No description available.

Wong, Wing Tak Jack. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 204-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

This is a study of muscle function in Juvenile Idiopathic Arthritis (JIA). Rheumatoid arthritis (RA) is a disease that primarily affects the synovial membrane of joints. Muscle weakness, atrophy and pain occur in adult RA. This may be a consequence of joint pain, stiffness and immobility. Muscle inflammation and neuropathy occur as complications in adults. Muscle function in JIA has been much less studied. The aim of the study was to examine whether muscle weakness and atrophy also occur in children with JIA. This was a longitudinal study over a two-year period, where muscle strength and thickness were measured repeatedly in a group of 20 children and teenagers with JIA. Muscle strength was measured using different methods and in several muscle groups. Muscle biopsies were obtained and nerve conduction velocity studies performed. The study concludes that, compared to healthy people, children and teenagers with JIA have as a group reduced muscle strength and muscle thickness. For most of these children and teenagers, muscle strength is only slightly lower than expected, but a few have marked muscle weakness. This is most apparent in patients with severe polyarthritis where the weakness seems to be widespread. Patients with isolated arthritis may also have greatly reduced strength and thickness of muscles near the inflamed joint. There is a risk of decreasing strength in patients with polyarthritis and in muscles near an active arthritis. Minor changes are common in muscle biopsies, and findings may indicate immunological activity in the muscles. Atrophy of type II fibres, as in adult RA, was not found in JIA. No patient had signs of neuropathy. / On the day of the public defence the status of article IV was: Submitted.

A research report submitted to the Faculty of Medicine, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Science in Medicine in Applied Physiology. Johannesburg, 1997. / A post-exercise reduction in blood pressure (BP) may be the primary reason that athletes suffer from exerclse-assoclated collapse (EAC) at the end ot ultra-endurance running ever.s. Plasma volume decreases, possibly caused by dehydration, may be the cause of the decrease til blood pressure, In order to determine whether there is a correlation between plasma volume changes and the post-exercise BP drop, this study evaluated alterations in pre- and post-race blood pressures and changes in blood and plasma volumes, It found that compared to resting values, systolic, dlastollc and mean arterial blood pressures (mmHg) fell significantly from 119 ± 4, mean ± standard deviation, 74 ± 8, and 88 ± 5 respectively to '106 ± 14, 62 ± 12 and 77 ± 10 (ps 0,05), whereas pulse pressure failed to change, Compared to pre-race values, plasma and blood volume were found not to have changed significantly, During the race plasma urea (U) and creatinine (C) concentrations increased significantly, whereas body mass and body mass index both fell significantly. Haernatocrlt, haemoglobin, mean cell volume, red blood cell number, mean cell haemoglobin concentration, the mean cell haemoglobin, plasma sodium, potassium, chloride and protein concentrations, the U:C ratio and osmolality remained constant. There were no significClnt correlations between changes in plasma or blood volume and changes in blood pressure, These data support the Idea that a post-race decrease in blood pressure does not result primarily from an intravascular fluid loss, It is likely therefore that athletes who collapse at the end of ultraendurance races due to EAC do so as a result of 'post-exercise hypotension' secondary to venous pooling, and not as a result of a reduction in plasma volume, / MT2017

Hemodynamics (the patterns of blood flow along blood vessels), such as laminar shear stress (LSS) and oscillatory shear stress (OSS), plays an important role in maintaining vascular homeostasis and also the pathogenesis of atherosclerosis. LSS, occurring mainly in the straight part of the vascular tree, is protective against the formation of atherosclerotic plagues, while OSS is the predominant hemodynamic pattern experienced by the branched regions and curvatures in the vascular system, in which most of atherosclerotic plaques are developed. Recent evidence indicates that the Hippo pathway is important in transducing mechanical stimulation. However, the exact role of Hippo signaling in hemodynamics and the development of atherosclerosis is unclear. Therefore, the