An alternative complementary approach was used to verify the antinociceptive effect of 4 CMMs aqueous extracts in a Drosophila adult model. Drosophila adults were subjected to CMM treatments and then placed on an in-house-designed heating device for noxious heat stimulation. Their behavioral outputs were quantified and expressed as heat avoidance index (AI) for revealing the degree of antinociceptive effect of CMMs. By comparing the AI value of non-CMM treated control group with CMM-treated groups at temperature challenge 32°C, it was found that an AI value of 0.2 was obtained for non-CMM-treated control group whereas CMMs-treated groups showed AI values ranged from 0.33 to 0.4. The increase of AI value in those CMM-treated groups means that Drosophila adults became more susceptible to noxious heat stimulation. This indicates that those identified CMMs by the larvae model possess strong and versatile antinoceiceptive activities in Drosophila adults. / In addition, reverse transcription PCR (RT PCR) analysis was performed to study the effects of CMMs on the mRNA expression of three nociceptive-related genes painless, nompC and CG4536. These three genes all belong to the Transient Receptor Potential (TRP) families and have been shown to be involved in heat response. The results indicate that the gene expression level for nompC was significantly down-regulated with fold changes ranging from 0.2 to 0.7 upon 2 hrs treatment of three aqueous CMM extracts Citrus aurantium, Angelica dahurica and Vitex trifolia. However, there is no significant difference in gene expression level for painless and CG4536. / In this study, it has been demonstrated that Drosophila are feasible to use for screening CMMs with antinociceptive activity. While the data of the relative gene expression level for those target genes observed in this study may also serve as biomarkers for providing more evidence to investigate drugs have antinociceptive effects. In the future, such information paves the way for further development in the study of antinociceptive drugs. / Nociception is the reception of signals in the central nervous system (CNS) triggered by specialized sensory receptors which received stimuli such as electrical, thermal, mechanical, or chemical and response to escape from danger. Similar to humans, the fruitfly Drosophila display evolutionarily conserved nociceptive response that makes it suitable for in vivo nociceptive study. In this study, Drosophila larvae were used as initial screening model to investigate the antinociceptive effect that was caused by 61 randomly selected Chinese Medicinal Materials (CMMs). Upon noxious heat stimulation, 73% of larvae in the control group produced a stereotypical rolling behavior within 1 s. Among those tested CMMs, the results indicated that 4 aqueous CMMs extracts from Citrus aurantium L. (family: Rutaceae), Angelica dahurica (Fish. ex Hoffm.) Benth. et Hook (family: Umbelliferae), Vitex trifolia L. var. simplicifolia Cham. (family: Verbenaceae) and Panax notoginseng (Burk.) F. H. Chen (family: Araliaceae) were found to have strong antinociceptive effect on Drosophila larvae since less than 40% of the larvae have produced stereotypical rolling behavior within 1 s upon noxious heat stimulation. / "September 2007." / Advisers: Ming Liang Song; Ho Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4768. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 134-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Kou Ying Chuck. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 186-199). / 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.

In conclusion, the results suggested the important role of Ncl in driving the optic vesicle formation during early eye development. / The eye is a complex sense organ. It develops from different embryonic origins that including neural ectoderm, surface ectoderm, neural crest and paraxial mesoderm. Morphogenetic waves occur during eye development involve timely interactions of transcription factors and inductive signaling to ensure the correct temporal and spatial development of different components. Genetic studies of congenital eye defects, especially mutation screening and gene targeting, have provided the information about the molecular regulation in the complex processes of eye development. However, our knowledge of the basic genetic pathways that regulate the normal embryonic eye formation is incomplete. / Though the developing eye is believed to be highly specialized extension from the developing neural tube, the formation of major eye structure involves independent coordination of inductive