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

Wu, Chi Chung. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 86-96). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- Neurodegenerative disorders 一 a brief overview --- p.1 / Chapter 1.2. --- Polyglutamine diseases --- p.1 / Chapter 1.3. --- Polyglutamine protein conformers and toxicity --- p.5 / Chapter 1.4. --- in vivo modeling of polyglutamine diseases in Drosophila / Chapter 1.4.1. --- GAL4/UAS transgene expression system in Drosophila --- p.13 / Chapter 1.4.2. --- Temporal control of transgene expression systemin Drosophila --- p.15 / Chapter 1.4.3. --- Drosophila as a model to study polyglutamine diseases --- p.16 / Chapter 1.5. --- in vitro polyglutamine diseases models --- p.19 / Chapter 1.6. --- Aim of study --- p.23 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- Drosophila culture and manipulation / Chapter 2.1.1. --- Drosophila culture --- p.25 / Chapter 2.1.2. --- Pseudopupil assay of adult retinal degeneration --- p.25 / Chapter 2.2. --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.2.1. --- Protein extraction from adult Drosophila heads --- p.26 / Chapter 2.2.2. --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.27 / Chapter 2.2.3. --- Western blotting --- p.28 / Chapter 2.2.4. --- Immunodetection --- p.29 / Chapter 2.3. --- Solubilization of SDS-insoluble protein --- p.31 / Chapter 2.4. --- Filter retardation assay --- p.31 / Chapter 2.5. --- Immunoprecipitation --- p.32 / Chapter 2.6. --- Nucleocytoplasmic fractionation --- p.33 / Chapter 2.7. --- PCR cloning / Chapter 2.7.1 . --- Drosophila DNA preparation --- p.34 / Chapter 2.7.2. --- Construction of pGEX4T3-MJDflQ27/81 expression plasmid --- p.34 / Chapter 2.8. --- in vitro aggregation assay / Chapter 2.8.1. --- Expression and purification of GST-MJDAQ27/81 protein --- p.36 / Chapter 2.8.2. --- in vitro aggregation --- p.37 / Chapter 2.8.3. --- Native slot-blot --- p.38 / Chapter 2.9. --- Reagents and buffers / Chapter 2.9.1. --- Reagents for Drosophila culture --- p.39 / Chapter 2.9.2. --- Reagents for SDS-PAGE --- p.39 / Chapter 2.9.3. --- Reagents for filter retardation assay --- p.42 / Chapter 2.9.4. --- Reagents for immunoprecipitation --- p.43 / Chapter 2.9.5. --- Reagents for nucleocytoplasmic fractionation --- p.43 / Chapter 2.9.6. --- Reagents for PCR cloning --- p.44 / Chapter 2.9.7. --- Reagents for in vitro aggregation assay --- p.46 / Chapter 3. --- Establishment of a GAL80ts-mediated transgenic Drosophila model of Machado-Joseph Disease (MJD) / Chapter 3.1. --- Introduction --- p.48 / Chapter 3.2. --- Results / Chapter 3.2.1. --- GAL80ts-mediated expression of expanded full-length MJD protein caused progressive neuronal degenerationin Drosophila --- p.49 / Chapter 3.2.2. --- Detection of SDS-insoluble expanded full-length MJD protein and its correlation with neuronal degeneration / Chapter 3.2.2.1. --- Progressive neuronal degeneration is not mediated by progressive accumulation of expanded full-length MJD protein --- p.51 / Chapter 3.2.2.2. --- SDS-soluble expanded full-length MJD protein does not correlate with progressive neuronal degeneration --- p.53 / Chapter 3.2.2.3. --- Progressive accumulation of SDS-insoluble expanded full-length MJD protein correlate with progressive neuronal degeneration --- p.55 / Chapter 3.3. --- Discussion --- p.57 / Chapter 4. --- Detection of conformational changes of expanded full-length MJD protein and its association with neuronal degeneration / Chapter 4.1. --- Introduction --- p.60 / Chapter 4.2. --- Results / Chapter 4.2.1. --- Expanded full-length MJD protein underwent conformational changes from monomer to fibrils and such conformational changes correlated with neuronal degeneration --- p.61 / Chapter 4.2.2. --- Mechanistic