present study was designed to investigate how the Hippo pathway responds to hemodynamic stimulation and the impact of this pathway in the development of atherosclerosis. / The central axis of the Hippo pathway consists of a chain of kinase cascade including serine/threonine-protein kinase 1/2 (LATS1/2) and downstream YAP/TAZ effectors. Activation of the Hippo pathway leads to phosphorylation, nuclear exportation and inhibition of YAP/TAZ transcription factors. The present study is probably for the first time to demonstrate that the Hippo pathway can be activated in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) subjected to LSS. Western blotting results showed that Hippo activation was most likely mediated by phosphorylation of Hippo kinase YAP. The results from the nuclear fractioning assay showed that the amount of the nuclear fraction of YAP/TAZ was reduced in HUVECs subjected to LSS. Repression of the established YAP/TAZ target genes CTGF and CYR61, and inhibition of Hippo reporter gene after LSS treatment, further supported the notion that activation of the Hippo pathway inhibits the YAP/TAZ downstream genes expression. In vivo experiments showed an increased YAP phosphorylation in the straight area of C57 mouse thoracic aortas as compared with aortic arch, which favors that LSS activates Hippo pathway under the physiological condition. To further understand whether different flow patterns have different impacts on the Hippo pathway, the effects of OSS were also studied. As expected, OSS inhibited the Hippo pathway through inducing the de-phosphorylation of YAP, and promoting the nuclear localization of YAP/TAZ. The induction of YAP/TAZ target genes (CTGF and CYR61) further confirms that OSS-induced inhibition of the Hippo pathway enhanced the YAP and TAZ transcription activity. / Since unfavorable hemodynamics is among the most important factors in the development of atherosclerotic plaques, I hypothesized that inhibition of the Hippo pathway is involved in the formation of atherosclerotic plaques. Adhesion molecules are a group of cell surface proteins that mediate cell-cell interaction. During the initiation phase of atherosclerosis, adhesion molecules are highly expressed on the surface of endothelial cells to enhance the attachment of monocytes to the vascular wall, leading to monocyte accumulation within the vascular wall. OSS up-regulates the expression of several adhesion molecules; however, the underlying mechanisms remain elusive. To identify the role of Hippo pathway in adhesion molecules expression, the gene expression of HUVECs with YAP/TAZ knockdown was investigated using real-time PCR. The expression of the four adhesion molecule, ICAM1, E-Selectin, MCP1 and CXCL1 were down-regulated by YAP and TAZ gene silencing. To further study the mechanism, the promoter region of the four target genes were amplified from HUVECs genomic DNA, and subcloned into PGL3 reporter plasmids. The promoter activity was tested by co-transfection of the reporter plasmids with different dosages of constructively active YAP and TAZ. The present results showed that activated YAP and TAZ dose-dependently increased the promoter activity of these four genes. CXCL1 was selected for further examination because it has been recently reported to play an important role in oxidized LDL-induced elevation of monocyte attachment to endothelial cells. Analysis of the CXCL1 promoter revealed that two TEAD1 binding sites located within the promoter region of CXCL1. The reporter gene assay results showed that TEAD1 induced the CXCL1 reporter activity, indicating that the CXCL1 promoter was regulated by YAP/TAZ/TEAD complex. The EMSA analysis further demonstrates that constitutively active YAP and TAZ induced the binding between TEAD1 and the CXCL1 promoter, suggesting a direct association between TEAD1 and CXCL1 promoter. / To further explore whether the Hippo pathway activation could regress atherosclerosis development, TAZ knockdown adenovirus was generated and administered to ApoE⁻/⁻ mice fed on Western diet. The formation and sizes of atherosclerotic plaques were visualized using oil red O staining. Monocyte infiltration was estimated using monocyte marker CD68. The expression of CXCL1 was detected using immunohistochemistry. The present results demonstrated that TAZ gene silencing significantly suppressed the expression of CXCL1 and reduced the sizes of atherosclerosis plagues as well as monocyte infiltration in the vascular wall of ApoE⁻/⁻ mice. / To further identify whether Hippo pathway could be a drug target for the treatment of