INTRODUÇÃO: A relevância da expressão MYCN em neuroblastomas sem amplificação, diferentemente do seu aumento quando amplificado, permanece controverso. Neste trabalho, avaliou-se a relação do nível de expressão do transcrito MYCN em neuroblastoma não amplificado com os fatores clínicos e biológicos de prognóstico. MÉTODOS: Neste estudo observacional realizado entre janeiro de 2000 e dezembro de 2004, foram aferidos os valores do nível de expressão MYCN em 29 amostras tumorais, pela técnica RQ-PCR, no Laboratório de Biologia Tumoral da Fundação Pró-Sangue de São Paulo. Seus resultados foram analisados em relação à idade ao diagnóstico, ao estadiamento tumoral, ao grupo de risco, à ocorrência de recaída tumoral e de óbito. RESULTADOS: Foram nove crianças com idade 1 ano, os valores da expressão do transcrito MYCN variaram de 0,041 e 27,569, mediana 3,193. O estadiamento foi: quatro estádio 1; três estádio 2; oito estádio 3 e 14 crianças estádio 4. Entre 20 crianças com classificação patológica, 11 foram favoráveis e nove desfavoráveis. Considerando a mediana dos valores expressos a estratificação em grupos de risco com aumento da expressão foram: cinco crianças baixo risco; quatro risco intermediário e cinco alto risco. Grupos de risco sem aumento da expressão foram: duas crianças baixo risco; quatro risco intermediário e nove alto risco. Vinte e oito crianças obtiveram remissão completa e entre elas 14 apresentaram doença progressiva, sendo que sete morreram. As variáveis clínicas e biológicas não apresentaram freqüências diferentes entre os grupos de risco sem e com aumento de expressão. Entre os grupo alto risco e não alto-risco as variáveis idade, recaída tumoral e óbito apresentaram resultado com significado estatístico quando não se considerou o valor da expressão e quando não houve o seu aumento. Entre os grupos alto risco e nãoalto risco com aumento da expressão apenas a idade apresentou resultado com significado estatístico. CONCLUSÃO: Em crianças com estádio clínico não avançado o nível de expressão parece exercer uma relevância clínica, sugerindo um efeito protetor quanto menor for o aumento da expressão MYCN. / INTRODUTION: MYCN expression value in non-amplified neuroblastosmas remains a controversial issue. In order to add contributions to this field, children with nonamplified neuroblastomas were studied regarding their expression and correlation with clinical and other biological factors. METHODS: Twenty nine tumor samples obtained from non-consecutive patients admitted from January, 2000 through December, 2004, had their MYCN transcript expression levels evaluated according to the RQ-PCR assay, at the Tumoral Biology of Laboratory of the \"Fundação Pró -Sangue Hemocentro de São Paulo\", and compared to the following other factor: age at onset; tumor stage; risk - group; tumoral relapse rate and death. RESULTS: nine under one-year-old children and 20 over one-year-old children, with MYCN transcription expression level between 0.041 and 27.569, mean 3.193. Four children were stage 1, three were stage 2, stage 3 in eight and stage 4 in 14 children. In 20 patients with pathological classifications, 11 were favorable and nine unfavorable histology. Children whose expression level was above the mean were stratified as follows according to risk groups: five low-risk; four intermediate-risk and five high-risk patients. The ones whose expression level was under the mean were two low-risk, four intermediate-risk end nine high-risk patients. Twenty eight children achieved complete remission, with 14 recurrences, with seven deaths. The only factor associated to highly expressed MYCN patients was tumoral state. CONCLUSION: In children with non-advanced-stage disease low levels of expression might be a relevant favorable prognostic factor.

by Cecilia Tsang Wai Ping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references. / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Xu Feng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 205-233). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Neste estudo foi avaliado o fotoenvelhecimento cutâneo dos membros superiores clinica, anatomo e imunohistoquimicamente, em 22 pacientes do sexo feminino, com idade acima de 50 anos, antes e após quatro sessões de esfoliação química com ácido pirúvico 80% com intervalo quinzenal. Clinicamente foram avaliados o envelhecimento cutâneo por meio da elastose clínica, rugas profundas e superficiais; lesões de queratose actinica e melanose solar. As biópsias foram avaliadas pela coloração hematoxilina-eosina e posteriormente coradas pelo marcador imunohistoquímico Ki-67, marcador de proliferação celular. A avaliação anatomopatológica foi embasada no estudo das alterações epidérmicas (hiperqueratose, presença de acantose ou atrofia, atipia celular e retificação da epiderme), enquanto a alteração dérmica foi avaliada pela degeneração basofílica do colágeno. As células coradas pelo marcador imunohistoquímico de proliferação celular, o Ki-67, foram contadas e comparadas antes e após as quatro esfoliações. Concluímos que o ácido pirúvico 80% se mostrou eficaz no tratamento clínico do fotoenvelhecimento (77,3%) e na queratose actinica (72,7%); resultados estatisticamente significantes, porém tanto a avaliação das lesões de melanose solar quanto a avaliação histopatológica epidérmica e dérmica não foram estatisticamente significantes. O Ki-67, marcador da proliferação celular se mostrou aumentado na maioria dos pacientes (52,63%), resultado este também estatisticamente não significante / This study assessed skin photoaging of the upper limbs by clinical, anatomical and immunohistochemical analyses in 22 female patients, aged over 50 years, before and after 4 sessions of chemical exfoliation with pyruvic acid at 80% within quartely intervals. From a clinically perspective, we assessed skin aging by clinical elastosis, deep or superficial wrinkles, actinic keratosis and solar melanosis damage before and after 4 exfoliations. Biopsies were assessed by hematoxylin-eosin staining and later stained with the immunohistochemical marker Ki-67, a marker of cellular proliferation. Clinical pathology analysis was based on the study of epidermal abnormalities (hyperkeratosis, acanthosis and atrophy, cellular atypia and epidermal rectification), whereas dermal abnormalities were assessed by collagen basophilic degeneration. The cells stained by the immunohistochemical marker of cellular proliferation Ki-67 were counted and compared before and after the 4 exfoliations. The purpose of the study was to investigate the use of pyruvic acid in the treatment of photoaging, assessing its clinical abnormalities, action in actinic keratosis and solar melanosis damage, as well as epidermal and dermal abnormalities in the clinical pathology and the repercussions in cell proliferation. Clinical pathology analysis was based on the study of epidermal abnormalities (hyperkeratosis, acanthosis and atrophy, cellular atypia and epidermal rectification), whereas dermal abnormalities were assessed by collagen basophilic degeneration. The cells stained by the immunohistochemical marker of cellular proliferation Ki67 were counted and compared before and after the 4 exfoliations. We concluded that pyruvic acid at 80% was effective for clinically managing photoaging (77,3%) and actinic keratosis (72,7%). These results were statistically significant whereas the results for dermal and epidermel histopathology assessment of solar melanosis damage were not. Ki-67, a marker of cell proliferation, was increased in most of the patients (52,63%), but did not result in statistically significant differences