studies of how conformational changes of expanded full-length MJD protein triggers neuronal degeneration / Chapter 4.2.2.1. --- Expanded full-length MJD protein gradually accumulated in the nucleus during the course of neurodegeneration --- p.62 / Chapter 4.2.2.2. --- Fibrillar expanded full-length MJD protein caused transcriptional dysregulation of endogenous Hsp70 gene --- p.66 / Chapter 4.2.3. --- Consolidation of the role of fibrillar expanded full-length MJD protein in neuronal degeneration --- p.67 / Chapter 4.3. --- Discussion --- p.72 / Chapter 5. --- Attempts to generate new conformation-specific antibody against recombinant expanded full-length MJD proteins / Chapter 5.1. --- Introduction --- p.75 / Chapter 5.2. --- Results / Chapter 5.2.1. --- Recombinant expanded full-length MJD protein underwent conformational changes during in vitro aggregation --- p.75 / Chapter 5.3. --- Discussion --- p.77 / Chapter 6. --- GENERAL DISCUSSION --- p.81 / Chapter 7. --- CONCLUSION --- p.84 / Chapter 8. --- REFERENCES --- p.86

Wu, Hoi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 92-104). / Abstracts in English and Chinese. / Thesis/ Assessment Committee --- p.i / Declaration --- p.ii / Acknowledgments --- p.vi / Table of Content --- p.vii / List of Abbreviations --- p.x / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Introduction of diabetes mellitus --- p.1 / Chapter 1.1.1 --- Definition and prevalence --- p.1 / Chapter 1.1.2 --- Diagnosis and classification --- p.2 / Chapter 1.1.3 --- Symptoms and complications --- p.4 / Chapter 1.1.4 --- Cause and risk factors --- p.5 / Chapter 1.1.5 --- Prevention and treatment --- p.9 / Chapter 1.2 --- The role of adipose tissue in pathophysiology of T2DM --- p.10 / Chapter 1.2.1 --- Randle's glucose-fatty acid hypothesis --- p.11 / Chapter 1.2.2 --- Ectopic fat storage hypothesis --- p.12 / Chapter 1.2.3 --- Adipose tissue as an endocrine organ --- p.13 / Chapter 1.2.4 --- Low-grade inflammation --- p.15 / Chapter 1.2.5 --- Endoplasmic reticulum (ER) stress --- p.17 / Chapter 1.3 --- Use of berberine in the treatment of T2DM --- p.18 / Chapter 1.3.1 --- Efficacy of berberine in treating diabetes --- p.18 / Chapter 1.3.2 --- Berberine on glucose and lipid metabolism of animals --- p.19 / Chapter 1.3.3 --- Inhibition of adipogenesis --- p.20 / Chapter 1.3.4 --- Activation of AMP-Activated Protein Kinase (AMPK) --- p.20 / Chapter 1.3.5 --- Mitochondrial inhibition --- p.21 / Chapter 1.4 --- Introduction of proteomics --- p.21 / Chapter 1.4.1 --- Why proteomics? --- p.22 / Chapter 1.4.2 --- Gel-based proteomics: Two-Dimensional Gel Electrophoresis --- p.23 / Chapter 1.4.3 --- Gel-free proteomics --- p.25 / Chapter 1.4.4 --- Mass spectrometry --- p.26 / Chapter 1.4.5 --- Proteomics as tool for diabetes research --- p.27 / Chapter 1.5 --- Objectives and significance --- p.32 / Chapter 2. --- Materials and Methods --- p.34 / Chapter 2.1 --- Drug preparation --- p.34 / Chapter 2.2 --- Animal experiment --- p.34 / Chapter 2.3 --- Comparison of proteome of visceral white adipose tissue: obese db/db micevs lean m+/db mice and BBR-treated vs control db/db mice --- p.36 / Chapter 2.3.1 --- Protein sample preparation from adipose tissue --- p.36 / Chapter 2.3.2 --- Protein quantitation --- p.37 / Chapter 2.3.3 --- 2D Gel electrophoresis --- p.37 / Chapter 2.3.4 --- Image analysis --- p.39 / Chapter 2.3.5 --- In-gel digestion and MALDI-ToF MS --- p.39 / Chapter 2.4 --- Cell culture experiment --- p.40 / Chapter 2.5 --- Oil Red O staining --- p.42 / Chapter 2.6 --- Glycerol determination --- p.42 / Chapter 2.7 --- Comparison of proteomes of BBR-treated and control 3T3-L1 adipocytes..