atherosclerosis, several known atherosclerosis risk factors and beneficial factors were tested for their effects on Hippo pathway. Four out of nine atheroprotective factors were found to suppress the TEAD reporter activity. On the other hand, four out of six risk factors were identified to increase the TEAD reporter activity. The present results suggest that atheroprotective LSS activates while atherogenic OSS inhibits the Hippo pathway. / The Hippo pathway could be activated by atheroprotective LSS, while it was inhibited by atherogenic OSS. Activation of the Hippo pathway inhibits the development of atherosclerosis at least in part through reducing the expression of adhesion molecules and decreasing macrophage accumulation in the vascular wall. The present new findings suggest that targeting this Hippo pathway is potentially useful for therapeutic intervention of atherosclerosis and associated vascular diseases. / 血流動力學（即血液在心血管系統中流動的模式），包括層流剪切應力（LSS）和振盪剪切應力（OSS）。血流動力學在血管穩態和動脈粥樣硬化的發展中起重要作用。LSS主要發生在血管系統的直線部分，可防止動脈粥樣硬化斑塊形成，而OSS是血管系統中分支和彎曲部分的主要血流模式，大部份粥樣硬化斑塊發生在此。最近的研究表明，Hippo通路在機械刺激傳導中起重要作用。然而，Hippo信號通路在血流動力學和動脈粥樣硬化發展中的確切作用目前尚不清楚。因此，本論文的目的是研究Hippo通路如何響應血流動力學刺激以及Hippo通路對動脈粥樣硬化的發展的影響。 / Hippo通路主要由一系列激酶鏈和下游YAP/ TAZ效應器組成，其中的激酶鏈包括絲氨酸/蘇氨酸－蛋白激酶1/2（LATS1/ 2）。激活Hippo通路可導致YAP和TAZ磷酸化，促進其出核運輸並抑制其轉錄激活的功能。本研究中，我們首次證明了在LSS條件下，人臍靜脈內皮細胞（HUVEC）和人主動脈內皮細胞（HAECs）中的Hippo通路被激活。Western雜交結果表明，Hippo通路的激活很可能是受YAP激酶的磷酸化調節。細胞核質分離結果顯示在LSS條件下，人臍靜脈內皮細胞中YAP/ TAZ的細胞核部分的量顯著減少。LSS处理后，YAP/ TAZ靶基因CTGF和CYR61的表達降低，其TEAD告基因受到抑制，这進一步證明激活Hippo通路可抑制YAP/ TAZ下游基因的表達。動物實驗結果顯示YAP磷酸化和YAP/ TAZ出核在C57小鼠的胸主動脈直的區域顯著高於彎的區域，這也表明在生理條件下，LSS可激活Hippo通路。為了進一步了解不同的血流模式是否對Hippo通路產生不同的影響，我們研究了OSS條件下的Hippo通路。正如所期，OSS通過誘導YAP的去磷酸化，促進YAP/ TAZ的核內分佈，從而抑制Hippo通路的活性。OSS誘導YAP/ TAZ靶基因（CTGF和CYR61）產生轉錄活性，證明了OSS可抑制Hippo通路。 / 不利的血流模式是動脈粥樣硬化斑塊產生的最重要因素之一，因此我們推測抑制Hippo通路或許可導致動脈粥樣硬化斑塊的形成。粘附分子是一類調節細胞間相互作用的細胞表面蛋白。在動脈粥樣硬化形成的初始階段，粘附分子會在內皮細胞的表面高表達，從而提高單核細胞附著和聚集於血管壁上。OSS上調粘附分子的表達，但機制仍不清楚。為了識別Hippo通路在粘附分子表達中的作用，本文用實時定量PCR檢測了YAP/ TAZ敲低了的人臍靜脈內皮細胞的基因表達水平。實驗結果顯示四種粘附基因，包括ICAM1，E-Selectin，MCP1和CXCL1通過YAP和TAZ的基因沉默而被下調。為了進一步研究此中機制，本文擴增了臍靜脈內皮細胞的基因組DNA中的四個靶基因啟動子區，並將其克隆到PGL3報告質粒。通過共轉染不同濃度的組成型激活的YAP和TAZ，本文測定了這四個啟動子的活性。結果顯示激活的YAP和TAZ能夠劑量依賴性上調這四個基因的啟動子活性。由於最近有報導說氧化低密度脂蛋白可誘導單核細胞附著於內皮細胞，而CXCL1在此誘導過程中具有重要作用，因此本文選擇CXCL1作進一步研究。通過分析CXCL1的啟動子，本文發現CXCL1的啟動子區域內有兩個TEAD1結合位點。報告基因檢測結果顯示TEAD1可誘導CXCL1報告活性，這表明CXCL1啟動子是通過YAP/ TAZ/ TEAD複合體來調節。EMSA分析進一步揭示了組成型激活的YAP和TAZ可誘導TEAD1和CXCL1啟動子之間的結合，表明TEAD1直接結合到CXCL1啟動子。 / 為進一步開拓Hippo通路是否是動脈粥樣硬化消退潛在的治療靶點，本文構建了TAZ敲低的腺病毒並將其轉入餵以西方飲食的ApoE⁻/⁻小鼠。通過油紅O染色來觀察動脈粥樣硬化斑塊的形態和大小。用單核細胞表面標記物CD68測定單核細胞對血管的浸潤。用免疫組化鑑定CXCL1的表達。結果表明TAZ基因沉默顯著抑制ApoE⁻/⁻小鼠血管壁CXCL1的表達，降低動脈粥樣硬化斑塊的大小及單核細胞浸潤。 / 為了進一步確定Hippo途徑是動脈粥樣硬化的藥物治療靶點，本文對幾個已知的動脈粥樣硬化的危險因子和有利因子對Hippo通路活性的影響進行了研究。出人意料的是，九個動脈粥樣硬化的保護因子中，有四個被確定為抑制TEAD報告基因活性。另一方面，六個危險因子中，有四個被鑑定為可提高TEAD報告活性。這些結果與上面的研究發現共同證明了LSS降低動脈粥樣硬化，激活Hippo通路，而OSS促進動脈粥樣硬化，抑制Hippo通路。 / 本研究確定了層流剪應力（LSS）激活Hippo通路，抑制動脈粥樣硬化；振盪剪切應力（OSS）抑制Hippo通路，導致動脈粥樣硬化。Hippo通路被激活能夠抑制動脈粥樣硬化的機制是通過降低粘附分子的表達，減少巨噬細胞在血管壁上的附著。這一新發現表明，靶向Hippo通路對介入治療動脈粥樣硬化以及相關的血管疾病具有潛在的應用意義。 / Wang, Li . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 129-138). / Abstracts also in Chinese. / Title from PDF title page (viewed on 08, November, 2016). / Wang, Li. / 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.

by Poon Lit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 97-113). / Title page --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of tables --- p.vii / List of figures --- p.viii / Abbreviations --- p.x / Abstract --- p.xi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Sacral agenesis --- p.2 / Chapter 1.1.1 --- Skeletal anomalies --- p.3 / Chapter 1.1.2 --- Neurological anomalies --- p.4 / Chapter 1.1.3 --- Other anomalies --- p.5 / Chapter 1.1.4 --- Etiology --- p.5 / Chapter 1.1.5 --- Pathogenetic mechanism of SA --- p.7 / Chapter 1.2 --- Retinoids --- p.8 / Chapter 1.2.1 --- RA in embryonic development --- p.9 / Chapter 1.2.2 --- Teratogenic effect of RA in embryonic development --- p.10 / Chapter 1.3 --- Strategy of the thesis --- p.13 / Chapter Chapter 2: --- General Materials and Methods --- p.15 / Chapter 2.1 --- Mouse maintenance and mating method --- p.16 / Chapter 2.2 --- All-trans RA preparation and injection --- p.16 / Chapter 2.3 --- Dissection of embryos --- p.16 / Chapter 2.4 --- Preparation of histological sections --- p.17 / Chapter 2.4.1 --- Dehydration and embedding --- p.17 / Chapter 2.4.2 --- Sectioning --- p.18 / Chapter 2.4.3 --- Haematoxylin and eosin staining --- p.18 / Chapter 2.5 --- Plasmid preparation --- p.19 / Chapter 2.5.1 --- Competent cells preparation --- p.19 / Chapter 2.5.2 --- Bacterial transformation --- p.19 / Chapter 