immunoprecipitation (ChIP) Analysis --- p.79 / Chapter 3.2.6 --- Western Blot Analysis --- p.79 / Chapter 3.2.7 --- Statistical Analysis --- p.79 / Chapter 3.3 --- RESULTS --- p.80 / Chapter 3.3.1 --- The Expression of miR-21 Is Up-regulated in TGF-β-induced Renal Fibrosis In Vitro --- p.80 / Chapter 3.3.2 --- The Up-regulation of miR-21 Is Mediated by TGF-β/Smad Signaling during Renal Fibrosis In Vitro --- p.82 / Chapter 3.3.2.1 --- The Up-regulation of miR-21 Depends On the Activation of TGF-β Signaling During Renal Fibrosis In Vitro --- p.82 / Chapter 3.3.2.2 --- The Up-regulation of miR-21 in Response to TGF-β1 Is Positively Mediated by Smad3, Negatively by Smad2 --- p.84 / Chapter 3.3.2.3 --- The Up-regulation of miR-21 in Response to TGF-β1 Is Physically Regulated by Smad3 in CHIP Assay --- p.86 / Chapter 3.3.3 --- miR-21 Plays an Important Role in TGF-β-induced Renal Fibrosis In Vitro --- p.89 / Chapter 3.3.3.1 --- The Role of miR-21 in Renal Fibrosis Is Identified by Transient Transfection with miR-21 Mimic or Anti-miR-21 --- p.89 / Chapter 3.3.3.2 --- The Role of miR-21 in Renal Fibrosis Is Identified by Applied Inducible-Stable Cell Lines which Is Overexpression of miR-21 or Knockdown of miR-21 in TECs --- p.92 / Chapter (1) --- Characterize the Inducible-Stable Cell Lines which Is Overexpression of miR-21 or Knockdown of miR-21 in TECs --- p.92 / Chapter (2) --- Overexpression of miR-21 Enhances the TGF-β-induced Renal Fibrosis In Vitro --- p.95 / Chapter (3) --- Knockdown of miR-21 Inhibits the TGF-β-induced Renal Fibrosis In Vitro --- p.99 / Chapter 3.4 --- DISCUSSION --- p.103 / Chapter 3.5 --- CONCLUSION --- p.106 / Chapter CHAPTER IV --- THE THERAPUTIC ROLE OF MIR-21 IN UNILATERAL URETERAL OBSTRUCTION (uuo)-INDUCED RENAL FIBROSIS IN VIVO --- p.107 / Chapter 4.1 --- INTRODUCTION --- p.107 / Chapter 4.2 --- MATERIAS AND METHODS --- p.109 / Chapter 4.2.1 --- Animal Model of Unilateral Ureteral Obstruction (UUO) --- p.109 / Chapter 4.2.2 --- Ultrasound-mediated Gene Transfer of Inducible miR-21 shRNA Plasmids Into the Ligated Kidneys --- p.109 / Chapter 4.2.3 --- Realtime RT-PCR --- p.110 / Chapter 4.2.4 --- Western Blot Analysis --- p.111 / Chapter 4.2.5 --- PAS Staining --- p.111 / Chapter 4.2.6 --- Immunohistochemistry Staining --- p.111 / Chapter 4.2.7 --- In Situ Hybridization --- p.111 / Chapter 4.2.8 --- Statistical Analysis --- p.112 / Chapter 4.3 --- RESULTS --- p.112 / Chapter 4.3.1 --- The Expression of miR-21 Is Up-regulated in Renal Fibrosis in UUO Mouse Model --- p.112 / Chapter 4.3.2 --- Induce miR-21 siRNA Plasmid into the Kidney by Using Ultrasound-microbubble-mediated Gene Transfer Technique --- p.114 / Chapter 4.3.2.1 --- Determine Transgene Expression --- p.114 / Chapter 4.3.2.2 --- Determine Gene Transfer Rate --- p.117 / Chapter 4.3.2.3 --- Determine Gene Transfer Safety --- p.118 / Chapter 4.3.3 --- Knockdown of miR-21 Prevents the Development of Renal Fibrosis in UUO Mice --- p.120 / Chapter 4.3.3.1 --- Delivery of miR-21 shRNA Plasmid Suppresses the Expression of miR-21 and TGF-β1 in UUO Mouse Model --- p.120 / Chapter 4.3.3.2 --- Knockdown of MiR-21 Suppresses the Deposition of Collagen I, Fibronectin and α-SMA in UUO