interactions and regional specifications; formation of neural connections between retina and optic tectum; and maturation to a functional eye. There is not much information about eye-specific expression in early embryonic period. In this study, microarray was used to profile the molecular changes occurring in the developing mouse eye between the stage of optic vesicle evagination at E9.5 and completion of basic eye formation at P0. Differentially expressed transcription factor and signaling molecules, including nucleolin gene (Ncl), in the early developing eye were displayed. Temporal expression patterns were confirmed by quantitative real time PCR and spatial expressions patterns were confirmed by the whole-mount in situ hybridization. siRNA and overexpression vector targeting nucleolin transcript was designed to study their roles in the early eye morphogenesis during mouse embryogenesis in vitro. The loss of function phenotype after nucleolin knockdown was demonstrated by the absence of early optic vesicles with normal neural tube in the developing mouse embryos. Ectopic optic vesicle in developing mouse embryo was resulted under overexpression of Ncl . With the aim to study the biological roles of Ncl in mouse embryonic eye development in vivo, both conventional and conditional knockout techniques were attempted. The expression and functional studies revealed that a new neural tube independent signaling pathway regulated in the induction and formation of optic vesicles in the early eye formation. / Tang, Ling Yin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3294. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 138-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

人類先天性巨結腸症（HSCR）主要表現為結腸末端的神經節缺失或稀少。 結腸末端的神經節來源於迷走源性神經脊細胞和骶源性神經脊細胞。迷走源性神經脊細胞是腸神經系統的主要來源，已被廣泛研究，而關於哺乳類包括人類的骶源性神經脊細胞的研究卻相當稀少。小鼠胚胎的骶源性神經脊細胞遷移途徑近期已被闡釋，其由神經管背側遷出並向腹側移動，於後腸附近聚集成旁神經節然後進入腸內。本研究運用一系列實驗鑒定了對小鼠骶源性神經脊細胞由旁神經節遷移至腸內過程有影響作用的因素。 / 研究發現骶源性神經脊細胞是沿著神經纖維並向口端方向遷移進腸內，同時由於迷走源性神經脊細胞於腸內向尾端遷移，它們在後腸末端相遇並相互作用，我們首先研究了這種相互作用以瞭解迷走源性神經脊細胞如何影響骶源性神經脊細胞的遷移。利用帶綠色螢光的小鼠骶源性神經脊細胞和可變螢光的小鼠迷走源性神經脊細胞（鐳射激發下由綠色變為紅色），以及鐳射共聚焦顯微鏡活細胞成像術，我們觀察了這兩種細胞在腸內相遇時的行為。當骶源性神經脊細胞和迷走源性神經脊細胞在神經纖維上相遇是，它們都停止了移動，不能前行。培養3天以後，骶源性神經脊細胞和迷走源性神經脊細胞在後腸中共同形成了神經細胞網路，其中骶源性神經脊細胞相對只占了小部分細胞。 / 由於骶源性神經脊細胞沿著由旁神經節發出的神經纖維遷移至腸內並且當在神經纖維上遇到迷走性源性神經脊細胞時便停止移動，我們進而研究了神經纖維在骶源性神經脊細胞遷移中的地位。活細胞成像術和免疫組化實驗表明旁神經節發出的神經纖維對骶源性神經脊細胞的遷移是非常重要的，它有助於細胞遷移同時，但當細胞到達纖維末端時它便也限制了細胞的遷移。儘管如此，體外實驗表明在一定培養條件下，骶源性神經脊細胞的遷移並不需要這些神經纖維。 / 由於有報導表明腸內微環境能影響腸神經脊細胞的遷移，我們利用2D電泳和質譜檢測了12.5天（骶源性神經脊細胞進入後腸之前）和13.5天（骶源性神經脊細胞進入後腸）胎鼠後腸中的蛋白表達情況。大多數鑒定到有差異表達的蛋白都與蛋白折疊、細胞生長和細胞骨架組織有關。我們選取了與細胞粘附和肌肉收縮有關的鈣離子依賴膜結合蛋白Anxa6，並結合腸內的平滑肌發育進行了進一步的研究。結果顯示在13.5天胎鼠中，旁神經節的口端方向有一段約600微米的腸的腹側的平滑肌還沒有發育，可能與腸神經脊細胞的遷移有關。但腸內平滑肌發育是否及如何影響腸神經脊細胞的遷移還需要進一步的研究。 / 綜上所述，骶源性神經脊細胞的遷移是一個複雜的過程，迷走源性神經脊細胞，神經纖維和腸內微環境都參與並能影響這個遷移過程。 / Hirschsprung’s disease (HSCR) in humans is characterized by the absence or reduction of enteric ganglia in the distal part of the colon. It is known that all enteric ganglia in the distal colon originate from neural crest cells (NCCs) at both vagal and sacral levels during embryonic development. Vagal NCCs have been well characterized as the main cellular source of the enteric nervous system (ENS), but however, information on the mammalian, including human, sacral NCCs is still scarce. Sacral NCCs in mouse embryos have been recently identified to be able to migrate from the dorsal neural tube to the mesenchyme, aggregate as pelvic ganglia adjacent to the hindgut and then enter the distal hindgut. In the present study, a series of experiments were performed to determine the factors that were involved in modulating their entry to the hindgut and their migration within the distal hindgut, using mouse embryos. / Having entered the hindgut, sacral NCCs migrated along nerve fibers in a caudal-to-rostral direction while vagal NCCs were colonizing the hindgut in a reverse, rostral-to-caudal direction. The migratory behaviors of the vagal and sacral NCCs were examined at the time when these two populations of NCCs met each other with live cell confocal imaging in the distal hindgut using GFP-expressing sacral NCCs (green fluorescent) and Ednrb-Kikume labeled vagal NCCs (red fluorescent) from transgenic mice. The rostral migration of sacral NCCs was observed to be temporarily affected when they met vagal NCCs on the nerve fiber. However, after 3 days in organotypic culture, sacral and vagal NCCs were found to intermingle with each other to form an interconnected cellular network in the hindgut with much greater cellular contribution from vagal NCCs than sacral NCCs. Hence, vagal NCCs were able to affect the migration and thus the final