2.5.3 --- Mini-scale preparation of plasmid DNA --- p.20 / Chapter 2.6 --- In situ hybridization --- p.21 / Chapter 2.6.1 --- Sample preparation --- p.21 / Chapter 2.6.2 --- Probe synthesis --- p.21 / Chapter 2.6.3 --- Hybridization --- p.23 / Chapter 2.6.4 --- Post-hybridization wash and antibody labeling --- p.24 / Chapter 2.6.5 --- Post-antibody wash and colour development --- p.25 / Chapter 2.6.6 --- Embryo powder preparation --- p.25 / Chapter Chapter 3: --- Time and Dose Responses to RA --- p.26 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.1.1 --- Time response --- p.27 / Chapter 3.1.2 --- Dose response --- p.29 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Dose response --- p.31 / Chapter 3.2.2 --- Time response --- p.31 / Chapter 3.2.3 --- Skeletal preparations and staining --- p.31 / Chapter 3.2.4 --- Early teratogenic responses to RA --- p.32 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- Dose response --- p.33 / Chapter 3.3.1.1 --- Tail length --- p.33 / Chapter 3.3.1.2 --- Vertebral pattern --- p.33 / Chapter 3.3.2 --- Time response --- p.36 / Chapter 3.3.2.1 --- Tail length --- p.36 / Chapter 3.3.2.2 --- Vertebral pattern --- p.36 / Chapter 3.3.2.3 --- Other anomalies associated with human SA --- p.40 / Chapter 3.3.3 --- Early teratogenic responses in RA-treated embryos --- p.41 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4: --- RA-induced Cell Death in Tail Bud --- p.51 / Chapter 4.1. --- Introduction --- p.52 / Chapter 4.1.1 --- Cell death --- p.52 / Chapter 4.1.2 --- Methods in cell death identification --- p.54 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Histology of tail bud region --- p.57 / Chapter 4.2.2 --- Detection of internucleosomal DNA fragmentation --- p.57 / Chapter 4.2.2.1 --- Sample collection --- p.57 / Chapter 4.2.2.2 --- DNA extraction --- p.58 / Chapter 4.2.2.3 --- Analysis of DNA fragmentation pattern by ethidium bromide staining --- p.59 / Chapter 4.2.2.4 --- Analysis of DNA fragmentation pattern by autoradiography --- p.59 / Chapter 4.2.3 --- TUNEL --- p.61 / Chapter 4.2.3.1 --- Sample collection / Chapter 57 4.2.3.2 --- In situ end-labeling --- p.62 / Chapter 4.2.4 --- In situ hybridization of Wnt-5A --- p.63 / Chapter 4.2.4.1 --- Sample collection --- p.63 / Chapter 4.2.4.2 --- Probe preparation --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Histological examination of cell death in tail bud --- p.65 / Chapter 4.3.2 --- Analysis of DNA fragmentation of tail bud cells --- p.66 / Chapter 4.3.3 --- TUNEL --- p.67 / Chapter 4.3.4 --- Wnt-5A gene expression --- p.68 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Retinoic Acid Receptors (RARs) --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.1.1 --- RARs and their isoforms --- p.76 / Chapter 5.1.2 --- Expression pattern of RARs during embryogenesis --- p.77 / Chapter 5.1.3 --- RAR mutants and functional redundancy of RARs --- p.78 / Chapter 5.1.4 --- RARs and SA --- p.79 / Chapter 5.2 --- Materials and methods --- p.80 / Chapter 5.2.1 --- In situ hybridization of RAR-α --- p.80 / Chapter 5.2.1.1 --- Sample collection --- p.80 / Chapter 5.2.1.2 --- Probe preparation --- p.80 / Chapter 5.2.2 --- In situ hybridization of RAR-β --- p.81 / Chapter 5.2.2.1 --- Sample collection --- p.81 / Chapter 5.2.2.2 --- Probe preparation --- p.81 / Chapter 5.2.3 --- In situ hybridization of RAR-γ --- p.82 / Chapter 5.2.3.1 --- Sample collection --- p.82 / Chapter 5.2.3.2 --- Probe preparation --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- RAR-α gene expression --- p.83 / Chapter 5.3.2 --- RAR-β gene expression --- p.84 / Chapter 5.3.3 --- RAR-γ gene expression --- p.86 / Chapter 5.4 --- Discussion --- p.88 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.92 / Chapter 6.1 --- Conclusion and future perspectives --- p.93 / References --- p.97 / Appendices --- p.114 / Figures --- p.118

by Ng Mei Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 173-183). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abbreviations --- p.3 / Abstracts --- p.4 / List of figures and tables --- p.7 / Contents Page No / General Introduction --- p.13 / Chapter CHAPTER ONE --- Biological Characterization of A431 Cells & SiHa Cells Under Different Microenvironments --- p.16 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.1.1 --- Microenvironment Surrounding Tumor Cells --- p.18 / Chapter 1.1.2 --- Hypoxic Environment --- p.20 / Chapter 1.1.2.1 --- The Hypoxic Chamber --- p.20 / Chapter 1.1.3 --- Reoxygenation --- p.22 / Chapter 1.1.4 --- Acidic Environment --- p.23 / Chapter 1.1.5 --- Glucose Depletion --- p.24 / Chapter 1.1.6 --- Irradiation --- p.25 / Chapter 1.2 --- Objectives --- p.26 / Chapter 1.3 --- Materials and Methods --- p.27 / Chapter 1.3.1 --- Materials --- p.27 / Chapter 1.3.2 --- Methods --- p.29 / Chapter 1.3.2.1 --- Cell Lines --- p.29 / Chapter 1.3.2.2 --- The Working Procedure for the Hypoxic Chamber --- p.30 / Chapter 1.3.2.3 --- "Aerobic, Hypoxic and Reoxygenated Conditions" --- p.33 / Chapter 1.3.2.4 --- Acidic Condition --- p.35 / Chapter 1.3.2.5 --- Glucose Depleted Condition --- p.36 / Chapter 1.3.2.6 --- Gamma-Irradiation --- p.37 / Chapter 1.3.2.7 --- Analysis of the Growth Pattern by MTT Assay and Cell Counting --- p.38 / Chapter 1.3.2.8 --- Cell Cycle Analysis --- p.39 / Chapter 1.3.2.9 --- Western Blot Analysis --- p.40 / Chapter 1.3.2.10 --- DNA Fragmentation Analysis --- p.42 / Chapter 1.4 --- Results --- p.43 / Chapter 1.4.1 --- Cell Proliferation Profile by MTT Assay --- p.43 / Chapter 1.4.1.1 --- Proliferation of cells under hypoxia --- p.43 / Chapter 1.4.1.2 --- Proliferation of cells under acidic pH environments --- p.49 / Chapter 1.4.1.3 --- Proliferation of cells under glucose depleted environment --- p.52 / Chapter 1.4.2 --- Distribution of cell cycles under different micro environments --- p.54 / Chapter 1.4.3 --- General Protein Expression Pattern by Western Blot Analysis --- p.57 / Chapter 1.4.4 --- Detection of Apoptosis by DNA Fragmentation Assay --- p.59 / Chapter 1.5 --- Discussion --- p.58 / Chapter CHAPTER TWO --- REACTION KINETICS OF A431 CELLS AND SiHa CELLS INDUCED BY EGF --- p.71 / Chapter 2.1 --- Introduction --- p.72 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.74 / Chapter 2.1.2 --- EGF Signaling Pathway --- p.76 / Chapter 2.2 --- Objectives --- p.79 / Chapter 2.3 --- Materials and Methods --- p.80 / Chapter 2.3.1 --- Materials --- p.80 / Chapter 2.3.2 --- Methods --- p.82 / Chapter 2.3.2.1 --- Cell Lines --- p.82 / Chapter 2.3.2.2 --- EGF Sensitivity Assay --- p.83 / Chapter 2.3.2.3 --- Combination Effect of Hypoxia and EGF --- p.83 / Chapter 2.3.2.4 --- Early Kinetics Analysis by Low EGF Concentration Treatment --- p.84 / Chapter 2.3.2.5 --- Late Kinetics Analysis by High EGF Concentration Treatment --- p.85 / Chapter 2.4 --- Results --- p.86 / Chapter 2.4.1 --- Sensitivity of A431 cells and SiHa cells to EGF by MTT Assay --- p.86 / Chapter 2.4.2 --- Early/Late Kinetics of EGF induced protein tyrosine phosphorylation Pattern --- p.90 / Chapter 2.4.3 --- Raf protein expression --- p.96 / Chapter 2.4.4 --- EGFR expression level --- p.100 / Chapter 2.5 --- Discussions --- p.104 / Chapter CHAPTER THREE --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A431 CELLS BY DIFFERENTIAL DISPLAY UNDER DIFFERENT TUMOR MICROENVIRONMENTS --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and Methods --- p.112 / Chapter 3.2.1 --- Materials --- p.112 / Chapter 3.2.2 --- Methods --- p.114 / Chapter 3.2.2.1 --- Spheroid Cells --- p.114 / Chapter 3.2.2.2 --- Identification of Differentally Expressed Genes by RT-PCR --- p.117 / Chapter 3.2.2.3 --- Ligation and Cloning of Differentially Expressed cDNA --- p.120 / Chapter 3.2.2.4 --- Screening and Sequencing of the cDNA Inserts --- p.121 / Chapter 3.2.2.5 --- Northern Blot Analysis --- p.123 / Chapter 3.3 --- Results --- p.124 / Chapter 3.4 --- Discussions --- p.161 / GENERAL CONCLUSION --- p.165 / REFERENCES --- p.167

子宮內膜異位症是一種常見的，但複雜的婦科疾病，發病機制不明。它的特點是異位生长的子宮內膜組織。目前已提出多种發病機制的相关理論。现广泛接受經典的Sampson经血逆行的理論，，子宮內膜細胞出现在腹腔中。然而，对于子宮內膜異位症的發展中出现的細胞和分子事件却研究甚少。血管生成在早期子宮內膜異位病灶的生長和发展中起關鍵作用。在缺氧條件下，低灌注的異位子宮內膜組織促进血管形成因子被激活，以建立新的血管來提供氧和營養物質。血管生成的確切病理機制目前仍不清楚。我們推測，氧化應激是子宮內膜異位症的血管生成和發展的關鍵。目前的研究只是表明氧化應激可能促進子宮內膜細胞的生長和粘附，加剧子宮内膜异位症。但是，子宮內膜異位症的氧化應激具体機制仍不清楚。 / 在這項研究中，我們旨在了解氧化應激在子宮內膜異位症的早期血管生成和發展的重要作用。我们利用實驗性子宮內膜異位症小鼠模型用来研究子宮内膜异位症潛在的氧化應激機制。活体成像显示在子宮內膜异位植入后2 -6小時可检测到高活性氧簇（ROS）。同時，植入位处的也可见巨噬細胞浸潤和HIF-1α的表達。緊接著1天之內血管生成細胞因子表達上調，植入后一周后即有新血管形成。這表明巨噬細胞可能在子宮內膜異位症的早期發展中啟動氧化應激和促進血管生成。為了驗證在氧化應激，血管生成，子宮內膜異位癥的發展中巨噬細胞的作用，，採用抗F4/80抗體造成巨噬細胞功能缺失和12/15 LOX基因敲除小鼠模型下的氧化應激失調兩種模型進行研究。 通過干擾和破壞巨噬細胞介導的氧化應激，在子宮內膜植入處ROS產物生成顯著減少。血管生成因子受到抑制且血管發育不全。子宮內膜異位病灶均小於對照組。這表明巨噬細胞是氧化應激中重要介質。他們在促進子宮內膜異位症的早期血管生成和發展中起重要作用。由PX-478，HIF-1α抑製劑介導的治療也在子宮內膜異位症模型中進行更深入地研究。在血管生成途徑中抑制HIF-1α可減小病灶的大小和抑制新血管形成，但不影響巨噬細胞浸潤和子宮內膜植入物中的活性氧的生產。這表明巨噬細胞和ROS作用於子宮內膜異位症的血管形成機制的上游。總之，我們證明了巨噬細胞介導的氧化應激在子宮內膜異位症的早期血管生成和發展中起重要作用。 / Endometriosis is a common but complex gynecological disorder of unknown pathogenesis. It is characterized by ectopic growth of endometrial tissues Many theories have been proposed to the pathogenesis of endometriosis. The classical Sampson’s theory of retrograde menstruation is widely accepted to determine the presence of endometrial cells in the peritoneal cavity. However, little is known on the cellular and molecular events in the development of endometriosis. Angiogenesis plays a key step in the early growth and survival of the endometriotic lesions. Under hypoxic condition, pro-angiogenic factors are activated in poorly perfused ectopic endometrial tissues in order to establish new blood vessel to supply oxygen and nutrients. The precise pathological mechanisms of this angiogenesis pathway are still unclear. We hypothesized that oxidative stress is critical for the angiogenesis and development of endometriosis. Current studies only suggested oxidative stress may increase growth and adhesion of endometrial cells, promoting endometriosis. However, the underlying mechanism of oxidative stress in endometriosis remains unclear. / In this study, we aimed to understand the important role of oxidative stress in early angiogenesis and development of endometriosis. Experimental endometriosis mouse model was used to determine the underlying mechanism of oxidative stress. By in vivo imaging, high reactive oxygen species (ROS) production in the endometrial implants was detected from 2 -6 hours of transplantation. Concurrently, macrophage infiltration and HIF-1α expression in the implants were anticipated. Subsequently, angiogenic cytokines were upregulated within 1 day and new blood vessels were formed at least 1 week after transplantation. This suggested that macrophage may initiate the oxidative stress surge and angiogenesis pathway in early development of endometriosis. To confirm the role of macrophage in the oxidative stress, angiogenesis and development of endometriosis, macrophage depletion by anti-F4/80 antibody and oxidative stress disruption by 12/15 Lox transgenic knockout mice were employed in the experimental endometriosis models. By depleting macrophage and disrupting macrophage to mediate oxidative stress, the ROS production was significantly decreased in the endometrial implants. Angiogenesis factors were suppressed and blood vessels were under-developed. The endometriotic lesions were smaller than control. This showed macrophage is the key mediator of oxidative stress. They played an important role on promoting early angiogenesis and development of endometriosis. Potential therapeutic treatment by PX-478, a HIF-1α inhibitor, was further investigated in the experimental endometriosis model. Inhibition of HIF-1α in the angiogenic pathway decrease the lesion size and new vessel formed, but macrophage infiltration and ROS production in the endometrial implants was not affected. This suggested that macrophage and ROS are the upstream mechanism of the angiogenic pathway in endometriosis. In conclusion, we demonstrated that oxidative stress mediated by macrophages play an important role in the early angiogenesis and development of endometriosis. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Man, Chi Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-82). / Abstracts also in Chinese. / Title --- p.i / Acknowledgment --- p.ii / List of publication and conferences --- p.iv / Additional achievements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xviii / Abstract --- p.xix / Chinese Abstract --- p.xxi / Chapter Chapter 1. --- Endometriosis / Chapter 1.1. --- Definition of Endometriosis --- p.1 / Chapter 1.1.1. --- Treatment of Endometriosis --- p.2 / Chapter 1.1.1.1. --- Expectant Management --- p.2 / Chapter 1.1.1.2. --- Medical Therapy --- p.2 / Chapter 1.1.1.2.1. --- Analgesics --- p.2 / Chapter 1.1.1.2.2. --- Hormones --- p.3 / Chapter 1.1.1.3. --- Surgery --- p.4 / Chapter 1.1.1.3.1. --- Surgical Treatment --- p.4 / Chapter 1.1.1.3.2. --- Conservative Surgery --- p.4 / Chapter 1.1.1.3.3. --- Definitive Surgery --- p.5 / Chapter 1.1.2. --- Pathogenesis and Pathophysiology of Endometriosis --- p.5 / Chapter 1.2. --- Oxidative stress --- p.7 / Chapter 1.2.1. --- Pro-oxidants --- p.7 / Chapter 1.2.2. --- Antioxidants --- p.8 / Chapter 1.2.2.1. --- Non-enzymatic Antioxidants --- p.9 / Chapter 1.2.2.2. --- Enzymatic Antioxidants --- p.9 / Chapter 1.2.2.2.1. --- Superoxide Dismutase --- p.10 / Chapter 1.2.2.2.2. --- Catalase --- p.10 / Chapter 1.2.2.2.3. --- Peroxiredoxins --- p.11 / Chapter 1.2.2.2.4. --- Glutathione Peroxidases --- p.11 / Chapter 1.2.3. --- Oxidative Stress and Diseases --- p.11 / Chapter 1.2.4. --- Endometriosis and Oxidative Stress --- p.12 / Chapter 1.2.5. --- Inflammatory Response --- p.13 / Chapter 1.2.6. --- Animal Models of Endometriosis --- p.14 / Chapter 1.3. --- Angiogenesis --- p.15 / Chapter 1.3.1. --- Angiogenesis