… --- p.43 / Chapter 2.7.1 --- Protein sample preparation from 3T3-L1 cells --- p.43 / Chapter 2.7.2 --- Protein quantitation --- p.43 / Chapter 2.7.3 --- 2D Gel electrophoresis --- p.44 / Chapter 2.7.4 --- Image analysis --- p.44 / Chapter 2.7.5 --- In-gel digestion and MALDI-ToF MS --- p.44 / Chapter 2.8 --- Western Immunoblotting --- p.44 / Chapter 2.8.1 --- Protein sample preparation of BBR-treated and control 3T3-L1 --- p.44 / Chapter 2.8.2 --- SDS-PAGE --- p.44 / Chapter 2.8.3 --- Protein blotting --- p.45 / Chapter 2.8.4 --- Membrane blocking and antibody incubations --- p.45 / Chapter 2.8.5 --- Detection of Proteins --- p.46 / Chapter 2.9 --- Statistical analysis --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.47 / Chapter 3.2 --- Effect of berberine on glucose metabolism of obese db/db mice --- p.53 / Chapter 3.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.55 / Chapter 3.4 --- Effect of berberine treatment on 3T3-L1 adipocytes --- p.61 / Chapter 3.4.1 --- Berberine treatment inhibited intracellular triglyceride accumulation in both mature and pre-mature 3T3-L1 adipocytes --- p.61 / Chapter 3.4.2 --- Berberine treatment enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.65 / Chapter 3.4.3 --- Color change in culture media after berberine treatment --- p.65 / Chapter 3.4.4. --- Comparison of protein profiles between berberine-treated and control 3T3-L1 adipocytes --- p.67 / Chapter 3.4.5 --- Western blotting --- p.73 / Chapter 4. --- Discussion --- p.75 / Chapter 4.1 --- Comparison of total protein profiles of visceral adipose tissue of obese db/db and lean m+/db mice --- p.75 / Chapter 4.2 --- "Berberine lowers body weight, reduces fasting blood glucose level and improves glucose-lowering ability of db/db mice" --- p.78 / Chapter 4.3 --- Comparison of the protein profiles of visceral adipose tissue of BBR-treated and control db/db mice --- p.79 / Chapter 4.4 --- Berberine inhibited lipid accumulation in mature and pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.5 --- Berberine enhanced lipolysis in mature 3T3-L1 adipocytes but inhibited lipolysis in pre-mature 3T3-L1 adipocytes --- p.84 / Chapter 4.6 --- Comparison of the protein profiles of BBR-treated and control 3T3-L1 adipocytes --- p.85 / Chapter 4.7 --- Western blotting --- p.88 / Chapter 4.8 --- General discussion --- p.89 / Chapter 5. --- References --- p.92

根據文獻研究記載，化療藥物可誘導癌細胞的凋亡，這是公認的化療療法的主要治療效果。作為一種程式性細胞死亡，積累的實驗證據表明，誘導所致的細胞凋亡是可逆轉的。這就引出了對於細胞凋亡恢復及其調節機制的相關問題。 / 在這項研究中，我們證明了在質膜不對稱的散失和半胱天冬酶（caspase）啟動後，HeLa細胞的凋亡的啟動可逆轉。我們發現，除了被廣泛研究的抗增殖作用外，金雀異黃素(genistein)可抑制細胞凋亡的復蘇。即時定量PCR發現抗凋亡基因MDM2和XIAP在凋亡逆轉過程中表達水準上調，金雀異黃素可抑制其表達水準的上調。金雀異黃素，MDM2蛋白抑制劑和XIAP抑制劑的利用，造成復原細胞內持續的半胱天冬酶活性和增強的細胞死亡效果。然而，半胱天冬酶抑制劑並不能挽救金雀異黃素的抑制作用。流式細胞儀的研究表明，金雀異黃素可以導致凋亡逆轉細胞持久磷脂醯絲氨酸（PS）外化和逆轉細胞的細胞壞死。抑制半胱天冬酶活性將金雀異黃素的主要作用轉移到壞死效果。這些結果揭示了金雀異黃素抑制細胞凋亡逆轉的兩個可能的機制。 / 金雀異黃素能維持現有的細胞凋亡信號從而增強細胞凋亡。它也可以破壞凋亡恢復過程，導致繼發性壞死。金雀異黃素對於細胞凋亡逆轉的抑制可與常規化療相結合，以提高治療結果. / It is well documented that chemotherapeutical agents could induce apoptosis of cancer cells, which is recognized as a major treatment effect of chemotherapy. Accumulating evidence indicates that chemopreventive agents like soybean isoflavone genistein could potentiate the antitumor effect of chemotherapeutic drugs both in vivo and in vitro. The mechanistic basis of this augmentation effect by genistein remains to be fully elucidated. / In this study, we demonstrated while low-concentration ethanol stressed cancer cells could recover, the presence of genistein promoted the cell death of stressed cancer cells that displayed apoptotic features. In HeLa cells, quantitative real-time PCR revealed the up-regulation of anti-apoptotic genes MDM2 and XIAP during the recovery process, and genistein suppressed their expression. The application of genistein, MDM2 inhibitor and XIAP inhibitor to the recovering HeLa cells caused persistent caspase activity