Mouse Model --- p.122 / Chapter 4.3.3.3 --- Knockdown of MiR-21 Suppresses the mRNA Levels of Collagen I, Fibronectin and α-SMA expression in UUO Mouse Model --- p.127 / Chapter 4.3.3.4 --- Knockdown of miR-21 Suppresses the Protein Levels of Collagen I, Fibronectin and α-SMA Expression in UUO Mouse Model --- p.129 / Chapter 4.3.4 --- Knockdown of miR-21 Attenuates the Progressive of Renal Fibrosis in UUO Mice --- p.131 / Chapter 4.3.4.1 --- Delivery miR-21 shRNA Plasmid Attenuates the Expression of miR-21 and TGF-β1 in Established UUO Mouse Model --- p.131 / Chapter 4.3.4.2 --- Knockdown of MiR-21 Attenuates the Deposition of Collagen I, Fibronectin and α-SMA in Established UUO Mouse Model --- p.133 / Chapter 4.3.4.3 --- Knockdown of MiR-21 Attenuates the mRNA Levels of Collagen I, Fibronectin and α-SMA in Established UUO Mouse Model --- p.138 / Chapter 4.3.4.4 --- Knockdown of miR-21 Attenuates the Protein Levels of Collagen I, Fibronectin and α-SMA Expression in Established UUO Mouse Model --- p.140 / Chapter 4.4 --- DISCUSSION --- p.143 / Chapter 4.5 --- CONCLUSION --- p.145 / Chapter CHAPTER V --- THE ROLE OF MIR-21 IN DIABETIC KIDNEY INJURY --- p.146 / Chapter 5.1 --- INTRODUCTION --- p.146 / Chapter 5.2 --- MATERIAS AND METHODS --- p.148 / Chapter 5.2.1 --- Cell Culture --- p.148 / Chapter 5.2.2 --- Construction of Inducible Stable Cell Lines of miR-21 Overexpression and Knockdown --- p.149 / Chapter 5.2.3 --- Animal Model of db/db Mice --- p.149 / Chapter 5.2.4 --- Ultrasound-mediated Gene Transfer of Inducible miR-21 shRNA Plasmids into the Kidneys of db/db Mice --- p.150 / Chapter 5.2.5 --- Realtime RT-PCR --- p.150 / Chapter 5.2.6 --- Western Blot Analysis --- p.150 / Chapter 5.2.7 --- PAS Staining --- p.151 / Chapter 5.2.8 --- Immunohistochemistry Staining --- p.151 / Chapter 5.2.9 --- Urine Albumin Excretion Measurement --- p.151 / Chapter 5.2.10 --- Construction of Plasmids and Luciferase reporter Assay --- p.152 / Chapter 5.2.11 --- Statistical Analysis --- p.152 / Chapter 5.3 --- RESULTS --- p.153 / Chapter 5.3.1 --- The Expression of miR-21 Is Increased Under Diabetic Conditions Both In Vitro and In Vivo --- p.153 / Chapter 5.3.1.1 --- The expression of miR-21 Is Increased in High Glucose Conditions in TECs and MCs --- p.153 / Chapter 5.3.1.2 --- The Expression of miR-21 Is Increased in Diabetic Kidney Injury in db/db Mouse Model --- p.155 / Chapter 5.3.2 --- The Expression of miR-21 Depends On The Activation of TGF-β/Smad Signaling Under Diabetic Conditions --- p.156 / Chapter 5.3.3 --- The Expression of MiR-21 Affects On Renal Fibrosis Under Diabetic Conditions In Vitro --- p.158 / Chapter 5.3.3.1 --- The Role of miR-21 in Renal Fibrosis Under Diabetic Conditions Is Identified in TECs --- p.158 / Chapter (1) --- Overexpression of miR-21 Enhances Renal Fibrosis in High Glucose Condition in TECs --- p.158 / Chapter (2) --- Knockdown of miR-21 Suppresses Renal Fibrosis in High Glucose Condition in TECs --- p.160 / Chapter 5.3.3.2 --- The Role of miR-21 in Renal Fibrosis Under Diabetic Conditions Is Identified in MCs --- p.162 / Chapter (1) --- Characterize the Inducible-Stable Cell Lines Which Is Overexpression of miR-21 or Knockdown of miR-21 