and Diseases --- p.16 / Chapter 1.3.2. --- Animal Models of Angiogenesis --- p.17 / Chapter 1.3.3. --- Endometriosis and Angiogenesis --- p.18 / Chapter 1.3.4. --- Anti-angiogenesis Therapy --- p.19 / Chapter 1.3.5. --- Rodent Models of Endometriosis --- p.21 / Chapter Chapter 2. --- Objective and Hypothesis --- p.23 / Chapter Chapter 3. --- Methodology / Chapter 3.1. --- Mouse Models of Endometriosis --- p.24 / Chapter 3.1.1. --- Animals --- p.24 / Chapter 3.1.2. --- Ovariectomy --- p.24 / Chapter 3.1.3. --- Transplantation of Endometrium --- p.25 / Chapter 3.1.3.1. --- Donor mice --- p.25 / Chapter 3.1.3.2. --- Recipient mice --- p.25 / Chapter 3.1.4. --- Luciferase ⁺/⁺ transgenic mice --- p.26 / Chapter 3.1.5. --- Macrophage Depletion Model --- p.26 / Chapter 3.1.6. --- 12/15 Lox Transgenic Knockout Mice --- p.26 / Chapter 3.1.6.1. --- 12/15 Lox Knockout to Wildtype --- p.27 / Chapter 3.1.6.2. --- Wildtype to 12/15 Lox Knockout --- p.27 / Chapter 3.1.7. --- HIF-1α Inhibition Model --- p.27 / Chapter 3.2. --- Growth and Development of Endometriotic Lesions --- p.28 / Chapter 3.2.1. --- Termination --- p.28 / Chapter 3.2.2. --- Lesion Size --- p.28 / Chapter 3.2.3. --- In Vivo Non-Invasive Imaging --- p.28 / Chapter 3.2.3.1. --- Luciferase Imaging --- p.28 / Chapter 3.2.3.2. --- Oxidative Stress Imaging --- p.29 / Chapter 3.2.3.3. --- Angiogenesis Imaging --- p.30 / Chapter 3.2.4. --- Histological Evaluation --- p.30 / Chapter 3.2.5. --- Immunostaining --- p.30 / Chapter 3.2.5.1. --- Immunohistochemistry on Oxidative Stress Markers --- p.30 / Chapter 3.2.5.2. --- Immunohistochemistry on Macrophages and Neutrophils --- p.31 / Chapter 3.2.5.2. --- Immunofluorescence on New Vessel Formation --- p.32 / Chapter 3.2.6. --- Terminal Deoxynucleotidyltransferase (TdT)-mediated dUTP End Labeling (TUNEL) Assay --- p.33 / Chapter 3.2.7. --- Quantitative real-time --- p.33 / Chapter 3.2.7.1. --- qPCR on Apoptotic Factors --- p.33 / Chapter 3.2.7.2. --- qPCR on Hypoxic Factors --- p.34 / Chapter 3.2.7.3. --- qPCR on Angiogenic Factors --- p.34 / Chapter 3.2.8. --- 8-Isoprostane --- p.34 / Chapter 3.3. --- Reproductive safety of PX-478 --- p.35 / Chapter 3.4. --- Data Analysis --- p.36 / Chapter Chapter 4. --- Results --- p.37 / Chapter 4.1. --- Endometriosis Growth and Development --- p.37 / Chapter 4.1.1. --- In vivo Non-Invasive Imaging --- p.37 / Chapter 4.1.2. --- Ectopic Endometriotic Lesions --- p.37 / Chapter 4.1.3. --- Histological Examination --- p.37 / Chapter 4.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.38 / Chapter 4.1.5. --- Real-time PCR --- p.38 / Chapter 4.1.6. --- Oxidative Stress --- p.38 / Chapter 4.1.6.1. --- Non-invasive Imaging (IVIS) --- p.38 / Chapter 4.1.6.2. --- Measurement of 8-isoprostane --- p.39 / Chapter 4.1.6.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.39 / Chapter 4.1.6.4. --- Immunohistochemistry (HIF-1α and VEGF) --- p.40 / Chapter 4.1.7. --- Angiogenesis --- p.40 / Chapter 4.1.7.1. --- Cellvizio Imaging --- p.40 / Chapter 4.1.7.2. --- Real-time PCR Analysis on Hypoxia & Angiogenic Markers --- p.40 / Chapter 4.1.7.3. --- Immunofluorescence --- p.41 / Chapter 4.2. --- Effects of Estrogen on Oxidative Stress and Angiogenesis --- p.41 / Chapter 4.3. --- Macrophage Depletion --- p.43 / Chapter 4.3.1. --- Lesion Growth and Development --- p.43 / Chapter 4.3.1.1. --- In Vivo Non-Invasive Imaging --- p.43 / Chapter 4.3.1.2. --- Ectopic Endometrium Lesions --- p.44 / Chapter 4.3.1.3. --- Histological Examination --- p.44 / Chapter 4.3.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.44 / Chapter 4.3.1.5. --- Real-time PCR --- p.45 / Chapter 4.3.2. --- Oxidative stress --- p.45 / Chapter 4.3.2.1. --- In Vivo Imaging --- p.45 / Chapter 4.3.2.2. --- 8-isoprostane --- p.45 / Chapter 4.3.2.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.45 / Chapter 4.3.2.4. --- Immunohistochemistry --- p.46 / Chapter 4.3.3. --- Angiogenesis --- p.46 / Chapter 4.3.3.1. --- Real-time PCR analysis on angiogenic markers --- p.46 / Chapter 4.3.3.2. --- Immunofluorescence --- p.47 / Chapter 4.4. --- 12/15 Lox Transgenic Knockout --- p.47 / Chapter 4.4.1. --- Lesion Growth and Development --- p.47 / Chapter 4.4.1.1. --- Ectopic Endometrium Lesions --- p.47 / Chapter 4.4.1.2. --- Histological Examination --- p.48 / Chapter 4.4.1.3. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.48 / Chapter 4.4.1.4. --- Real-time PCR --- p.49 / Chapter 4.4.2. --- Oxidative Stress --- p.50 / Chapter 4.4.2.1. --- 8-isoprostane --- p.50 / Chapter 4.4.2.2. --- Real-time PCR Analysis on Hypoxic Markers --- p.50 / Chapter 4.4.2.3. --- Immunohistochemistry --- p.51 / Chapter 4.4.3. --- Angiogenesis --- p.51 / Chapter 4.4.3.1. --- Real-time PCR Analysis on Angiogenic Markers --- p.51 / Chapter 4.4.3.2. --- Immunofluorescence --- p.53 / Chapter 4.5. --- HIF-1α inhibition --- p.53 / Chapter 4.5.1. --- Lesion Growth and Development --- p.53 / Chapter 4.5.1.1. --- In Vivo Imaging --- p.53 / Chapter 4.5.1.2. --- Ectopic Endometrium Lesions --- p.54 / Chapter 4.5.1.3. --- Histological examination --- p.54 / Chapter 4.5.1.4. --- Immunohistochemistry (Macrophage and Neutrophil) --- p.54 / Chapter 4.5.1.5. --- Real-time PCR --- p.55 / Chapter 4.5.2. --- Oxidative stress --- p.55 / Chapter 4.5.2.1. --- In Vivo Imaging --- p.55 / Chapter 4.5.2.2. --- 8-isoprostane --- p.55 / Chapter 4.5.2.3. --- Real-time PCR Analysis on Hypoxic Markers --- p.55 / Chapter 4.5.2.4. --- Immunohistochemistry --- p.56 / Chapter 4.5.3. --- Angiogenesis --- p.56 / Chapter 4.5.3.1. --- Real-time PCR Analysis on Angiogenic Markers --- p.56 / Chapter 4.5.3.2. --- Immunofluorescence --- p.57 / Chapter 4.5.4. --- Safety Assessment of PX-478 --- p.58 / Chapter Chapter 5. --- Discussion --- p.59 / Chapter 5.1. --- Animal Model and Transplantation Method --- p.59 / Chapter 5.2. --- Oxidative Stress and Its Role in Early Development of Endometriosis --- p.59 / Chapter 5.3. --- Macrophage and Oxidative Stress in Endometriosis --- p.60 / Chapter 5.4. --- Potential Antioxidative Therapy for Endometriosis Treatment --- p.61 / Chapter 5.5. --- Safety --- p.62 / Chapter 5.6. --- Limitation and Further Studies --- p.63 / Chapter Chapter 6. --- Conclusions --- p.65 / Chapter Chapter 7. --- References --- p.66

Addendum attached to back facing leaves. Includes bibliographical references (leaves 172-199). The findings establish leukemia inhibitory factor, and possibly oncostatin M, as new members of a small but growing class of cytokines produced in an intracellularly active form and also suggest that the production of alternate transcripts and intercellularly-retained proteins may be a common and important feature of cytokines of the IL-6 and other families.

This doctoral dissertation presents a series of studies that were conducted to investigate the relationship between morphological, as well as functional, changes and clinical disability in multiple sclerosis (MS) using magnetic resonance imaging (MRI) and functional MRI (fMRI). / The extent of macroscopic brain tissue damage, as seen on T2-weighted MRI scans, is poorly correlated with measures of functional impairment in MS. We hypothesized that this might be due to the failure to take lesion location into account. By combining sophisticated lesion segmentation tools with the statistical and stereotaxic techniques of functional neuroimaging, we have shown a relationship between lesion location and the extent and type of physical and cognitive disability. / Brain atrophy is another manifestation of MS. We conducted the first large-scale study of focal cortical atrophy in MS that uses cortical thickness measurements across the entire cortex. We present evidence that cortical atrophy occurs relatively early in the course of the disease, despite the lack of severe disability in MS patients, as assessed by the Expanded Disability Status Scale (EDSS), and follows a pattern of focal thinning that is more pronounced in areas that are heavily inter-connected with other brain regions, such as anterior cingulate cortex and association areas, suggesting that interruption of white matter tracts by MS plaques might play a causative role in cortical atrophy. / Finally, we conducted an fMRI study of working memory in controls, cognitively unimpaired and impaired MS patients that revealed significant differences in the regions that were activated between the groups. Most interestingly, while both cognitively unimpaired MS patients and control subjects significantly activated the left dorsolateral prefrontal cortex and the left thalamus, cognitively impaired MS patients failed to significantly activate these areas. Levels of deactivation within the medial prefrontal/anterior cingulate cortices and posterior cingulate cortex were inferior in MS patients than in controls. This study suggests that with an increased white matter lesion volume there is an increased damage to a number of afferents and efferents to and from the thalamus (cortico-basal ganglia-thalamo-cortical loops and other thalamo-cortical projections) that ultimately causes the observed cognitive deficits. These cognitive deficits seem also to be dependent on a reduced capacity of MS patients to show task-related deactivations.