and enhanced cell death. However, the death-promoting effect of genistein was not rescued by caspase inhibitor. Flow cytometry study indicated that genistein treatment could lead to persistent phosphatidylserine (PS) externalization and necrotic events in the recovering HeLa cells. Caspase activity inhibition shifted the major effect of genistein to secondary necrosis. / These results suggested two possible mechanisms through which genistein promoted cell death in stressed HeLa cells. Genistein could maintain the existing apoptotic signal to enhance apoptotic cell death. It could also disrupt the recovering process in caspase-independent manner, which lead to secondary necrosis. These effects may account for the enhanced antitumor effect of chemotherapeutic drugs when they were combined with genistein. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xie, Xin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 79-90). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in also in Chinese. / Cover Page / Statement --- p.i / Thesis Committee members --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Table of contents --- p.vi / List of abbreviations --- p.ix / List of figures and tables --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction to general cancer biology --- p.1 / Chapter 1.1.1 --- Overview of cancer --- p.1 / Chapter 1.1.1.1 --- Classification of cancer --- p.1 / Chapter 1.1.1.2 --- Risk factors of carcinogenesis --- p.2 / Chapter 1.1.1.3 --- Cancer prevention and therapies --- p.4 / Chapter 1.1.2 --- Models of cancer development --- p.6 / Chapter 1.1.2.1 --- Multistage model of carcinogenesis --- p.6 / Chapter 1.1.2.2 --- Colorectal cancer as an example of multistep / multigene carcinogenesis --- p.7 / Chapter 1.1.2.3 --- Driving force for cancer development --- p.9 / Chapter 1.1.3 --- Properties of cancer cells --- p.11 / Chapter 1.2 --- Apoptosis and its roles in cancer development and treatment --- p.14 / Chapter 1.2.1 --- Overview of apoptosis --- p.14 / Chapter 1.2.2 --- Molecular mechanism of apoptosis --- p.15 / Chapter 1.2.3 --- Positive and negative regulation of apoptosis --- p.18 / Chapter 1.2.4 --- Apoptotic defects in cancer development --- p.20 / Chapter 1.2.5 --- Apoptosis in cancer treatment --- p.23 / Chapter 1.3 --- The reversibility of apoptotic events --- p.25 / Chapter 1.4 --- Genistein and its relevance to cancer therapy --- p.27 / Chapter 1.5 --- Objectives of the study --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Cancer cell lines --- p.30 / Chapter 2.1.2 --- Cell culture media and additives --- p.30 / Chapter 2.1.3 --- Biochemical kits --- p.30 / Chapter 2.1.4 --- Chemicals and reagents --- p.30 / Chapter 2.1.5 --- Antibodies --- p.31 / Chapter 2.1.6 --- Primers used for quantitative real-time PCR --- p.32 / Chapter 2.1.7 --- Buffers and solutions --- p.32 / Chapter 2.2 --- Methods and procedures --- p.33 / Chapter 2.2.1 --- Cell culture establishment and cryopreservation --- p.33 / Chapter 2.2.2 --- Living cell staining and imaging --- p.34 / Chapter 2.2.3 --- MTT cell viability assay --- p.34 / Chapter 2.2.4 --- BrdU cell proliferation assay --- p.35 / Chapter 2.2.5 --- LDH cytotoxicity assay --- p.35 / Chapter 2.2.6 --- Quantitative real-time PCR --- p.36 / Chapter 2.2.7 --- Western blotting --- p.37 / Chapter 2.2.8 --- Annexin V/ Propidium Iodide Assay --- p.38 / Chapter 2.2.9 --- Trypan Blue Dye Exclusion Assay --- p.39 / Chapter 2.2.10 --- Cleaved-Caspase 3 Immunostaining --- p.39 / Chapter 2.2.11 --- Statistical Analysis --- p.39 / Chapter Chapter 3 --- Results --- p.40 / Chapter 3.1 --- Low concentration ethanol stressed cancer cells displayed apoptotic features and the stressed cell could recover after stress removal --- p.40 / Chapter 3.1.1 --- Morphological