location of sacral NCCs within the hindgut. / Since sacral NCCs have been observed to enter the hindgut by migrating on the nerve fibers extending from pelvic ganglia, the role the nerve fibers in migration was then examined. Results obtained from time-lapse confocal live cell imaging and immunohistochemical localization indicated that nerve fibers extending from pelvic ganglia were very important for sacral NCCs migration. It was found that these nerve fibers could both assist in sacral NCC migration and also restrain their migration once the cells reached the distal tip of the fibers. However, under specific in vitro conditions, sacral NCCs were still able to migrate without the presence of nerve fibers. / The gut microenvironment surrounding the migrating NCCs has also been reported to affect NCCs migration. Therefore, protein molecules with differential expression levels prior to and after the entry of sacral NCCs to the distal hindgut between E12.5 to E13.5 were examined with 2-dimensional gel electrophoresis and mass spectrometry. The proteins identified with significant changes of expression (more than 1.5 folds) were grouped according to their predicted biological functions and involved in protein folding, cell growth and cytoskeletal organization. Among them, Anxa6, a calcium-dependent membrane binding protein related to cell adhesion and muscle contraction, was further examined for its relationship with the muscle development in the hindgut at E12.5 to E14.5. The results showed that a segment of the hindgut (about 600μm) rostral to pelvic ganglia exhibited an incomplete layer of smooth muscle at E13.5. Whether Anxa6 and the smooth muscle are involved in the sacral NCC migration is worth further investigations. / In summary, sacral NCCs migration is a complex process regulated by their interactions with nerve fibers, vagal neural crest cells and possibly molecules in the hindgut microenvironment through which they migrate. / 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. / Chen, Jielin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 201-214). / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of contents --- p.XII / Abbreviation --- p.XIII / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The enteric nervous system (ENS) --- p.1 / Chapter 1.1.1 --- Embryonic origin and development of the ENS --- p.2 / Chapter 1.1.2 --- Hirschsprung’s disease (HSCR) --- p.5 / Chapter 1.2 --- Enteric neural crest cells (ENCCs) --- p.7 / Chapter 1.2.1 --- Vagal neural crest cells (NCCs) --- p.8 / Chapter 1.2.2 --- Sacral neural crest cells (NCCs) and pelvic ganglia --- p.10 / Chapter 1.2.3 --- Interactions between neural crest cells (NCCs) --- p.14 / Chapter 1.3 --- Microenvironment within the gut --- p.16 / Chapter 1.3.1 --- Effect of molecules on ENCCs migration --- p.16 / Chapter 1.3.2 --- Effect of tissue age on ENCC migration --- p.19 / Chapter 1.3.3 --- Absence of ENCCs facilitates ENCC colonization --- p.20 / Chapter 1.4 --- Objectives of the present study --- p.22 / Chapter Figures and Legends --- p.25 / Chapter Chapter 2 --- Migratory behaviors of sacral and vagal neural crest cells in the distal hindgut --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.36 / Chapter 2.2.1 --- Mouse strains --- p.36 / Chapter 2.2.2 --- Isolation of gut tubes and pelvic ganglia --- p.36 / Chapter 2.2.3 --- Photo-conversion of Ednrb-kikume labeled neural crest cells within the gut --- p.37 / Chapter 2.2.4 --- Preparation of general culture medium and organ culture agarose gel --- p.38 / Chapter 2.2.5 --- Organotypic culture --- p.39 / Chapter 2.2.6 --- Time-lapse live cell confocal microscopic imaging --- p.39 / Chapter 2.2.7 --- Whole mount gut preparations for immunohistochemical staining --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Conversion of green fluorescent, Ednrb-kikume labeled vagal NCCs into red fluorescent --- p.42 / Chapter 2.3.2 --- Ednrb-kikume labeled cells were Sox10 immunorecative --- p.42 / Chapter 2.3.3 --- No discernible photo-toxicity after photo-conversion --- p.43 / Chapter 2.3.4 --- Sacral NCCs migration was hindered by vagal NCCs when they met on the nerve fiber --- p.43 / Chapter 2.3.5 --- Vagal NCCs migrated toward each other to form an interconnected network --- p.45 / Chapter 2.3.6 --- Sacral NCCs contributed much fewer cells than vagal NCCs in the terminal hindgut --- p.46 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.4.1 --- Ednrb-kikume mouse is a potentially ideal animal model for studies of NCCs migratory behaviors --- p.47 / Chapter 2.4.2 --- Migration of sacral NCCs in the hindgut was affected by vagal