讀寫障礙是最普遍的一種學習障礙（80%)，影響全球大約一成的學童。讀寫障礙患者於閱讀及書寫能力方面出現困難，而這並非因為患者本身的智力、學習動機或學習機會引致。對於引至讀寫障礙的理仍未清楚，但在西方人士的遺傳研究方面已發現多個與讀寫障礙相聯的基因位點及基因。本研究針對其中4個基因位點（DYX1 ’ DYX2 ’ DYX3 ’ DKX8)及其覆蓋的11基因測試了 131讀寫障礙的中國人家庭與讀寫障礙的關聯性。是項研究從國際人類基因組單體型圖(HapMap)中選擇標籤單核苷酸多型性(Tag-SNPs)及選擇以往報告與讀寫障礙有關的單核苷酸多型性進行測試。並在DYXZa基因（rs3743205 ’ padjusted =0.0072' OR = 0.08 ( 95% CI: 0.01 - 0.64 ))私MRPL19 (風險單体型rs2422229-rs7570229,風險單体型T-G, Padjusted=0.0020, OR = 2.345 (95% CI: 1.402 - 3.923))發現與讀寫障礙有正關聯性。單核苷酸多態性亦與閱讀的幾個特徵相關：DYX1C1 ( rs3743205 )與快速命名（Digit Rapid Naming ) ’語音記憶（Non-word repetition)，字型結構的左右逆轉（Left-Right Reversal )相關；KIAA0319 ( rs2760157 rs807507 )與語音意識（Onset Detection )相關；MRPL19 ( rs2422229-rs7570229 ) 與字型結構的部首位置(Radical Position)相關；SFPQand ZMYM4 ( rs3738697 - rs12093076 )與詞彙決策（Lexical Decision )相關。本研究是首個對於讀寫障礙的中國人族群進行的的基因研究（Lim et al. 2011) ’結果有助提供我們了解讀寫障礙在使用不同語言的族群的情況。 / 在過往歐洲的研究樣本中’位於KAA0319基因5'-上游的基因變異重複地被找出與讀寫障礙有關的閱讀特徵有關。過往研究亦指出位於KIAA0319的假定調控區上的單核苷酸多態性(SNP)顯示與KIAA0315的基因表達有關。唯本項研究於這區域並無發現陽性結果。為了找出這區域中與讀寫障礙有關聯但未被本研究策略選擇使用基因組單體型圖的標籤-單核苷酸多態性(Hapmap Tag-SNPs)的基因變異，本研究對KAM0319基因的5'-上游進行了基因组重测序。其中發現的3個短序變異（-121的rs6456625，-128到- 1 5 4 的r s 7 1 8 1 5 1 4 3 及- 1 5 7 的6 > A )出現了不同的榮光素酶報告基因活動，當中單体型A-DEL-A的活動訊號最高，而G-INS-G則最低。然而，它們的等位基因和單体型基因出現率於讀寫障礙樣本與對照組沒有顯著不同。 / 至今有關對KIAA0319基因抑制的行為研究仍然不足。本研究亦對位於果繩的CG7565基因，即KIAA0319的同源基因’進行特性分析。CG7565在果繩的發展階段出現了不同的表達水平及基因剪接形式。本研究使用了UAS-RNAi糸統和飛行模擬器對CG7565基因抑制的果繩的行為變化作出了分析，結果顯示於泛神經基因抑制晰/+; e/aV-Gal4/+; 3707/+及晰/+; e/av-Gal4/+; 8396/+的果蠅視覺模式記憶出現了缺陷。當CG7565在果繩大腦中央複合區的神經元F5(扇形體)和R2/R4m(摘球体)被基因抑制時，果繩視覺模式記憶亦出現了缺陷。是次有關M/PL119的遺傳關聯研究跟以往MRPL19中5'-上游與讀寫障礙的關聯報導的一致’顯示這可能是真正的致病序列變異的位置。在M/PL119上的假定調控區進行基因突變分析顯示，在其中一個讀寫障礙的樣本中發現一個新的序列變化(-647 T>G)，而在對照組則沒有發現此變化。計算機預測模型分析估計這個序列變化會取消了熱休克轉錄因子1的結合位點。攜帶了 G等位基因的調控區會增加榮光素酶的活動。這種變異的作用必須得到進一步的證實。我們亦觀察到在其中兩個讀寫障礙樣本中出現了非孟德爾遺傳，在一個個体身上帶有3或4種單倍型的基因。基因拷貝數目變異或基因轉換可能是一個引至這種現象的因素。 / Developmental dyslexia is a learning disability characterized by difficulties in acquisition of reading and writing skills not due to intelligence, motivation or schooling. Being the most common form of learning disability (80%), it affects 10% of schoolchildren worldwide. Research delineating genetic factors in developmental dyslexia identified loci and candidate genes in Caucasian populations, although disease mechanisms are still unknown. Four loci covering eleven genes (DYX1, DYX2, DYX3, DYXS) were tested for association in 131 Chinese families with dyslexic children in our study. Tagged-SNPs selected from International HapMap Consortium and reported SNPs were used as markers for this study. Positive associations with dyslexia were found in two genes, DYX1C1 (rs3743205, padjusted=0.0072, OR =0.08 (95% CI: 0.01 - 0.64)) and MRPL19(rs2422229-rs7570229, risk haplotype T-G,Padjusted=0.0020, OR = 2.345 (95% CI: 1.402 一 3.923)), in our study. SNPs associated with several reading-related traits were also identified: DYX1C1(rs3743205) associated with Rapid Naming (Digit Rapid Naming), Phonological Memory (Non-word repetition),Orthographic skill (Left-Right Reversal); KIAA0319 (rs2760157-rs807507) with honological awareness (Onset Detection); MRPL19 (rs2422229-rs7570229) with Orthographic knowledge (Radical Position); SFPQ and ZMYM4 (rs3738697 - rs12093076) with Orthographic knowledge (Lexical Decision). This is the first genetic study in Chinese dyslexia (Lim et al.2011), and results provide knowledge into dyslexia in populations using different languages. / Variants located 5' upstream of KIAA0319 were consistently reported for association with DD reading-related traits in European samples. A SNP in the putative promoter of KIAA0319 showed functional significance in KIAA0319 expression. However, no positive result of this region is found in this study. Resequencing of the 5' upstream of KIAA0319 was done to reveal potentially associated variants not selected using current strategies in genetic association (Hapmap Tagged-SNPs). A short sequence fragment of 3 variants (-121 rs6456625, -128 to -154 rs71815143 and -157 G>A) show differential luciferase activities, haplotype A-del-A have highest signal, G-Ins-G the lowest. However, allele and haplotype frequencies in dyslexia samples were not significantly different from controls. / Direct behavioral study of KIAA0319-knockdown is still inadequate. A homolog of KIAA0319, CG7565 in Drosophila, was characterized. Differential gene expression and splicing forms were observed during Drosophila development stages. Using UAS-RNAi system and flight simulator to study behavioral change in CG7565-knockdown Drosophila showed pan-neural knockdown lines w/+; elav-Gal4/+; 3707/+ and w/+; elav-Gal4/+; 8396/+ are defective in visual pattern memory. The study of neuronal specific knockdown showed this memory was impaired when CG7565 was selectively knocked down in F5 neuron (fan-shaped body) and R2/R4m (ellipsoid body) of the central complex in Drosophila brain. / Our genetic association study of MRPL19 agrees with reports of the association of 5' upstream of MRPL19 with DD, showing that true causative sequence variants may lie here. Mutational analyses of the putative promoter of MRPL19 revealed a novel sequence change T>G at -647 in a dyslexic sample not found in controls. In-silico analysis indicates a binding site of heat shock factor-1 that is predicted to be abolished by this variant. Luciferase activity increased in the promoter carrying the G allele. The role of this variant must be confirmed. Non-Mendelian Inheritance was observed in 2 individual dyslexic samples with 3 and 4 types of haplotypes. Copy number variation or gene conversion may be a factor. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lim, King Poo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 201-225). / Abstracts also in Chinese. / Dedication --- p.I / Abstract --- p.II / 摘要 --- p.IV / Acknowledgements --- p.V / Table of contents --- p.VI / List of Figures --- p..XI / List of Tables --- p.XIV / List of Abbreviations --- p.XVII / Chapter 1. --- Chapter 1 --- p.1 / Genetic Association of dyslexia-candidate genes in Chinese children with dyslexia --- p.1 / Chapter 1.1. --- Introduction --- p.2 / Chapter 1.2. --- Prevalence --- p.4 / Chapter 1.3. --- Definition --- p.7 / Chapter 1.4. --- Theories of developmental dyslexia --- p.9 / Chapter 1.4.1. --- Phonological deficit theory --- p.9 / Chapter 1.4.2. --- Double deficit hypothesis --- p.10 / Chapter 1.4.3. --- Cerebellar deficit theory --- p.11 / Chapter 1.4.4. --- Magnocellular deficit theory --- p.12 / Chapter 1.4.5. --- Deficits in Chinese people with dyslexia --- p.12 / Chapter 1.5. --- Neurobiological aspects of Dyslexia --- p.15 / Chapter 1.5.1. --- Postmortem studies --- p.15 / Chapter 1.5.2. --- Structural Neuroimaging studies of dyslexia --- p.16 / Chapter 1.5.3. --- Functional Neuroimaging studies --- p.17 / Chapter 1.5.4. --- fMRI results in Chinese --- p.18 / Chapter 1.6. --- Genetics of Dyslexia --- p.19 / Chapter 1.6.1. --- Familial studies --- p.19 / Chapter 1.6.2. --- Twin studies --- p.21 / Chapter 1.6.3. --- Mode of Inheritance --- p.24 / Chapter 1.6.4. --- Genetic mapping of disease gene --- p.26 / Linkage analysis --- p.26 / Association study --- p.29 / Molecular genetic findings in dyslexia --- p.33 / Chapter 1.6.5. --- Statement of Research Rationale --- p.47 / Chapter 1.6.6. --- Objectives --- p.48 / Chapter 1. --- Chapter 2 --- p.49 / Genetic association of dyslexia-candidate genes in Chinese children with dyslexia --- p.49 / Chapter 2.1. --- Introduction --- p.50 / Chapter 2.2. --- Materials and methods --- p.51 / Chapter 2.2.1. --- Subjects --- p.51 / Chapter 2.2.2. --- DNA extraction and genotyping --- p.54 / Chapter 2.2.3. --- SNP marker selection --- p.55 / Chapter 2.2.4. --- Statistical analyses --- p.57 / Chapter 2.3. --- Results --- p.59 / Chapter 2.3.1. --- DYX1C1 --- p.59 / Single marker analysis --- p.59 / Haplotype analyses --- p.62 / Chapter 2.3.2. --- KIAA0319 --- p.67 / Association of KIAA0319 with Chinese dyslexic children --- p.67 / Association of KIAA0319 with reading related traits --- p.67 / Chapter 2.3.3. --- DCDC2 --- p.74 / Association of DCDC2 with Chinese dyslexic children --- p.74 / Chapter 2.3.4. --- MRPL19 and C2orf3 --- p.78 / Haplotypes located within 5' upstream of MRPL19 are significantly associated with DD --- p.78 / Association of the 5' upstream variants with reading related traits --- p.79 / Chapter 2.3.5. --- KIAA0319L and its surrounding genes --- p.85 / Association of KIAA03190L and its surrounding genes with Chinese dyslexic children --- p.85 / Chapter 2.3.6. --- Gene-Gene interaction analyses --- p.88 / Chapter 2.3.7. --- Parent-of-origin analysis --- p.92 / Chapter 2.4. --- Discussion --- p.94 / Chapter 2.4.1. --- DYX1C1 variant associated with DD and reading skills --- p.94 / Chapter 2.4.2. --- KIAA0319 associated with phonological awareness in Chinese --- p.99 / Chapter 2.4.3. --- DCDC2 is not associated with DD in Chinese children --- p.108 / Chapter 2.4.4. --- Association of MRPL19 and C2ORF3 in a Chinese sample --- p.111 / Chapter 2.4.5. --- Association of KIAA03190L and its surrounding genes with Chinese children with dyslexia --- p.115 / Chapter 2.4.6. --- Gene-Gene interaction --- p.118 / Chapter 2.4.7. --- Parent-of-origin --- p.119 / Chapter 2.5. --- Summary --- p.122 / Chapter 3. --- Chapter 3 --- p.126 / Resequencing analyses and characterization of 5' upstream of KIAA0319 --- p.126 / Chapter 3.1. --- Introduction --- p.127 / Chapter 3.2. --- Materials and Methods --- p.129 / Chapter 3.2.1. --- DNA samples --- p.129 / Chapter 3.2.2. --- DNA re-sequencing --- p.129 / Chapter 3.2.3. --- KIAA0319 Promoter constructs --- p.131 / Chapter 3.2.4. --- Luciferase Reporter Assays --- p.133 / Chapter 3.2.5. --- In-silico sequence analyses --- p.133 / Chapter 3.3. --- Results --- p.134 / Chapter 3.4. --- Discussion --- p.143 / Chapter 4. --- Chapter 4 --- p.148 / Characterization of CG7565, a homolog of KIAA0319, in a Drosophila model --- p.148 / Chapter 4.1. --- Introduction --- p.149 / Chapter 4.2. --- Methods and Materials --- p.152 / Chapter 4.2.1. --- Drosophila stock --- p.152 / Chapter 4.2.2. --- Sequence analyses --- p.153 / Chapter 4.2.3. --- RNA extraction and quantitative reverse-transcription PCR (RT-PCR) --- p.153 / Chapter 4.2.4. --- Behavioral Assays --- p.156 / Visual pattern memory assays --- p.156 / Optomotor Response Assays --- p.158 / Visual Discrimination Analyses --- p.159 / Chapter 4.3. --- Results --- p.160 / Chapter 4.4. --- Discussion --- p.174 / Chapter 5. --- Chapter 5 --- p.181 / Mutational analyses of 5' region of MRPL19 in children with dyslexia --- p.181 / Chapter 5.1. --- Introduction --- p.182 / Chapter 5.2. --- Materials and Methods --- p.183 / Chapter 5.2.1. --- DNA samples --- p.183 / Chapter 5.2.2. --- High resolution melting analyses (HRM) --- p.183 / Chapter 5.2.3. --- MRPL19 promoter constructs --- p.185 / Chapter 5.2.4. --- In-silico sequence analyses --- p.186

Co-culture of eosinophils and BEAS-2B cells was found to increase the release of cytokine IL-6 and chemokines MIG, MCP-1, IL-8 and IP-10 and up-regulate the corresponding genes expression in BEAS-2B cells or eosinophils. Interaction of eosinophil-BEAS-2B cells could also elevate adhesion molecules ICAM-1, VCAM-1, ICAM-3, and CD49d expression on the surface of BEAS-2B cells, and CD18 and ICAM-3 on eosinophils, and up-regulate ICAM-1 gene expression in BEAS-2B cells. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha could induce or further induce ICAM-1 expression on eosinophils and BEAS-2B cells upon their interaction. Moreover, activities of both NF-kappaB and p38 MAPK in BEAS-2B cells were markedly elevated after co-cultured with eosinophils. / Freshly isolated eosinophils from human peripheral blood and confluent BEAS-2B cells were co-cultured together in tissue culture plate for a pre-determined time period. Cytokines including interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-10, IL-12p70, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma and chemokines regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein (MCP)-1, IL-8, and interferon inducible protein (IP)-10 in culture supernatant were evaluated by protein array and quantified by cytometric bead array (CBA) kit of Th1/Th2 cytokines, inflammatory cytokines, and human chemokines using flow cytometry and enzyme linked immunosorbent assay (ELISA) kit. / In order to investigate the immunopathological mechanism in allergic asthma of eosinophils interacting with bronchial epithelium in inflammation site, a in vitro system of co-culture of human bronchial epithelial cells and eosinophils was set up to mimic the inflammatory reaction. / In summary, co-culture of epithelial cells, BEAS-2B cells, and eosinophils could activate NF-kappaB and p38 MAPK signal transduction pathways to induce inflammatory cytokine IL-6, and chemokines IL-8, MCP-1, MIG and IP-10 release in culture supernatant, and up-regulated the expression of surface adhesion molecules ICAM-1, VCAM-1, ICAM-3 and CD49d protein on BEAS-2B, and CD18 and ICAM-3 on eosinophils. (Abstract shortened by UMI.) / In this study, co-culture of a human epithelial cell line, BEAS-2B cells, and peripheral eosinophils was adopted as an in vitro model to investigate the effect of interaction of epithelial cells and eosinophils in airways on pathophysiology of asthma. / Wang Chengbin. / "July 2005." / Advisers: Wai kei Lam; Chun kwok Wong; Yaping Tian. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3723. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 119-134). / 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. / Abstract in English and Chinese. / School code: 1307.