changes and apoptotic marker activation in low concentration ethanol stress --- p.40 / Chapter 3.1.2 --- In situ study of morphological changes and caspase 3 activation in HeLa --- p.44 / Chapter 3.2 --- Genistein promoted the cell death of stressed cancer cells at non-cytotoxic concentration towards unstressed cells --- p.46 / Chapter 3.2.1 --- Dose-dependent response of genistein on stressed and unstressed cells --- p.46 / Chapter 3.2.2 --- In HeLa cells, genistein suppressed the recovery from stress treatment at non-cytotoxic concentration --- p.48 / Chapter 3.2.3 --- Genistein promoted both apoptosis and necrosis in stressed cells. . --- p.49 / Chapter 3.3 --- Genes involved in the recovery from stress treatment were influenced by genistein --- p.53 / Chapter 3.3.1 --- Stressed HeLa cells were more sensitive to the inhibition of de novo synthesis --- p.53 / Chapter 3.3.2 --- Expression profiles of genes involved in recovery and the influence of genistein --- p.55 / Chapter 3.4 --- Like genistein, MDM2 and XIAP inhibitor potentiated the cell death and caused persistent caspase-3 activity in stressed cells --- p.58 / Chapter 3.4.1. --- Stressed HeLa cells were much more sensitive to the inhibition of XIAP and MDM2 --- p.58 / Chapter 3.4.2 --- The presence of inhibitor at non-cytotoxic concentration to unstressed cells suppressed the recovery of the stressed cells --- p.60 / Chapter 3.4.3 --- Genistein, MDM2 inhibitor and XIAP inhibitor caused persistent apoptotic signals in recovering cells. --- p.61 / Chapter 3.5 --- The death-promoting effect by genistein could be caspase-independent --- p.64 / Chapter 3.6 --- Caspase activity abrogation shifted genistein’s action profile --- p.66 / Chapter Chapter 4 --- Discussion and prospect --- p.70 / Chapter 4.1 --- The apoptotic features were induced by low concentration ethanol stress --- p.70 / Chapter 4.2 --- The apoptotic features caused by ethanol stress were reversible --- p.71 / Chapter 4.3 --- Genistein showed death-promoting effects on the recovering cells --- p.72 / Chapter 4.4 --- The genes (XIAP and MDM2) that were involved in the recovery process may function to terminate apoptotic signal --- p.73 / Chapter 4.5 --- Genistein suppressed the upregulation of anti-apoptotic genes and promoted the expression of pro-apoptotic genes --- p.74 / Chapter 4.6 --- The XIAP and MDM2 activity were essential for the recovery from stress --- p.75 / Chapter 4.7 --- Caspase inhibition increased the secondary necrosis in recovering cells with genistein treatment --- p.76 / Chapter 4.8 --- Hypothetic mechanism of genistein’s inhibitory effect on the recovery of stressed cells --- p.77 / Chapter 4.9 --- Summary and prospects --- p.78 / Reference list --- p.79

以客觀存在之腎臟損害和功能異常的臨床表現為診斷依據的腎臟疾病，業已成為全世界所共同面臨的危害人類健康的一項主要健康問題。現今，人群中有超過十分之一的人患有慢性腎臟疾病（CKD）, 該病的患病率不亞於糖尿病 (James et al., 2010)。慢性腎髒病是一種難以治癒的疾病。儘管存在有效的治療方法，但在全世界範圍內，慢性腎髒病仍舊是導致終末期腎臟疾病和死亡的首要原因。據2010年全球疾病負擔的研究報導 (Lozano et al., 2013)，在引起全球死亡總數原因列表中，慢性腎髒病由1990年的第27位（年齡標準化的每100 000人的年死亡率為15.7）躍升至2010年的第18位（每100 000人的年死亡率為16.3）。慢性腎髒病的攀升趨勢僅次於HIV和AIDS。這種狀況已經引起高度關注，應對措施的實施刻不容緩！因此，迫切需要慢性腎髒病早期診斷和治療的有效方案。 / 纖維化是慢性腎髒病這一以終末期腎髒病（ESRD）為結局的疾病腎臟損傷和進展的主要病理特徵。現已確立转化生长因子β/Smads 信号蛋白（TGF-β/Smad）在腎臟纖維化發病機制中具有主要的作用。在過去近20年對於转化生长因子β/Smads 信号蛋白在慢性腎髒病發病機制中的作用研究揭示：Smad3在腎臟纖維化中起致病作用，而Smad2和Smad7具保護作用。鑒於转化生长因子β/Smads 信号蛋白在免疫調節中也發揮關鍵作用，直接針對转化生长因子β/Smads 信号蛋白的干預方案可能引起免疫損傷的副作用。因而，針對转化生长因子β/Smads 信号蛋白特異性下游靶目標的療法是對抗慢性腎髒病更好的選擇。 / 自首個微小核糖核酸的發現至今，已歷經20年。微小核糖核酸在基因表达的转录后起著重要調節作用。現已明確，在人類疾病包括腎臟病的發生和/或進展中存在微小核糖核酸的表達或功能的失調。最近的研究明確的表明，微小核糖核酸與腎臟疾病的發病機制密切相關。另外，已有確鑿的證據顯示，一些微小核糖核酸正是转化生长因子β/Smads信号蛋白的特異性下游。並且我們發現，不僅在转化生长因子β和血管緊張素II誘導的體外纖維化反應中，而且在小鼠體內梗阻性和高血壓性腎病模型中，微小核糖核酸-433-Azin1軸不但在腎臟纖維化中起重要作用，並且和转化生长因子β/Smad3信号蛋白密切相關。據重要意義的是，藉助安全、有效的超聲微泡介導的基因轉染方法，通過逆轉失調的微小核糖核酸-433-Azin1軸，有效的抑制了小鼠腎臟疾病的進展、腎組織的損傷并減少了蛋白尿的排泄。