NCCs --- p.49 / Chapter 2.4.3 --- Migratory behaviors of vagal NCCs --- p.52 / Chapter 2.4.4 --- Vagal NCCs potentially preferred to move on nerve fibers --- p.53 / Chapter 2.4.5 --- Sacral NCCs contributed much less to the cellular network than vagal NCCs --- p.53 / Chapter 2.5 --- Summary --- p.55 / Chapter Table 2-1 --- Primary and secondary antibodies used in the experiments --- p.56 / Chapter Figures and Legends --- p.57 / Chapter Chapter 3 --- The relationship between nerve fiber extension and sacral neural crest cell migration in vitro --- p.81 / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials and methods --- p.86 / Chapter 3.2.1 --- Mouse strains --- p.86 / Chapter 3.2.2 --- Preparation of fibronectin (FN) coated coverslips and confocal dishes --- p.86 / Chapter 3.2.3 --- Preparation of media --- p.87 / Chapter 3.2.4 --- Isolation of pelvic ganglia --- p.87 / Chapter 3.2.5 --- In vitro culture of pelvic ganglia in 4-well plates or confocal dishes --- p.88 / Chapter 3.2.6 --- Live cell imaging using Nikon live cell imaging system --- p.88 / Chapter 3.2.7 --- WGA treatments on the pelvic ganglia culture --- p.89 / Chapter 3.2.8 --- Effect of embryonic cell proliferation medium and stem cell proliferation medium on pelvic ganglia growth in vitro --- p.90 / Chapter 3.2.9 --- Immunohistochemical staining --- p.90 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Sacral NCCs and nerve fibers from the pelvic ganglia were in close association in vitro --- p.92 / Chapter 3.3.2 --- Migratory behaviors of sacral NCCs on the nerve fiber in vitro --- p.92 / Chapter 3.3.3 --- WGA treatments affected the growth of nerve fibers and sacral NCCs migration in vitro --- p.93 / Chapter 3.3.4 --- Sacral NCCs migrated without nerve fibers when cultured in proliferation media --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter 3.4.1 --- In vitro culture of pelvic ganglion --- p.97 / Chapter 3.4.2 --- Migratory behaviors of sacral NCCs in vitro --- p.98 / Chapter 3.4.3 --- Sacral NCCs migration was affected by the extension of nerve fibers from pelvic ganglia in vitro --- p.101 / Chapter 3.4.4 --- Nerve fibers from the pelvic ganglia were not necessary for sacral NCCs migration in vitro --- p.103 / Chapter 3.5 --- Summary --- p.106 / Chapter Figures and Legends --- p.107 / Chapter Chapter 4 --- Differentially expressed protein molecules in the distal hindgut before and after the entry of sacral neural crest cells --- p.128 / Chapter 4.1 --- Introduction --- p.128 / Chapter 4.2 --- Materials and methods --- p.132 / Chapter 4.2.1 --- Mouse strain --- p.132 / Chapter 4.2.2 --- Preparation of solutions for 2-dimensional (2D) gel electrophoresis --- p.132 / Chapter 4.2.3 --- Preparation of solutions for mass spectrometry --- p.133 / Chapter 4.2.4 --- Isolation of the distal hindgut and protein extraction --- p.133 / Chapter 4.2.5 --- Measurement of protein concentration --- p.134 / Chapter 4.2.6 --- 2D gel electrophoresis --- p.135 / Chapter 4.2.7 --- Mass spectrometry --- p.138 / Chapter 4.2.8 --- SDS-PAGE and Western blot --- p.139 / Chapter 4.2.9 --- Immunohistochemical staining of gut tubes and embryos --- p.140 / Chapter 4.2.10 --- Distal hindgut model reconstruction --- p.141 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- E13.5 was the critical stage at which sacral NCCs started to enter the hindgut --- p.143 / Chapter 4.3.2 --- Protein molecules identified by 2D electrophoresis and mass spectrometry --- p.144 / Chapter 4.3.3 --- Western blot analysis and immunostaining confirmed expression levels of Anxa6 --- p.145 / Chapter 4.3.4 --- Smooth muscle actin (SMA) and Anxa6 partially co-localized within the E13.5 hindgut --- p.146 / Chapter 4.3.5 --- Expression of SMA and Anxa6 before and after sacral NCC entry to the distal hindgut --- p.146 / Chapter 4.3.6 --- Reconstruction of distal hindgut images from serial sections with SMA and Anxa6 immunoreactivities --- p.147 / Chapter 4.4 --- Discussion --- p.149 / Chapter 4.4.1 --- Tissue age affected enteric NCC colonization --- p.149 / Chapter 4.4.2 --- Proteomics used in modern biological research --- p.150 / Chapter 4.4.3 --- Molecules differentially expressed in the distal hindgut at E12.5 and E13.5 --- p.151 / Chapter 4.4.4 --- Anxa6 and SMA expression in the distal hindgut --- p.153 / Chapter 4.4.5 --- The role of the smooth muscle development in sacral NCC entry into the hindgut --- p.155 / Chapter 4.5 --- Summary --- p.157 / Chapter Table 4-1 --- Identification of proteins by MALDI-TOF analysis and the MASCOT search program --- p.158 / Chapter Table 4-2 --- Differentially expressed proteins identified