Research interest in cerebral small vessel disease, which is manifested as lacunar infarct and white matter changes, has surged in the last decade. Small vessel disease has been increasingly recognized via neuroimaging to be highly prevalent among the elderly and more importantly; it is associated with cognitive impairment. Since the population worldwide is ageing, the cognitive burden associated with small vessel disease is foreseen to rise. This burden will be particularly great in China where the population is vast. However, data of cognitive impairment related to small vessel disease among Chinese is scarce. / The methods and results of these studies will be presented in the thesis. In brief, the author concluded that (1) among Chinese stroke patients with relevant subcortical lacunar infarct, underlying intracranial large artery disease should be looked for before attributing that the lacunar infarct is due to small vessel disease because of its not uncommon association with lacunar infarcts among Chinese; (2) half of the patients with stroke associated with small vessel disease complain of varying severity levels of cognitive impairment 3 months poststroke and executive dysfunction also affects functional activities; (3) thalamic lacunar infarct and left frontal lobe atrophy have small yet significant influences on cognitive performances; (4) cerebral atrophy predicts prestroke cognitive impairment; and (5) Chinese frontal assessment battery is a moderately valid, while executive clock drawing test is not a valid test in the evaluation of executive dysfunction among Chinese with small vessel disease. / This thesis aimed to present studies that were conducted by the author among Chinese stroke patients on this particular field. The aims of the studies were to evaluate the (1) frequency of relevant intracranial large artery disease among Chinese stroke patients having subcortical lacunar infarcts; (2) frequency and impact of cognitive impairment after stroke associated with small vessel disease; (3) neuroimaging determinants of cognitive performances after stroke associated with small vessel disease; (4) determinants of prestroke cognitive impairment in stroke associated with small vessel disease; and the (5) validity of frontal assessment battery and executive clock drawing test in assessing executive dysfunction among Chinese patients with small vessel disease. / Mok Chung Tong Vincent. / "April 2005." / Adviser: Lawrence Ka Sing Wong. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3695. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 180-197). / 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. / School code: 1307.

Tsang, Wan Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-165). / Abstracts in English and Chinese. / Abstract --- p.iii / Abstract in Chinese (摘要） --- p.V / Acknowledgement --- p.vi / Table of Content --- p.vii / List of Figures and Tables --- p.xiv / List of Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.Page / Chapter 1.1 --- Developmental Dyslexia --- p.1 / Chapter 1.1.1 --- Study of Developmental Dyslexia --- p.1 / Chapter 1.1.2 --- Genetic Basis of Developmental Dyslexia --- p.2 / Chapter 1.1.3 --- Susceptibility Gene for Developmental Dyslexia --- p.3 / Chapter 1.2 --- Dyslexia-Susceptibility Gene KIAA0319-Like --- p.5 / Chapter 1.2.1 --- Association Studies of KIAA0319-Like --- p.5 / Chapter 1.2.2 --- KIAA0319-Like Gene and Protein --- p.6 / Chapter 1.2.3 --- Functional prediction of KIAA0319-Like Protein --- p.10 / Chapter 1.3 --- Potential Interacting Partners of KIAA0319-Like Protein --- p.12 / Chapter 1.3.1 --- Nogo Receptor 1 (NgR) --- p.12 / Chapter 1.3.2 --- SH2B Adaptor Protein 1 (SH2) --- p.13 / Chapter 1.3.3 --- FEM-1-like death receptor binding protein (FEM) --- p.14 / Chapter 1.3.4 --- Adaptor-related protein complex 2，mu 1 subunit (AP2) --- p.14 / Chapter 1.4 --- Notch Signaling Pathway and KIAA0319-Like --- p.16 / Chapter 1.5 --- Previous Research on KIAA0319-Like --- p.18 / Chapter 1.6 --- Research Objectives --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.Page / Chapter 2.1 --- Gene Cloning --- p.21 / Chapter 2.1.1 --- Cloning of Human KIAA0319-Like into expression vectors --- p.21 / Chapter 2.1.2 --- Gel Extraction of PCR Product --- p.22 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.22 / Chapter 2.1.4 --- Ligation of gene products with vector --- p.23 / Chapter 2.1.5 --- "Cloning of Human SH2, FEM, AP2 and NgR gene" --- p.23 / Chapter 2.1.7 --- Preparation of chemically competent E.coli strain DH5a --- p.26 / Chapter 2.1.8 --- Transformation of the ligation product into competent cells --- p.26 / Chapter 2.1.9 --- Diagnostic PCR for confirmation of successful ligation --- p.27 / Chapter 2.1.10 --- Small scale preparation of bacterial plasmid DNA --- p.27 / Chapter 2.1.11 --- DNA sequencing of the cloned plasmid DNA --- p.28 / Chapter 2.1.12 --- Large scale preparation of target recombinant plasmid DNA --- p.28 / Chapter 2.2 --- Cell Culture --- p.30 / Chapter 2.2.1 --- Culture medium --- p.30 / Chapter 2.2.2 --- Cell lines --- p.30 / Chapter 2.2.3 --- Freezing and thawing cells --- p.31 / Chapter 2.3 --- DNATransfection --- p.32 / Chapter 2.3.1 --- Transfection of HEK293 Cells with LipofectaminéёØ Reagent --- p.32 / Chapter 2.3.2 --- Transfection of Neuronal Cells with Tranfas´tёØ Reagent --- p.32 / Chapter 2.4 --- Immunocytochemical Staining --- p.33 / Chapter 2.5 --- Immunohistochemical Staining --- p.34 / Chapter 2.6 --- Western Blotting --- p.36 / Chapter 2.6.1 --- Collection of Cell Lysate --- p.36 / Chapter 2.6.2 --- Collection of Mouse Tissue Lysate --- p.36 / Chapter 2.6.3 --- Protein concentration determination by Bradford Protein Assay --- p.36 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.37 / Chapter 2.6.5 --- Semidry Transfer to PVDF Membrane --- p.37 / Chapter 2.6.6 --- Blocking and Antibody Probing --- p.38 / Chapter 2.6.7 --- Enhanced chemiluminescence (ECL) --- p.38 / Chapter 2.6.8 --- Synthetic peptide based neutralization --- p.39 / Chapter 2.7 --- Quantitative Real-Time PCR --- p.40 / Chapter 2.7.1 --- RNA extraction from Cell culture --- p.40 / Chapter 2.7.2 --- Genomic DNA Elimination and Reverse Transcription --- p.40 / Chapter 2.7.3 --- Quantitative Real-Time PCR --- p.41 / Chapter 2.8 --- Coimmunoprecipitation --- p.42 / Chapter 2.9 --- Luciferase Reporter Assay --- p.43 / Chapter 2.10 --- Wound-Healing Assay --- p.44 / Chapter Chapter 3 --- Distribution of KIAA0319-Like Protein --- p.Page / Chapter 3.1 --- Study of the Distribution of KIAA0319-Like Protein --- p.45 / Chapter 3.1.1 --- Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.1.2 --- Affinity Purification of Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.2 --- C57/BL6 Mouse Tissue Expression Pattern of KIAA0319-Like --- p.48 / Chapter 3.3 --- C57/BL6 Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.50 / Chapter 3.4 --- Immunostaining of Sagittal Section of C57/BL6 Mouse Brain --- p.52 / Chapter 3.5 --- Mouse Brain Co-staining with KIAA0319-Like Protein and Markers --- p.54 / Chapter 3.6 --- Subcelluar Localization of KIAA0319-Like Protein in Human Neurons --- p.57 / Chapter 3.7 --- Discussion --- p.59 / Chapter 3.7.1 --- Affinity Purification of Anti-KIAA0319-Like Antisera --- p.59 / Chapter 3.7.2 --- Mouse Tissue Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.3 --- Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.4 --- Brain Cell Types expressing KIAA0319-Like Protein --- p.61 / Chapter 3.7.5 --- Subcellular Localization of KIAA0319-Like Protein --- p.62 / Chapter 3.7.6 --- Future Perspectives --- p.63 / Chapter Chapter 4 --- KIAA0319-Like Modulates Neuronal Migration --- p.Page / Chapter 4.1 --- Study of the effect of KIAA0319-Like on Neuronal Migration --- p.64 / Chapter 4.2 --- Establish Stable Cell Lines with Altered KIAA0319-Like Expression --- p.65 / Chapter 4.2.1 --- Cloning of siRNA into Lentiviral Vector pLVTHM --- p.65 / Chapter 4.2.2 --- Lentivirus packaging in HEK293 Cells --- p.67 / Chapter 4.2.3 --- Checking of Lentivirus by HEK293 Cells Transduction --- p.70 j / Chapter 4.2.4 --- Lentiviral Transductions to Neuronal Cells --- p.70 / Chapter 4.2.5 --- Fluorescent-Activated Cell Sorting of Transduced Cells --- p.70 / Chapter 4.2.6 --- KIAA0319-Like Expression Changes in Stable Cell Lines --- p.71 / Chapter 4.3 --- Effects of KIAA0319-Like Overexpression on Neuronal Migration --- p.75 / Chapter 4.4 --- Effects of KIAA0319-Like Knockdown on Neuronal Migration --- p.77 / Chapter 4.5 --- Effect of Anti-KIAA0319-Like Antibody on Neuronal Migration --- p.79 / Chapter 4.6 --- Discussion --- p.81 / Chapter 4.6.1 --- Stable Cell line with altered KIAA0319-Like Level --- p.81 / Chapter 4.6.2 --- Disruption of KIAA0319-Like expression affects Cell Migration --- p.81 / Chapter 4.6.3 --- Antibody Blockade of KIAA0319-Like Inhibits Cell Migration --- p.81 / Chapter 4.6.4 --- Possible existence of Multiple Regulatory Pathways --- p.82 / Chapter 4.6.5 --- Possible Limitations of Expression alternations by Transduction --- p.83 / Chapter 4.6.6 --- Possible Susceptibility Polymorphism of KIAA0319-Like --- p.83 / Chapter 4.6.7 --- Future Perspectives X --- p.84 / Chapter Chapter 5 --- Interacting Partners of KIAA0319-Like Protein --- p.Page / Chapter 5.1 --- Study of Interacting Partners of KIAA0319-Like Protein --- p.85 / Chapter 5.1.1 --- Identification of Potential Interacting Partners of KIAA0319-Like --- p.85 / Chapter 5.1.2 --- Identification of Domains involved in Interactions --- p.86 / Chapter 5.1.3 --- Subcloning of KIAA0319-Like Protein Domain Deletion Mutant --- p.86 / Chapter 5.2 --- Interaction between NgR and KIAA0319-Like Protein --- p.92 / Chapter 5.2.1 --- Reciprocal Coimmunoprecipitation of NgR for KIAA0319-Like --- p.92 / Chapter 5.2.2 --- Colocalization of NgR and KIAA0319-Like Protein --- p.94 / Chapter 5.2.3 --- Interaction between NgR and KIAA0319-Like Deletion Mutants --- p.97 / Chapter 5.3 --- Interaction between SH2 and KIAA0319-Like Protein --- p.99 / Chapter 5.3.1 --- Coimmunoprecipitation between SH2 and KIAA0319-Like --- p.» 99 / Chapter 5.3.2 --- Colocalization of SH2 and KIAA0319-Like Protein --- p.101 / Chapter 5.3.3 --- Interaction between SH2 and KIAA0319-Like Deletion Mutants --- p.103 / Chapter 5.4 --- Interaction between FEM and KIAA0319-Like Protein --- p.105 / Chapter 5.4.1 --- Coimmunoprecipitation between FEM and KIAA0319-Like --- p.105 / Chapter 5.4.2 --- Colocalization of FEM and KIAA0319-Like Protein --- p.107 / Chapter 5.4.3 --- Interaction between FEM and KIAA0319-Like Deletion Mutants --- p.109 / Chapter 5.5 --- Interaction between AP2 and KIAA0319-Like Protein --- p.111 / Chapter 5.5.1 --- Coimmunoprecipitation between AP2 and KIAA0319-Like --- p.111 / Chapter 5.5.2 --- Colocalization of AP2 and KIAA0319-Like Protein --- p.113 / Chapter 5.5.3 --- Interaction between AP2 and KIAA0319-Like Deletion Mutants --- p.115 / Chapter 5.6 --- Discussion --- p.117 / Chapter 5.6.1 --- The Interaction of KIAA0319-Like with NgR and SH2 Protein --- p.118 / Chapter 5.6.2 --- Interaction between FEM and KIAA0319-Like Protein --- p.121 / Chapter 5.6.3 --- Interaction between AP2 and KIAA0319-Like Protein --- p.121 / Chapter 5.6.4 --- Future Perspectives --- p.122 / Chapter Chapter 6 --- KIAA0319-Like Association with Notch Pathway --- p.Page / Chapter 6.1 --- Study of KIAA0319-Like association with Notch Pathway --- p.123 / Chapter 6.1.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.123 / Chapter 6.2 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in HEK293 Cells --- p.126 / Chapter 6.3 --- Effects of Increasing KIAA0319-Like Protein Level in Culture Medium on y-Secretase Activity in HEK293 Cells --- p.128 / Chapter 6.4 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in SH-SY5Y Cells --- p.130 i / Chapter 6.5 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in Neuro-2a Cells --- p.132 / Chapter 6.6 --- Effect of Notch Blockade on SH-SY5Y Cells Migration Rate --- p.134 / Chapter 6.7 --- Effect of KIAA0319-Like Expression changes on Notch Inhibited SH-SY5Y Cells Migration Rate --- p.136 / Chapter 6.8 --- Discussion --- p.139 / Chapter 6.8.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.139 / Chapter 6.8.2 --- Cell-Type Specific association of KIAA0319-Like expression level and y-Secretase activity --- p.139 / Chapter 6.8.3 --- Entry Point of KIAA0319-Like to the Notch Signaling Pathway --- p.140 / Chapter 6.8.4 --- Functional relationship between KIAA0319-Like and Notch --- p.141 / Chapter 6.8.5 --- Future Perspectives --- p.142 / Chapter Chapter 7 --- Discussions and Conclusions --- p.Page / Chapter 7.1 --- Discussions --- p.143 / Chapter 7.1.1 --- Further Evidence for KIAA0319-Like Association with Dyslexia --- p.143 / Chapter 7.1.2 --- Possible Pathways connecting KIAA0319-Like with Cell Motility --- p.145 / Chapter 7.1.3 --- Nogo Receptor Pathway association with Cell Motility --- p.146 / Chapter 7.1.4 --- Notch Signaling Pathway association with Cell Motility --- p.146 / Chapter 7.1.5 --- Combined Effects of NgR and Notch Pathway on Cell Migration --- p.149 / Chapter 7.1.6 --- Cellular Compartment for Interactions of KIAA0319-Like --- p.151 / Chapter 7.2 --- Conclusions --- p.153 / Chapter Chapter 8 --- References --- p.156 / Chapter Chapter 9 --- Appendix --- p.166

血管紧张素II(Ang II)在慢性肾脏病中起重要的致病作用，尽管体外研究证实TGF-β/Smad3起正调控，Smad7起负调控作用，但Smad3在Ang II 诱导的肾脏损害中的作用仍不清楚。因此，本论文在Smad3基因敲除的小鼠中通过Ang II诱导的高血压肾损伤模型研究TGF-β/Smad3通路的作用及机制。如第三章所述，敲除Smad3的小鼠不发生Ang II诱导的高血压肾损伤如尿白蛋白，血肌酐升高，肾脏炎症（如IL-1, TNFα上调，F4/80+ 巨噬细胞浸润）及肾脏纤维化（包括α-SMA+肌成纤维细胞聚集，和胶原基质沉积）。敲除Smad3对高血压肾病起保护作用是因为抑制了肾脏TGF-β1表达及Smurf2 依赖的Smad7泛素化降解，从而抑制TGF-β/Smad3介导的肾脏纤维化和NF-B介导的炎症。 / 越来越多的证据显示Ang II产生和降解的平衡在高血压肾病的发展中起重要作用。在这篇论文中，我们假设ACE2的降解可能会引起Ang II代谢通路的失衡，从而加重其介导的高血压肾病。这一假设在第四章得到验证，在单侧输尿管梗阻小鼠模型敲除ACE2加重肾内Ang II介导的肾脏纤维化和炎症。这一变化与肾内高水平的Ang II和降低的血管紧张素1-7，上调的血管紧张素受体1，及激活的TGF-β/Smad3 和 NF-κB 信号通路有关。另外，升高的Smurf2介导的Smad7泛素化降解加重了敲除ACE2 基因后Ang II介导的肾脏纤维化和炎症。 / 因为Smad7 是TGF-β/Smad和NF-κB通路的负调控因子，因此论文进一步提出假设过表达Smad7能够阻止Ang II介导的肾脏纤维化炎症。如第五章所述，ACE2基因敲除的小鼠肾内升高的Smurf2介导了肾脏Smad7 的泛素化降解， 加重了Ang II 介导的肾脏损伤如白蛋白尿，血肌酐的升高，及肾脏纤维化和炎症，这与激活的Ang II/TGF-β/Smad3/NF-κB信号有关。相反，过表达Smad7能够阻断TGF-β/Smad3 介导的肾脏纤维化和 NF-κB介导的肾脏炎症以缓解ACE2敲除小鼠中Ang II诱导的肾脏损伤。 / 总之，Smad3在Ang II诱导的高血压肾脏病中起关键作用，Smad7具有肾脏保护作用。 ACE2敲除引起Ang II产生和降解的失衡从而增加肾内Ang II的产生，加重TGF-β/Smad3介导的肾脏纤维化和NF-κB介导的肾脏炎症，而这可以被Smad7缓解。 本论文得出结论针对TGF-β/Smad3 和NF-κB通路，通过过表达Smad7可能为高血压肾脏病和慢性肾脏病提供新的治疗策略。 / Angiotensin II (Ang II) plays a pathogenic role in chronic kidney disease (CKD). Although in vitro studies find that Ang II mediates renal fibrosis via the Smad3-dependent mechanism, the functional role of Smad3 in Ang II-mediated kidney disease remains unclear. Therefore, this thesis examined the pathogenesis role and mechanisms of TGF-β/Smad3 in Ang II-mediated hypertensive nephropathy in Smad3 Knockout (KO) mice. As described in Chapter III, Smad3 deficiency protected against Ang II-induced hypertensive nephropathy as demonstrated by lowering levels of albuminuria, serum creatinine, renal inflammation such as up-regulation of pro-inflammatory cytokines (IL-1β, TNFα) and infiltration of CD3+ T cells and F4/80+ macrophages, and renal fibrosis including α-SMA+ myofibroblast accumulation and collagen matrix deposition (all p<0.01). Inhibition of hypertensive nephropathy in Smad3 KO mice was associated with reduction of renal TGF-β1 expression and Smurf2-associated ubiquitin degradation of renal Smad7, thereby blocking TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. / Increasing evidence shows that the balance between the generation and degradation of Ang II is also important in the development of hypertensive nephropathy. In this thesis, we also tested a hypothesis that enhanced degradation of ACE2 may result in the imbalance between the Ang II generation and degradation pathways, therefore enhancing Ang II-mediated hypertensive nephropathy and CKD. This hypothesis was examined in a mouse model of unilateral ureteral obstructive nephropathy (UUO) induced in ACE2 KO mice. As described in Chapter IV, loss of ACE2 increased intrarenal Ang II-mediated renal fibrosis and inflammation in the UUO kidney. These changes were associated with higher levels of intrarenal Ang II, reduced Ang 1-7, up-regulated AT1R, and activation of TGF-β/Smad3 and NF-κB signalling. In addition, enhanced Smurf2-associated ubiquitin degradation of Smad7 was another mechanism by which loss of ACE2 promoted Ang II-mediated renal fibrosis and inflammation. / Because Smad7 is a negative regulator for TGF-β/Smad and NF-κB signalling, this thesis also examined a hypothesis that overexpression of renal Smad7 may be able to prevent Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in ACE2 KO mice. As described in Chapter V, mice null for ACE2 resulted in degradation of renal Smad7 via the Smurf2 -- dependent mechanism (all p<0.01). Enhanced Ang II-mediated renal injury in ACE2 KO mice such as albuminuria, serum creatinine, and renal fibrosis and inflammation was associated with enhanced activation of Ang II/TGF-β/Smad3/NF-κB signalling. In contrast, overexpression of Smad7 was able to rescue AngII-induced progressive renal injury in ACE2 KO mice by blocking TGF-β/Smad3 and NF-κB-dependent renal fibrosis and inflammation. In conclusion, Smad3 plays an essential role in Ang II-induced hypertensive nephropathy, while Smad7 is reno-protective. Loss of ACE2 results in the imbalance between the Ang II generation and degradation pathways and thus enhances intrarenal Ang II-induced, TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation, which can be rescued by Smad7. Results from this thesis indicate that targeting TGF-β/Smad3 and NF-κB pathways by overexpressing Smad7 may represent a novel therapy for hypertensive nephropathy and CKD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Zhen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-209). / Abstracts also in Chinese. / ABSTRACT --- p.i / DECLARATION --- p.v / ACKNOWLEDGEMENTS --- p.vi / LIST OF PUBLICATION --- p.viii / TABLE OF CONTENTS --- p.ix / LIST OF ABBREVIATIONS --- p.xiv / LIST OF FIGURES AND TABLES --- p.xvii / CHAPTER I --- p.1 / INTRODUCTION --- p.1 / Chapter 1.1 --- RAS (Renin-Angiotensin system) --- p.2 / Chapter 1.1.1 --- Circulating RAS --- p.2 / Chapter 1.1.2 --- Tissue RAS --- p.5 / Chapter 1.1.2.1 --- Angiotensinogen --- p.6 / Chapter 1.1.2.2 --- Renin Receptors --- p.7 / Chapter 1.1.2.3 --- ACE and ACE2 --- p.9 / Chapter 1.1.2.4 --- Angiontensin II and Its Receptors --- p.10 / Chapter 1.1.2.5 --- AT2 Receptors --- p.11 / Chapter 1.1.2.6 --- Chymase-Alternative Pathways of Ang II Generation --- p.13 / Chapter 1.1.2.7 --- Ang (1-7) Receptor (MAS) --- p.13 / Chapter 1.2 --- Ang II and Renal Injury --- p.15 / Chapter 1.2.1 --- Pressure Dependent Renal Injury Induced by Ang II --- p.15 / Chapter 1.2.2 --- Ang II induces production of cytokines and growth factors --- p.16 / Chapter 1.2.3 --- Ang II and Renal Fibrosis --- p.17 / Chapter 1.2.4 --- Signalling Mechanisms Involved in Ang II-Induced Renal Fibrosis --- p.18 / Chapter 1.2.5 --- Ang II in Renal Inflammation --- p.22 / Chapter 1.3 --- TGF-β/Smad Signalling Pathway in Renal Disease --- p.24 / Chapter 1.3.1 --- Mechanisms of TGF-β/Smad Activation --- p.24 / Chapter 1.3.1.1 --- Cross-talk Between Smads and Other Signalling Pathways in Renal Fibrosis --- p.26 / Chapter 1.3.1.2 --- Activation of R-Smads (Smad2 and Smad3) --- p.28 / Chapter 1.3.2 --- Inhibitory Role of Smad7 in Renal Fibrosis and Inflammation --- p.30 / Chapter CHAPTER II --- p.32 / MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- MATERIALS --- p.33 / Chapter 2.1.1 --- Regents and Equipments --- p.33 / Chapter 2.1.1.1 --- Regents and Equipments for Cell Culture --- p.33 / Chapter 2.1.1.2 --- General Reagents and Equipments for Real-time PCR --- p.34 / Chapter 2.1.1.3 --- General Reagents and Equipments for Masson Trichrome Staining --- p.34 / Chapter 2.1.1.4 --- General Reagents and Equipments for Immunohistochemistry --- p.35 / Chapter 2.1.1.5 --- General Reagents and Equipments for Western Blot --- p.35 / Chapter 2.1.1.6 --- General Reagents and Equipments for ELISA --- p.37 / Chapter 2.1.1.7 --- Measurement of Blood Pressure in Mice --- p.37 / Chapter 2.1.1.8 --- Reagents and Equipment for Genotyping --- p.37 / Chapter 2.1.2 --- Buffers --- p.38 / Chapter 2.1.2.1 --- Immunohistochemistry Buffers --- p.38 / Chapter 2.1.2.2 --- Buffers for Western Blotting --- p.40 / Chapter 2.1.2.3 --- ELISA Buffers --- p.44 / Chapter 2.1.2.4 --- Primer Sequences --- p.46 / Chapter 2.1.2.5 --- Primary Antibodies --- p.47 / Chapter 2.1.2.6 --- Secondary Antibodies --- p.48 / Chapter 2.2 --- METHODS --- p.49 / Chapter 2.2.1 --- Animal --- p.49 / Chapter 2.2.1.1 --- Genotypes of Gene KO Mice --- p.49 / Chapter 2.2.1.2 --- Animal Model of Unilateral Ureteral Obstruction (UUO) --- p.50 / Chapter 2.2.1.3 --- Animal Model of Angiotensin II (Ang II)-Induced Hypertensive Nephropathy --- p.50 / Chapter 2.2.1.4 --- Measurement of Ang II and Ang 1-7 --- p.51 / Chapter 2.2.2 --- Cell Culture --- p.51 / Chapter 2.2.3 --- Microalbuminuria and Renal Function --- p.51 / Chapter 2.2.3.1 --- Urine Collection --- p.51 / Chapter 2.2.3.2 --- Plasma Collection --- p.52 / Chapter 2.2.3.3 --- Microalbuminuria --- p.52 / Chapter 2.2.3.4 --- Creatinine Measurement --- p.52 / Chapter 2.2.4 --- Real-time PCR --- p.53 / Chapter 2.2.4.1 --- Total RNA Extraction --- p.53 / Chapter 2.2.4.2 --- Reverse Transcription --- p.53 / Chapter 2.2.4.3 --- Real-time PCR --- p.54 / Chapter 2.2.4.4 --- Analysis of Real-time PCR --- p.54 / Chapter 2.2.5 --- Western Blot --- p.55 / Chapter 2.2.5.1 --- Protein Preparation --- p.55 / Chapter 2.2.5.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.56 / Chapter 2.2.5.3 --- Protein Transfer (Wet Transfer) --- p.56 / Chapter 2.2.5.4 --- Incubation of Antibodies --- p.56 / Chapter 2.2.5.5 --- Scanning and Analysis --- p.57 / Chapter 2.2.5.6 --- Stripping --- p.57 / Chapter 2.2.6 --- Histochemistry --- p.57 / Chapter 2.2.6.1 --- Tissue Fixation --- p.57 / Chapter 2.2.6.2 --- Tissue Embedding and Sectioning --- p.58 / Chapter 2.2.6.3 --- Preparation of Paraffin Tissue Sections for PAS Staining --- p.58 / Chapter 2.2.6.4 --- PAS Staining --- p.58 / Chapter 2.2.7 --- Immunohistochemistry --- p.59 / Chapter 2.2.7.1 --- Tissue Embedding and Sectioning --- p.59 / Chapter 2.2.7.2 --- Antigen-Antibody Reaction and Immunostaining --- p.59 / Chapter 2.2.7.3 --- Semi-quantification of Immunohistochemistry --- p.60 / Chapter 2.2.8 --- Statistical Analysis --- p.60 / Chapter CHAPTER III --- p.62 / ROLE OF SMAD3 IN ANGIOTENSIN II-INDUCED RENAL FIBROSIS AND INFLAMMATION --- p.62 / Chapter 3.1 --- INTRODUCTION --- p.63 / Chapter 3.2 --- MATERIALS AND METHODS --- p.64 / Chapter 3.2.1 --- Generation of Smad3 KO Mice --- p.64 / Chapter 3.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.64 / Chapter 3.2.3 --- Histology and Immunohistochemistry --- p.65 / Chapter 3.2.4 --- Renal Function and Proteinuria --- p.65 / Chapter 3.2.5 --- Western Blot Analysis --- p.65 / Chapter 3.2.6 --- Real-time RT-PCR --- p.65 / Chapter 3.2.7 --- In Vitro Study of Mesangial Cells from Smad3 WT and KO Mice --- p.66 / Chapter 3.2.8 --- Statistical Analysis --- p.66 / Chapter 3.3 --- RESULTS --- p.66 / Chapter 3.3.1 --- Smad3 KO Mice Prevents Ang II-induced Renal Injury Independent of Blood Pressure --- p.66 / Chapter 3.3.2 --- Smad3 KO Mice Are Resistant to Renal Fibrosis in a Mouse Model of Ang II -Induced Hypertension --- p.70 / Chapter 3.3.3 --- Smad3 KO Mice Are Resistant to Renal Inflammation in a Mouse Model of Ang II-Induced Hypertension --- p.76 / Chapter 3.3.4 --- Smad3 Deficiency Inhibits Ang II-induced Renal Fibrosis and Inflammation In Vitro --- p.82 / Chapter 3.3.5 --- Smad3 Mediates Ang II-Induced Renal Fibrosis by the Positive Feedback Mechanism of TGF-β/Smad Signalling --- p.87 / Chapter 3.3.6 --- Enhancing NF-κB Signalling via the Smurf2-associated Ubiquitin Degradation of Smad7 In Vivo and In Vitro --- p.92 / Chapter 3.4 --- DISCUSSION --- p.101 / Chapter 3.5 --- CONCLUSION --- p.106 / Chapter CHAPTER IV --- p.107 / LOSS OF ANGIOTENSIN-CONVERTING ENZYME 2 ENHANCES TGF-β/SMAD-MEDIATED RENAL FIBROSIS AND NF-κB-DRIVEN RENAL INFLAMMATION IN A MOUSE MODEL OF OBSTRUCTIVE NEPHROPATHY --- p.107 / Chapter 4.1 --- INTRODUCTION --- p.108 / Chapter 4.2 --- MATERIALS AND METHODS --- p.109 / Chapter 4.2.1 --- Generation of ACE2 KO Mice --- p.109 / Chapter 4.2.2 --- Mouse Model of Unilateral Ureteral Obstruction (UUO) --- p.109 / Chapter 4.2.3 --- Histology and Immunohistochemistry --- p.110 / Chapter 4.2.4 --- Western Blot Analysis --- p.110 / Chapter 4.2.5 --- Real-time RT-PCR --- p.110 / Chapter 4.2.6 --- Measurement of Ang II and Ang 1-7 --- p.110 / Chapter 4.2.7 --- Statistical Analysis --- p.111 / Chapter 4.3 --- RESULTS --- p.111 / Chapter 4.3.1 --- ACE2 KO Mice Accelerate Renal Fibrosis and Inflammation Independent of Blood Pressure in the UUO Nephropathy --- p.111 / Chapter 4.3.2 --- Loss of ACE2 Enhances Ang II, Activation of TGF-β/Smad and NF-κB Signalling Pathways --- p.128 / Chapter 4.3.3 --- Loss of Renal Smad7 Is an Underlying Mechanism Accounted for the Progression of TGF-β/Smad-mediated Renal Fibrosis and NF-κB-Driven Renal Inflammation in the UUO Nephropathy in ACE2 KO Mice --- p.140 / Chapter 4.4 --- DISCUSSION --- p.143 / Chapter 4.5 --- CONCLUSION --- p.147 / CHAPTER V --- p.148 / PROTECTIVE ROLE OF SMAD7 IN HYPERTENSIVE NEPHROPATHY IN ACE2 DEFICIENT MICE --- p.148 / Chapter 5.1 --- INTRODUCTION --- p.149 / Chapter 5.2 --- MATERIALS AND METHODS --- p.151 / Chapter 5.2.1 --- Generation of ACE2 KO Mice --- p.151 / Chapter 5.2.2 --- Mouse Model of Ang II-Induced Hypertension --- p.151 / Chapter 5.2.3 --- Smad7 Gene Therapy --- p.151 / Chapter 5.2.4 --- Histology and Immunohistochemistry --- p.152 / Chapter 5.2.5 --- Western Blot Analysis --- p.153 / Chapter 5.2.6 --- Real-time RT-PCR --- p.153 / Chapter 5.2.7 --- Measurement of Ang II and Ang 1-7 --- p.153 / Chapter 5.2.8 --- Statistical Analysis --- p.153 / Chapter 5.3 --- RESULTS --- p.154 / Chapter 5.3.1 --- Deletion of ACE2 Accelerates Ang II-Induced Renal Injury --- p.154 / Chapter 5.3.2 --- Renal Fibrosis and Inflammation are Enhanced in ACE2 KO Mice with Ang II-Induced Renal Injury --- p.156 / Chapter 5.3.3 --- Enhanced Activation of TGF-β/Smad3 and NF-κB Signalling Pathways are Key Mechanism by Which Deletion of ACE2 Promotes Ang II-Induced Renal Injury --- p.163 / Chapter 5.3.4 --- Loss of Renal Smad7 Mediated by Smurf2-ubiquintin Degradation Pathway Contributes to Ang II-Induced Hypertensive Nephropathy in ACE2 KO Mice --- p.166 / Chapter 5.3.5 --- Overexpression of Smad7 is able to Rescue Ang II-induced Renal Injury in ACE2 KO Mice by Blocking Both TGF-β/Smad3 and NF-κB-dependent Renal Fibrosis and Inflammation --- p.168 / Chapter 5.4 --- DISCUSSION --- p.180 / Chapter 5.5 --- CONCLUSION --- p.182 / Chapter CHAPTER VI --- p.183 / SUMMARY AND DISCUSSION --- p.183 / Chapter 6.1 --- Smad3 Plays a Key Role in Ang II-Induced Hypertensive Nephropathy --- p.185 / Chapter 6.2 --- The Intrarenal Ang II Plays a Key Role in the Progress of Ang II-Mediated Renal Injury --- p.185 / Chapter 6.3 --- A Novel Finding of Ang II-Smad3-TGF-β-Smad3 amplification loop in Ang II-mediated Renal Fibrosis --- p.186 / Chapter 6.4 --- Smurf2-associated Ubiquitin-Proteasome Degradation of Smad7 Contributes to the Progression of Ang II-mediated Renal Injury in ACE2 KO Mice --- p.187 / Chapter 6.5 --- Smad7 Protects against Ang II-Mediated Hypertensive Kidney Disease by Negatively Regulating TGF-β/Samd and NF-κB Signalling --- p.187 / REFERENCE --- p.189

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