因此，微小核糖核酸-433-Azin1軸不但可以作為慢性腎臟病早期診斷的生物學標記，也能夠成為遏制甚至逆轉慢性腎髒病的治療靶點。 / 近年來，從基礎到臨床旨在預防和治療慢性腎髒病的研究已取得碩果累累。基於以转化生长因子β/Smad3信号蛋白特異性微小核糖核酸為把目標的治療策略，預見了遏制慢性腎髒病治療的未來希望。然而， 消除慢性腎髒病這一世界性的人類健康威脅，仍需付諸長期不懈的努力。 / Kidney disease is diagnosed with objective clinic manifestation of kidney damage and renal dysfunction has been recognized as a major global health burden. Nowadays, more than one out of ten people have chronic kidney disease (CKD) and the overall prevalence is not less than that of diabetes (James et al., 2010). CKD is a kind of persistent ailment. In spite of the availability of medical treatments, CKD continues to be a leading cause of end stage of renal disease (ESRD) and death worldwide. Reported in the 2010 Global Burden of Disease study (Lozano et al., 2013), CKD was ranked from 27th in 1990 (age-standardised annual death rate of 15.7 per 100 000) and rose to 18th in 2010 (annual death rate 16.3 per 100 000) in the list of causes of total number of global deaths. The growing momentum of CKD was just second to that for HIV and AIDS. This situation has claimed our serious attention and the prompt action is imperative. Thus, effective early diagnosis biomarker and treatment of CKD are urgent needed. / Fibrosis is the key feature during renal lesion formation and progression in CKD which will be ended in ESRD eventually. It has been established that Transforming growth factor β/Combination of the Drosophila protein "Mothers against decapentaplegic" (MAD) and C. elegant protein SMA (TGF-β/Smad) signaling plays a central role in the pathogenesis of renal fibrosis. For last two decades, dissection of the critical role of TGF-β/Smad signaling in the fibrogenesis of CKD has unveiled that Smad3 plays a pathogenic but Smad2 and Smad7 play a protective role in renal fibrosis. As TGF-β/Smad signaling also plays a crucial function in immunity, targeting TGF-β/Smad signaling may cause adverse effect. Thus, targeting specific downstream of T GF-β/Smad signaling should be a better alternative to fight against CKD. / A score of years has elapsed from the first microRNA discovery. MicroRNAs are important post-transcriptional regulators of gene expression. It is now widely acknowledged that the dysregulation of microRNA expression or action underlies the onset and/or development of various human diseases including kidney disease. Recently, researches have substantial evidences that microRNAs are tightly associated with the pathogenesis of kidney disease. In addition, eye-catching tangible evidences showed that several microRNAs are specific downstream of TGF-β/Smad3 signaling. Moreover, we found that not only in TGF-β and angiotensin II induced fibrotic response in vitro, but also in mouse models of obstructive and hypertensive nephropathy, microRNA-433-Azin1 axis is vital in renal fibrosis and is closely related with TGF-β/Smad3 signaling. Last but not least, our study suggests that, using a safe, effective, ultrasound microbubble-mediated gene transfer therapeutic method can significantly halt the progression of kidney lesions and reduce renal tissue damage and the excretion of albuminuria by balancing the microRNA-433-Azin1 axis. Hence, microRNA-433-Azin1 axis may act either as biomarkers favorable early diagnosis or therapeutic targets to halt or even reverse CKD. / The bench and bedside research on preventing and managing CKD have gained fruitful results in recent decades. Therapeutic strategy against TGF-β/Smad3 specific microRNA brings us a bright future in combating against CKD. However, more endeavors are necessary to eliminate the enormous burden of CKD worldwide. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Rong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 182-202). / Abstracts also in Chinese.