by 2-D electro-phoresis and MALDI-TOF/TOF and their predicted biological functions --- p.159 / Figures and Legends --- p.160 / Chapter Chapter 5 --- Conclusions and discussion --- p.182 / Chapter 5.1 --- Vagal NCCs hindered sacral NCC migration when they coalesced on the nerve fiber --- p.182 / Chapter 5.2 --- Nerve fibers from pelvic ganglia were important but not necessary for sacral NCCs migration in vitro --- p.186 / Chapter 5.3 --- Possible involvement of smooth muscle development in modulating sacral NCCs migration --- p.189 / Chapter 5.4 --- Future prospects --- p.191 / Chapter 5.4.1 --- Interactions of vagal and sacral NCCs within the hindgut of mouse embryos --- p.191 / Chapter 5.4.2 --- Role of nerve fibers for sacral NCCs migration ex vivo --- p.192 / Chapter 5.4.3 --- Role of Anxa6 in muscle development of the gut and NCCs migration --- p.193 / Chapter Appendix I --- Solutions used in 2-D electrophoresis --- p.195 / Chapter Appendix II --- Solutions for Colloidal Coomassie staining --- p.198 / Chapter Appendix III --- Procedures for embryo processing --- p.199 / Chapter Appendix IV --- Other solutions --- p.200 / References --- p.201

Mok Wing Fai Simon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 91-97). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.iv / Table of content --- p.v / List of Figures --- p.viii / List of Tables --- p.x / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Origin of the Neural Crest Cells / Chapter 1.1.1 --- Formation of the Neural Tube --- p.1 / Chapter 1.1.2 --- The Neural Crest cells and the Vagal Neural Crest Cells --- p.2 / Chapter 1.1.3 --- The migration profiles of Neural Crest Cells Originated from the Axial level other than Vagal Neural Crest --- p.4 / Chapter 1.1.4 --- Development of the Gastrointestinal Tract and the Enteric Nervous System --- p.5 / Chapter CHAPTER TWO: --- MIGRATION OF NEURAL CREST CELLS IN NORMAL ICR AND DOM MUTANT MOUSE EMBRYOS / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Pregnant mice --- p.39 / Chapter 2.2.2 --- The Handling Medium --- p.39 / Chapter 2.2.3 --- The Culture Medium --- p.40 / Chapter 2.2.4 --- Preparation of Wheat Germ Agglutinin-Gold Conjugates (WGA-Au) --- p.42 / Chapter 2.2.5 --- "Preparation of 1,´1ة-dioctadecyl-´3ة 3,3 '3,3 226}0ة-tetramethyl indocarbocyanine perchlorate (Di-I) " --- p.43 / Chapter 2.2.6 --- Preparation of Carnoýةs Solution --- p.43 / Chapter 2.2.7 --- Preparation of Paraformaldehyde --- p.43 / Chapter 2.2.8 --- Pregnont Dominant Megacolon (Dom) Mice --- p.44 / Chapter 2.2.9 --- DNA Extraction for Genotyping of Dom Embryos --- p.45 / Chapter 2.2.10 --- Primers Used in PCR for Genotyping of Dom Embryos --- p.45 / Chapter 2.2.11 --- PCR Reagent System --- p.46 / Chapter 2.2.12 --- 10XTBE --- p.46 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of Embryos from Pregnant Mice --- p.47 / Chapter 2.3.2 --- In situ labeling of exogenous dye --- p.48 / Chapter 2.3.3 --- Whole Embryo Culture --- p.49 / Chapter 2.3.4 --- Morphological Examination of Cultured Embryos --- p.49 / Chapter 2.3.5 --- Histological Examination of Cultured embryos --- p.50 / Chapter 2.3.6 --- Genotyping of Dom F1 Generation --- p.51 / Chapter 2.3.7 --- Genotyping of Dom Embryos by PCR --- p.52 / Chapter 2.3.8 --- Gel Electrophoresis --- p.52 / Chapter 2.3.9 --- Counting of WGA-Au Labelled Cells --- p.53 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Genotyping --- p.54 / Chapter 2.4.2 --- Examination on Gross morphology of Control and Experimental Embryos --- p.54 / Chapter 2.4.3 --- Morphological Examination of DOM Mutant Embryo after culture --- p.57 / Chapter 2.4.4 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration in Mouse Embryos --- p.62 / Chapter 2.4.5 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration in DOM Embryos --- p.64 / Chapter 2.4.6 --- Distribution of Labelled Cells in ICR Embryos after WGA-Au Labelling --- p.65 / Chapter 2.4.7 --- Distribution of WGA-Au Labelled Cells in DOM Embryos --- p.69 / Chapter CHAPTER THREE: --- DISCUSSION / Chapter 3.1 --- Development of embryos in vitro --- p.78 / Chapter 3.2 --- Comparison of the Two Exogenous Dyes --- p.80 / Chapter 3.3 --- Migration Pathway of the Vagal and Trunk Neural Crest Cells --- p.81 / Chapter 3.4 --- Counting of Labelled Cells in DOM Embryos --- p.83 / Chapter 3.5 --- Initial Stage of Vagal and Trunk Neural Crest Cells Migration of Different Genotypes of the DOM Embryos --- p.84 / Chapter 3.6 --- Differences in Distribution of WGA-Au Labelled Cells in Different Genotypes of DOM Embryos --- p.85 / Chapter CHAPTER FOUR: --- CONCLUSION --- p.88 / REFERENCES --- p.91 / "FIGURES, LEGEND TABLE AND APPENDIX"