in MCs --- p.162 / Chapter (3) --- Knockdown of miR-21 Suppresses Renal Fibrosis in High Glucose Condition in MCs --- p.165 / Chapter 5.3.4 --- The Expression of miR-21 Affects On Renal Inflammation Under Diabetic Conditions In Vitro --- p.167 / Chapter 5.3.4.1 --- The role of miR-21 in Renal Inflammation Under Diabetic Conditions Is Identified in TECs --- p.167 / Chapter 5.3.4.2 --- The Role of miR-21 in Renal Inflammation Under Diabetic Conditions Is Identified in MCs --- p.169 / Chapter 5.3.5 --- Knockdown of miR-21 Suppresses the Renal Fibrosis and Inflammation in db/db Mice --- p.172 / Chapter 5.3.5.1 --- Delivery of miR-21 siRNA suppresses the Expression of miR-21 in db/db Mice --- p.172 / Chapter 5.3.5.2 --- Knockdown of miR-21 Improves the Microalbuminuria in db/db Mice --- p.174 / Chapter 5.3.5.3 --- Knockdown of miR-21 Suppresses the Renal Fibrosis in db/db Mice --- p.176 / Chapter 5.3.5.4 --- Knockdown of miR-21 Suppresses the Renal Inflammation in db/db Mice --- p.183 / Chapter 5.3.6 --- Identification of Smad7 Is A Directly Target of miR-21 Both In Vitro and In Vivo --- p.187 / Chapter 5.3.6.1 --- The Expression of miR-21 Negatively Regulates the Smad7 Expression Under Diabetic Conditions Both in vitro and in vivo. --- p.187 / Chapter 5.3.6.2 --- Knockdown of miR-21 Blocks the Smad7-mediated TGF-β and NF-κB Signaling Pathways. --- p.190 / Chapter 5.3.6.3 --- Smad7 Is A Directly Target of miR-21. --- p.192 / Chapter 5.4 --- DISCUSSION --- p.194 / Chapter 5.5 --- CONCLUSION --- p.197 / Chapter CHAPTER VI --- SUMMARY AND CONCLUSION --- p.198 / Chapter 6.1 --- SUMMARY AND DISCUSSION --- p.200 / Chapter 6.1.1 --- The Up-regulation of miR-21 Was Observed in TGF-β- Induced Renal Fibrosis and Under Diabetic Conditions Both In Vitro and In Vivo. --- p.200 / Chapter 6.1.2 --- The Expression of miR-21 Is Regulated by TGF-β/Smad3 Signaling. --- p.200 / Chapter 6.1.3 --- The Expression of miR-21 Plays a Critical Role in Renal Fibrosis and Inflammation. --- p.201 / Chapter 6.1.4 --- MiR-21 Directly Targets on Smad7 to Regulate Renal Fibrosis and Inflammation. --- p.202 / Chapter 6.1.5 --- The Therapeutic Effect of miR-21 on Renal Fibrosis and Inflammation Is Developed in UUO and db/db Mouse Models. --- p.203 / Chapter 6.1.6 --- The Potential Clinical Use by Targeting On miR-21 --- p.204 / Chapter 6.2 --- CONCLUSION --- p.205 / REFERENCES --- p.206

by Simon Chan Siu Hoi. / Spine title varies. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 130-148). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Abbreviations --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / Page / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Growth Hormone --- p.1 / Chapter 1.2 --- Growth Hormone Receptor --- p.3 / Chapter 1.2.1 --- Cytokine/Hematopoietin Receptor Superfamily --- p.3 / Chapter 1.2.2 --- Tissue Distribution of GHR --- p.6 / Chapter 1.2.3 --- Biosynthesis and Degradation of GHR --- p.7 / Chapter 1.2.4 --- Regulation of GHR Level --- p.8 / Chapter 1.2.5 --- The GHR Protein --- p.10 / Chapter 1.2.6 --- The GHR Gene --- p.15 / Chapter 1.2.7 --- GHR Dimerization --- p.16 / Chapter 1.2.8 --- Mechanism of Signaling by GHR --- p.19 / Chapter 1.2.9 --- GH Binding Protein --- p.21 / Chapter 1.2.10 --- GHR Related Dwarfism --- p.23 / Chapter 1.3 --- Objectives of the Present Investigation --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Fish Growth Hormone Radioactive Labeling --- p.27 / Chapter 2.1.1 --- Preparation of Iodogen-Coated Tubes --- p.27 / Chapter 2.1.2 --- Packing of the Sephadex G-75 Column --- p.28 / Chapter 2.1.3 --- Iodination of brGH and Purification of the Iodinated brGH --- p.28 / Chapter 2.1.4 --- Determination of the Specific Radioactivity and Percentage of 125I Incorporation --- p.29 / Chapter 2.1.5 --- Reagents and Buffers Used --- p.30 / Chapter 2.2 --- Integrity of 125I-brGH --- p.30 / Chapter 2.2.1 --- HPLC of brGH --- p.31 / Chapter 2.2.2 --- HPLC of 125I-brGH after Iodination --- p.31 / Chapter 2.2.3 --- HPLC of 125I-brGH after Receptor Binding --- p.31 / Chapter 2.3 --- Preparation of Membranes from Fish Tissues --- p.32 / Chapter 2.3.1 --- Preparation of Snakehead Fish Liver Membranes --- p.32 / Chapter 2.3.2 --- Reagents and Buffers Used --- p.33 / Chapter 2.4 --- Protein Determination of Membrane Preparations --- p.34 / Chapter 2.4.1 --- The BCA Protein Reaction Scheme --- p.34 / Chapter 2.4.2 --- BCA Protein Determination Protocol --- p.34 / Chapter 2.5 --- Receptor Binding Studies --- p.35 / Chapter 2.5.1 --- Association and Dissociation Studies --- p.36 / Chapter 2.5.2 --- pH Dependence Study --- p.36 / Chapter 2.5.3 --- Membrane Protein Dependence Study --- p.37 / Chapter 2.5.4 --- Ca2+ Dependence Study --- p.37 / Chapter 2.5.5 --- Tissue Distribution Study --- p.37 / Chapter 2.5.6 --- Displacement and Specificity Studies --- p.38 / Chapter 2.5.7 --- Dithiothreitol (DTT) Dependence Study --- p.39 / Chapter 2.5.8 --- p-Chloromercuribenzene Sulfonate (PCMBS) Pretreatment: Dose Dependence Study --- p.39 / Chapter 2.5.9 --- Scatchard Analysis of the PCMBS Pretreated and Control Snakehead Fish Liver Membranes --- p.40 / Chapter 2.5.10 --- Reversibility of the PCMBS Effect --- p.40 / Chapter 2.5.11 --- Reagents and Buffers Used --- p.41 / Chapter 2.6 --- Crosslinking Studies --- p.41 / Chapter 2.6.1 --- Crosslinking Performed on Snakehead Fish Liver Membranes --- p.41 / Chapter 2.6.2 --- Crosslinking Performed on Solubilized Snakehead Fish Liver Membranes --- p.42 / Chapter 2.6.3 --- Gel Filtration Chromatography of the Crosslinked Comp)lexes --- p.43 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of the Crosslinked Complexes --- p.43 / Chapter 2.6.5 --- Reagents and Buffers Used --- p.45 / Chapter 2.7 --- Western Blot Analysis of Snakehead Fish Liver GHR --- p.46 / Chapter 2.7.1 --- SDS-PAGE of Snakehead Fish Liver Membranes --- p.46 / Chapter 2.7.2 --- Transfer of Proteins onto Polyvinylidene Fluoride (PVDF) Membrane --- p.46 / Chapter 2.7.3 --- Antibody Development of PVDF Membrane --- p.47 / Chapter 2.7.4 --- Reagents and Buffers Used --- p.48 / Chapter 2.8 --- Solubilization of Snakehead Fish Liver Membranes and Solubilized Receptor Binding Studies --- p.48 / Chapter 2.8.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.49 / Chapter 2.8.2 --- Solubilized Receptor Binding Assay --- p.49 / Chapter 2.8.3 --- "Solubilization of Snakehead Fish Liver Membranes: Detergent Concentration, pH, Temperature and Time Dependence" --- p.50 / Chapter 2.8.4 --- Solubilized Receptor Binding Study: Interference of Detergent --- p.50 / Chapter 2.8.5 --- Reagents and Buffers Used --- p.51 / Chapter 2.9 --- Purification of Snakehead Fish Liver GHR by Affinity Chromatography --- p.51 / Chapter 2.9.1 --- Affinity Column Preparation --- p.52 / Chapter 2.9.2 --- Snakehead Fish Liver GHR Purification --- p.52 / Chapter 2.9.3 --- Reagents and Buffers Used --- p.53 / Chapter Chapter 3 --- Results: fGH Labeling and Integrity Determination --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Experimental Results --- p.55 / Chapter 3.2.1 --- Iodination of fGH --- p.55 / Chapter 3.2.2 --- Integrity of 125I-fGH --- p.55 / Chapter 3.3 --- Discussion --- p.61 / Chapter Chapter 4 --- Results: Membrane Receptor Binding Studies --- p.62 / Chapter 4.1 --- Introduction --- p.62 / Chapter 4.2 --- Experimental Results --- p.63 / Chapter 4.2.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.64 / Chapter 4.2.1.1 --- Association and Dissociation Studies --- p.64 / Chapter 4.2.1.2 --- pH Dependence Study --- p.67 / Chapter 4.2.1.3 --- Membrane Protein Dependence Study --- p.70 / Chapter 4.2.1.4 --- Ca2+ Dependence Study --- p.73 / Chapter 4.2.2 --- Localization and Specificity of Snakehead Fish GHR --- p.76 / Chapter 4.2.2.1 --- Tissue Distribution Study --- p.76 / Chapter 4.2.2.2 --- Displacement and Specificity Studies --- p.78 / Chapter 4.2.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.81 / Chapter 4.2.3.1 --- Effect of DTT: Concentration Dependence Study --- p.81 / Chapter 4.2.3.2 --- Effect of PCMBS: Concentration Dependence Study --- p.84 / Chapter 4.2.3.3 --- Scatchard Analysis of Control and PCMBS- pretreated Membranes --- p.86 / Chapter 4.2.3.4 --- Reversibility of the PCMBS Effect --- p.88 / Chapter 4.3 --- Discussion --- p.90 / Chapter 4.3.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.90 / Chapter 4.3.2 --- Localization and Specificity of Snakehead Fish GHR --- p.93 / Chapter 4.3.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.96 / Chapter Chapter 5 --- Results: Crosslinking and Western Blot Analysis --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.1.1 --- Crosslinking Studies --- p.101 / Chapter 5.1.2 --- Western Blot Analysis --- p.103 / Chapter 5.2 --- Experimental Results --- p.104 / Chapter 5.2.1 --- Crosslinking Studies --- p.104 / Chapter 5.2.2 --- Western Blot Analysis --- p.105 / Chapter 5.3 --- Discussion --- p.112 / Chapter Chapter 6 --- Results: Affinity Purification of Snakehead Fish Liver GHR --- p.115 / Chapter 6.1 --- Introduction --- p.115 / Chapter 6.1.1 --- Membrane Solubilization and Solubilized GHR Binding Studies --- p.115 / Chapter 6.1.2 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.116 / Chapter 6.2 --- Exp erimental Results --- p.117 / Chapter 6.2.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.117 / Chapter 6.2.2 --- Interference of Detergents in the Solubilized Receptor Binding Assay --- p.118 / Chapter 6.2.3 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.120 / Chapter 6.3 --- Discussion --- p.122 / Chapter Chapter 7 --- General Discussion --- p.125 / References --- p.130