The neuropathology of Tourette Syndrome (TS) is poorly characterized. This thesis provides the first quantitative stereologic immunohistochemical study of the basal ganglia in TS. TS is a childhood neuropsychiatric disorder characterized by motor and vocal tics. Previous imaging studies found alterations in caudate (Cd) and putamen (Pt) volumes. To investigate possible alterations in cell populations, postmortem basal ganglia tissue from individuals with TS and normal controls (NC) was analyzed using unbiased stereological techniques. A markedly higher (>160% of control) total neuron number and density was found in the internal segment of the globus pallidus (GPi) of TS (p<0.025). An increased number (>220% of control) and proportion of these GPi neurons were positive for the calcium-binding protein parvalbumin (PV) in the tissue from TS subjects (p<0.025). In contrast, a lower number (<60% of control) of neurons was observed in the external segment (GPe) (p<0.025). In addition, there was a lower density of PV-positive interneurons in both Cd (<50% of control) and Pt (<65% of control) (p>0.025). The imbalance in striatal and GPi inhibitory neuron distribution suggests that the functional dynamics of cortico-striato-thalamic circuitry are fundamentally altered in severe, persistent TS.

Urotensin-II (U-II) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study, we hypothesized that selective overexpression of UT in an SMC-specific fashion would increase atherosclerotic lesion formation in a hypercholesterolemic mouse model. The objectives were to demonstrate the role of UT in this mouse model of atherosclerosis, and to elucidate some of the mechanism involved in the process. We used four strains of mice; wildtype (WT), UT+ (a transgenic strain expressing human UT driven by the alpha-SM22 promoter), ApoE knockout (ko), and UT+/ApoE ko. All animals were fed a high-fat diet for 12 weeks. Western blot analysis revealed a significant increase in UT expression in UT+ and ApoE ko mice (P&lt;0.05). Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT+/ApoE ko but not in UT + mice when compared to wild type mice (P&lt;0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT +, ApoE ko and UT+/ApoE ko compared to WT mice (P&lt;0.05). Oral administration of the UT receptor antagonist SB-657510A for 10 weeks in a group of ApoE ko mice fed a high fat diet resulted in a significant reduction of lesion (P&lt;0.001). Immunohistochemistry revealed the presence of strong expression of UT and U-II proteins in the atheroma of UT+, ApoE ko and UT+/ApoE ko mice, particularly in foam cells. SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P&lt;0.05). The present findings suggest that the use of UT receptor antagonists may reduce lesion formation through reduced foam cell formation and lipid uptake, demonstrating an important role for UT in the pathogenesis of atherosclerosis.

This Master's dissertation explored whether a rapid orienting of attention toward or away from social stress information during a restful state, relates to the magnitude of glucocorticoids (GC) released in response to a stressor, the Trier Social Stress Test (TSST). It also assessed whether childhood rearing in a low socioeconomic status (SES) context mediates this relationship. Subjects rested for 45 minutes during which time they completed a modified version of Posner's attention paradigm, comprising social stress words. Immediately following, participants were exposed to the stressor. Results indicated that a rapid attentional engagement toward social stress words associated with pronounced GC responses to the TSST. Fast engagers displayed lower self-esteem and did not differ in terms of their past SES. These findings demonstrate that attentional biases for social stress information at rest combine with diminished self-esteem to predict the magnitude of GC released during psychological stress irrespective of early SES conditions.