糖調節蛋白78 (Glucose-regulated protein 78),也稱之Bip,是70kDa熱休克蛋白家族成员之一。已有的研究表明，Bip 是一個具有多功能的蛋白，參與眾多的生物調控過程，包括蛋白折疊，調節鈣平衡，以及作為內質網緊張（ER stress) 的感應器。有研究表明，Bip可以在細胞膜上定位，作為Nodal信號通路的一個輔助受體發揮作用。大量的研究表明，Bip在疾病和代謝方面也發揮重要作用。它參與胰島素的生物合成，並可以提高長期高血糖下β細胞的功能。同時具有抗細胞凋亡的作用。然而Bip在胚胎髮育中的生物功能卻知之甚少。 / 高等脊椎動物腎臟發育中經歷形成3種腎臟形式：前腎，中腎和後腎。腎單位是這3種形式的基本結構和功能單位。在兩棲類，前腎在胚胎時期發揮作用，在胚胎的兩側各只有一個腎單位。這使得爪蟾成為前腎研究的一個非常好的模型。 / 在此項研究中，我們採用非洲爪蛙作為動物模型來研究Bip在胚胎髮育過程中，尤其是在前腎發育中的生物功能。Bip是一個母性因子，在尾芽期，Bip 表達在粘液腺，前腎，肝以及耳囊。 Bip在前腎清晰明確的表達，表明Bip可能在前腎的發育中發揮作用。我們利用BipMO來進行敲低功能實驗，免疫印記顯示BipMO能阻斷帶Flag標記Bip的翻譯。通過原位雜交技術檢測前腎的不同標記基因的表達發現，敲低Bip抑制前腎的形成，表明Bip的正常表達是前腎發育所必須的。 / 為了研究Bip調節前腎的發育的分子機制， 我們使用Affmetrix基因芯片分析在Bip敲低情況下的不同時期胚胎中基因的表達譜，發現在Bip敲低表達的胚胎中，視黃酸信號通路的一些重要的組分的表達受到抑制。爪蛙胚胎原腸胚的動物帽細胞具有多能性， 使用激活素和視黃酸一同處理動物帽細胞可以誘導其分化成為原腎組織。在此體外分化體系中敲低Bip表達，前腎標記基因表達降低，顯示在這一體外系統中前腎的分化受到抑制。該實驗結果與體內實驗結果一致。在體外培養的HEK293T細胞中敲低Bip，抑制視黃酸處理後視黃酸信號通路螢光素報告的活性。 lhx1是前腎發育早期表達標記之一，對於前腎原基的初始化具有重要的作用，同是它也是視黃酸信號通路的靶基因。共同註射BipMO和lhx1表明，前腎的異常可以明顯降低，顯示lhx1可以部份拯救由於Bip缺失所造成的腎臟發育缺陷。該實驗表明Bip通過調節視黃酸信號通路，來調控lhx1的表達前腎的形成。我們進一不發現，敲低Bip後，前腎異常形成的區域內，細胞凋亡增加，增殖減少。該結果在細胞水平上解釋了Bip敲低表達時前腎形成異常的一個原因。 / 综述所述，Bip正確表達对胚胎前肾的发育極為重要。它胚胎发育过程中通过視黃酸信号通路調控lhx1的表達，從而对前肾的形成发挥重要作用。 / Glucose-regulated protein 78 (Grp78), also known as Bip, belongs to heat shock protein 70kDa family. It has been implicated in various biological processes including protein folding, regulation of calcium homeostasis, and serving as a sensor of ER (Endoplasmic Reticulum) stress. Moreover, it can localize in cell membrane, acting as co-receptor of nodal signaling. It is essential for insulin biosynthesis. In addition, Bip plays important roles in a number of diseases. For example, BIP can improve β-cell function in the prolonged hyperglycemia. Knockdown of BIP in β-cell can induce apoptosis. However, little is known about its function during embryonic development. / In high vertebrate, three sets of nephric forms develop successively during embryonic kidney development. They are pronephros, mesonephros, and metanephros. Nephron is the basic structural and functional unit of all these three forms. In amphibian, the pronephros performs function at the embryonic stages, which has only one nephron on either side of the body. It makes Xenopus a very good model for pronephros study. / In this study, we took advantage of Xenopus leavis as an animal model to investigate Bip function during embryonic development, especially its role in pronephros development. We first examined the expression of Bip in developing embryos. Whole mount in situ hybridization showed that Bip was expressed in the cement gland, pronephros, liver and ear vesicle during tailbud stages. It was expressed in the pronephros strongly and clearly which suggested that Bip might play roles in pronephros development. We performed loss-of-function experiment by using morpholino oligonucleotide (MO) knock down translation of endogenous Bip expression. Depletion of Bip impaired formation of pronephros revealed by reduction expression of different pronephros maker genes. The pluripotent animal caps can differentiate into pronephros tissue when treated with activin and all-trans retinoic acid (atRA) in vitro kidney induction assay. In line with our in vivo observation, knockdown of Bip inhibited pronephros differentiation that can normally achieved by combined effects of activin and atRA in animal cap assay. / In order to investigate the molecular mechanisms as how Bip regulated pronephros development, we performed Affymetrix DNA microarray assay to generate gene expression profile in Bip morphants. We found that some components of RA signaling were inhibited when Bip was knockdown. Moreover, knockdown of Bip caused reduction of RA target genes expression after treatment with RA. Consistent with above observations, luciferase activities of RA signaling reporter was reduced in HEK293T cells when BIP expression was depleted by RNAi. lhx1 is one of RA target genes and has been implicated playing essential roles in pronephros development. The inhibition of pronephros formation induced by Bip depletion can be partially rescued by co-overpression, suggesting 1) lhx1 is downstream of Bip in the regulatory network of pronephros formation; and 2) Bip regulates pronephros formation through RA signaling via lhx1. We also found increased apoptosis and decreased cell proliferation at pronephros-forming region in Bip morphants. That could explain the reason of pronephros malformation when Bip is downregulated. / Taken together, Bip is essential for pronephros development. It functions through RA signaling during the complex developmental processes. / 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. / Shi, Weili. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 125-143). / Abstracts also in Chinese.

A poluição atmosférica é um importante fator de risco à saúde humana, com maior vulnerabilidade em pessoas com doenças pré-existentes, idosos, crianças e fetos. A teoria da origem desenvolvimentista da saúde e doença se baseia em evidências que alterações no desenvolvimento fetal que podem levar a prematuridade, baixo peso ao nascer e predispor ao surgimento de síndrome metabólica, hipertensão e doenças renais na idade adulta. Há evidências também que alterações do ambiente intrauterino na gestação afetam o número de néfrons ao nascimento, o que predispõe à hipertensão na idade adulta. Estudos experimentais prévios comprovam que o ar de São Paulo apresenta uma concentração de poluentes capaz de desencadear, além de lesões cardio-respiratórias, alterações reprodutivas como baixo peso ao nascer e prematuridade. O objetivo deste estudo foi avaliar de maneira objetiva se a exposição aos níveis ambientais de poluição atmosférica no período gestacional tem o potencial de promover alterações de volume renal e número de glomérulos ao nascimento, o que, com base na teoria da origem fetal das doenças, poderia ser um fator predisponente para doenças renais e hipertensão no adulto. Para tanto, neste estudo, foram estudados rins fetos de camundongos Balb/c no 18º dia pós-concepção, expostos aos níveis ambientais de poluição do ar em São Paulo, em duas câmaras de exposição uma recebendo ar filtrado (F) e outra ar não filtrado (P). Os rins dos fetos foram avaliados morfologicamente por métodos estereológicos para estimativa do volume renal total e de cada compartimento (córtex e medula) e número de néfrons. A concentração do material particulado na câmara filtrada foi significantemente menor (71%, p<0.001) que na câmara não filtrada. O peso fetal e dos rins foi 30% menor no grupo NF em relação ao grupo F (p=0.007 e p=0,0112 respectivamente). iv Doutorado Nilsa Regina Damaceno-Rodrigues 2012 O córtex e a medula renais dos fetos do grupo NF apresentaram redução de volume: córtex: 4,46±0,55 x 2,44±0,79 (p=0,0003); medula: 2,35±0,43 x 1,24±0,61 (p=0,0034). O número absoluto de néfrons no rim dos fetos NF (3301,0±1720,8) foi significantemente menor (p=0,0082) que nos fetos F (7199,5±2023,5). Estes dados mostram que a exposição pré-natal aos níveis ambientais de poluição em São Paulo provoca uma alteração no desenvolvimento renal, que pode levar à doenças no adulto / Air pollution is an important risk factor to human health, with greater vulnerability in people with pre-existing diseases, elderly, children and fetuses. The theory of developmental origins of health and illness proposes that changes in fetal development that can lead to prematurity, low birth weight and predisposition to chronic diseases in adulthood. There is also evidence that changes in the intrauterine environment during pregnancy affect the number of nephrons at birth, which predisposes to hypertension in adulthood. Previous experimental studies show that the air of Sao Paulo has a concentration of pollutants that can trigger cardio-respiratory lesions and reproductive hindrances as low birth weight and prematurity. Our proposal was to evaluate objectively whether exposure to ambient levels of air pollution during pregnancy has the potential to promote changes in renal volume and number of glomeruli at birth, which could be a predisposing factor for adult renal illness and hypertension based on the theory of fetal origin of the disease. Therefore, in this study, we used kidneys of fetal Balb/c mice on day 18 post-conception (dpc), exposed to ambient levels of air pollution in Sao Paulo in two exposure chambers: one receiving filtered air (F) and other non-filtered air (P). The kidneys of the fetuses were morphologically evaluated by stereological methods for estimation of total renal volume and the volume of each compartment (cortex and medulla) and the number of nephrons. The concentration of particulate matter in the filtered chamber was significantly lower (71%, p<0.001) than in the unfiltered chamber. Fetal weight and kidney weight were 30% lower in the unfiltered chamber (p=0.007 and p=0.0112 respectively). The renal cortex and medulla of exposed fetuses showed volume reduction; cortex: 4.46 ± 0.55 x 2.44 ± Doutorado Nilsa Regina Damaceno-Rodrigues 2012 0.79 (p=0.0003); medulla: 2.35 ± 0.43 x 1.24 ± 0.61 (p=0.0034). The absolute number of nephrons in the kidney of exposed fetuses (3301,0±1720,8) was significantly lower (p=0.0082) when compared to the filtered air group (7199,5±2023,5). These data show that pre-natal exposure to ambient levels of air pollution in Sao Paulo entails changes in the development of the kidney, that can lead to illness in adult age.

Glial cells represent the most abundant cell population in the central nervous system (CNS), and yet, have historically been thought of as merely support cells for neurons. Over the past few decades, however, the number of identified roles that glial cells play in the CNS has expanded at an exponential rate, revealing new and exciting functions in neuron-glial communication. At synapses, astrocytes are now recognized as part of a "tripartite" complex with pre- and postsynaptic structures and can modulate synaptic transmission and plasticity. Accumulating evidence has also revealed new roles for oligodendrocytes in regulating axon diameter and integrity, and ion channel clustering. Despite our knowledge of the physiological connections between neurons and glia, relatively little is known about the morphological interplay of these cells during development and in the mature brain. The results presented in this thesis reveal the extent and time-course of rapid remodelling of astrocytes and oligodendrocytes in close proximity to dendritic spines and axons respectively. These findings provide further evidence that glia play an important role in regulating the structural plasticity of the brain. The methodology developed also provides a powerful system for the study of neuron-glial structural dynamics and may contribute to the development of novel therapeutic strategies for diseases affecting the central nervous system.

Myelin plays an essential function in the behaviour of higher organisms by increasing the speed of axonal conduction. Indeed, myelin deficiency or damage can lead to serious motor and sensory dysfunction. In the central nervous system, myelin is produced by a specialized macroglial cell known as the oligodendrocyte. Although much is known about oligodendrocytes with respect to their role in myelin production, many details regarding their development and maintenance still remain unclear. These details may be of great importance for fully understanding the fundamental properties of oligodendrocytes and for devising strategies for treating myelin-related diseases. In this thesis, we report the development of a system using organotypic hippocampal slices, viral gene delivery methods, immunostaining techniques and confocal imaging that can be used to study the properties of oligodendrocytes and myelin. This system preserves many features of the intact brain and can be used to investigate cells of the oligodendrocyte lineage at different developmental stages. Individual oligodendrocytes were targeted using this system and we showed that they can be induced to express various proteins, such as proteolipid protein and neurofascin-155, that localized to specific compartments of oligodendrocytes. This system was then used to address the importance of glutamate receptor signalling on myelin. Studies were performed at the light microscopic level using agonists and antagonists of ionotropic glutamate receptors. Myelin showed progressive pathology over the course of hours following exposure to glutamate receptor agonists and, interestingly, glutamate signalling was not essential for myelin maintenance over a 48 hour period. This thesis work thus describes the use of a novel system that can help analyze both the cellular and molecular aspects of oligodendrocyte development and maintenance.

The ability of Candida albicans to change morphology plays several roles in its virulence and as a human commensal. The yeast-to-hyphae transition is tightly regulated by several sets of activating and repressing pathways. The DNA-binding proteins Rfg1p, Nrg1p and the global repressor Tup1p are part of the repressors found to regulate this morphogenesis. Knowledge of these repressors is based on extrapolations from homology to S. cerevisiae and from expression studies of mutants in inducing conditions, all of which are indirect means of determining a protein's function. We proposed a genome-wide location study of the Nrg1 and Rfg1 transcription factors to obtain direct data to identify their in vivo targets. Our results suggest different avenues for Nrg1p function and a regulation behaviour diverging from the previously suggested model: Nrg1p acts not only as a repressor but also as a transcription activator. Furthermore it regulates its target genes through binding in their coding regions instead binding to the expected regulatory elements on promoters.