My thesis is divided into 4 major sections. The first section is devoted in part to a description of the biochemical abnormalities in the metabolism of homocarnosine (y-aminobutyryl-L-histidine, HCarn) occurring in a patient with homocarnosinosis. The patient studied and two of her siblings have a progressive neurological disorder with grossly elevated concentrations of HCarn in their CSF. HCarn content was four times higher in a biopsy from the patient's frontal cortex than in biopsied cortex from a large group of control subjects. Using new techniques for the measurement of the HCarn synthesizing and catabolizing enzymes, it was found that the activity of HCarn-Carn synthetase was not increased in the patient's biopsy whereas homocarnosinase activity was undetectable. It is concluded that the elevated HCarn in brain and CSF in the homocarnosinosis patient is due to a deficiency of brain homocarnosinase activity. The first description of the regional distribution of the two HCarn metabolizing enzymes in human brain was also obtained. The remainder of the first section deals with a description of the neuropharmacological properties of HCarn. Intraventricular injection of HCarn in the rat produced hyperexcitability and in high doses, convulsions, whereas unilateral intra-striatal injection of HCarn resulted in contralateral myoclonus. The results of these experiments are consistent with the possibility that HCarn may be involved in the neuronal excitability of brain. The second section describes experiments which test the hypothesis that the content of the inhibitory neurotransmitter GABA is altered in the autopsied brains of some patients dying with schizophrenia. The mean content of GABA was reduced by 20-25% in nucleus accumbens, caudate nucleus, frontal cortex and thalamus of the schizophrenic patients as compared to a control group. However, the differences were found to be statistically significant for only the caudate and thalamus. Extraneous factors such as age of patient at death and prolonged drug treatment did not readily explain the observed reduction in GABA content. The results of the investigation suggest an association between a deficiency of GABAergic function in certain brain areas with some forms of schizophrenia. The third section describes experiments which test the hypothesis that a deficiency of aspartate found in the cerebellar cortex of some patients with dominantly inherited cerebellar disorders might be due to reduced activity of two enzymes involved in the synthesis of aspartate, namely, aspartate aminotransferase and pyruvate carboxylase. No deficiency of either enzyme was observed in the cerebellar specimens studied. The results of this investigation suggest that the aspartate deficiency in cerebellar cortex found in some dominantly inherited cerebellar disorders does not result from a deficiency of either of these two brain enzymes. In the final section, experiments are described which study the effects of chronic administration of Y-vinyl GABA and of hydrazine on the contents of GABA and other amino compounds in rat brain. Both of these compounds are presently under consideration for use in clinical trials on patients with disorders involving a brain GABA deficiency. Chronic administration of either y-vinyl GABA or of hydrazine markedly increased brain GABA content in the rat. Prolonged treatment with y-vinyl GABA, but not hydrazine, produced a decrease in the activity of glutamic acid decarboxylase (GAD) in rat brain. Since GAD is localized to a large extent in nerve endings, the possibility exists that y-vinyl GABA might reduce the amount of GABA available for release at synapses, a potentially undesirable effect. The contents of many brain amino compounds other than GABA were markedly altered by both drugs. Since the potential harmful effects of these unexpected biochemical alterations in brain are unknown, the nonspecific effects of Y-vinyl GABA and hydrazine are disturbing. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Following reports by Shapira et al. that α₂Macro-globulin (α₂M) is abnormal in cystic fibrosis (CF), the author set out to examine the properties of α₂M isolated from the plasma of children with CF and from the plasma of age/sex matched controls. To do so, a technique capable of isolating pure, physiologically "active" α₂M from small plasma samples had to be developed. By a two-step chromatographic technique, involving Cibacron Blue Sepharose chromatography and immuno-adsorption, the author was able to isolate "active" CF and control α₂M of at least 98 percent purity from 5 ml of plasma, regardless of plasma haptoglobin type. Having accomplished this, comparative studies of CF and control α₂M were undertaken. Four parameters were investigated: (1) the molar protease binding of α₂M (2) the interaction of α₂M -bovine cationic trypsin (BCT) complexes with the low molecular weight substrate BAEE, (3) the stability of formed α₂M-BCT complexes, and (4) the subunit structure of α₂M. Contrary to the reports of Shapira and his colleagues, this author found no differences between the subunit structure of CF and control α₂M nor between the abilities of CF and control α₂M to interact with BCT. Based upon these findings, the author believes that no firm evidence exists to implicate an α₂M defect in the pathogenesis of cystic fibrosis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate