In recent years, numerous studies have linked cryopreservation with increased occurrence of mutations, DNA fragmentation and the event of apoptosis in biological objects. However, the evidence emerged from such studies is somewhat inconclusive. The current study, therefore, aimed to analyse the DNA damage response (DDR) from the cryopreserved cells in order to characterise the nature of the putative DNA damage. The study set out to investigate the effects of different cryopreservation parameters on the genome in terms of double strand breaks (DSBs), single strand breaks (SSBs), and various forms of sequence alteration using 5¼ hour post fertilisation (hpf) zebrafish (Danio rerio) embryos. The experimental conditions under which the investigation was carried out were short term chilling at 0˚C, treatment with two cryoprotective agents (CPA), namely, MeOH and Me2SO, and cooling to -35˚C. Assays for detecting DSB-activated DDR proteins and SSB-activated DDR proteins in 5¼ hpf zebrafish (Danio rerio) were developed and then utilised to investigate the occurrence of DSBs and SSBs in the genome of the embryos treated with the experimental conditions. The study then analysed the expression profiles of a set of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) pathways as indicators of the occurrence of various forms of sequence alterations in the genome of the embryos treated with the experimental conditions. It was found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER, respectively. CPA treatment also down-regulated the NER and the MMR mechanisms. Cooling, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling also up-regulated the NER and the BER mechanisms in the embryos. The overall finding of the study indicated that the experimental conditions increased the occurrence of various single stranded DNA lesions in the genome of the embryos. The present study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the current knowledge of the effects of cryopreservation on the genome of biological objects.

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

Cell-to-cell communication is one of the fundamental requisites of making multicellular organisms. G protein-coupled receptors (GPCRs) are one of the most abundant receptor-types within vertebrates. They canonically mediate their signal via hetrotrimeric G proteins, and G protein signaling is regulated by regulators of G protein-signaling (RGS). One of these RGS proteins, RGS4, is preferentially expressed in the central nervous system of humans and has been strongly connected to dopaminergic signaling, along with a number of severe neuronal diseases. rgs4 is not well studied in the model organism Danio rerio, the zebrafish, with only two publications. In this project, a newly constructed transgenic line, tg(rgs4:mCherry), with the fluorophore mCherry regulated by the promoter element of rgs4 was characterized in order to investigate fidelity to endogenous rgs4 expression and the utility of the transgenic line. The mCherry expression is apparent by 48 hours post fertilization, and expression is found mainly in neuronal tissue. Cell bodies are visible only in some labeled areas, while other areas show a more diffuse signal indicative of projections. There is only one transgenically labeled area that also unambiguously expresses rgs4; the pronephric tubule. This line is therefore not particularly well suited for rgs4-specifc studies, but this does not discredit the fidelity of the construct. A transgenic line made with a site-directed technique would most likely confer the fidelity of the promoter to the expression of the fluorophore. A way of increasing the labeling resolution includes exchanging the mCherry fluorophore for one with stronger signal and a lower tendency to aggregate, e.g. eGFP. Increasing the resolution of the characterization, e.g. to the level of sub-nuclei or neuronal types, would serve to enhance the utility of the line. As it is, the tg(rgs4:mCherry) zebrafish line has limited uses, and yet it is not without them.

Zebrafish (Danio rerio) show a unique pigmentation pattern comprised of three pigment cell types: melanophores, iridophores and xanthophores. Other pigmented cells include the retinal pigmented epithelium (rpe) which absorbs excess light in the eye and maintain the extracellular environment around the photoreceptors. While previous mutations in mitfa showed a role in regulating trunk melanophores, the rpe was not affected. TALENs and CRISPR-Cas9 systems were used to generate mutant zebrafish for tfec, a transcription factor expressed in both neural crest and rpe. Embryos with tfec mutations showed a loss of iridophore pigmentation, and delays in the pigmentation of xanthophores and rpe, showing positive regulation of multiple pigment cells. Double mutants for tfec and mitfa displayed greater losses of iridophore, xanthophore and rpe pigmentation with noncircular globes, suggesting cooperative roles for these transcription factors.

Submitted by PPG Biotecnologia Farmac?utica (mpbf@pucrs.br) on 2017-10-05T18:16:51Z No. of bitstreams: 1 DAIANE_BRIDI-DIS.pdf: 2089124 bytes, checksum: c80421954ce799cf7f5b1206f22ec59a (MD5) / Rejected by Caroline Xavier (caroline.xavier@pucrs.br), reason: Devolvido devido ? falta de capa institucional no arquivo PDF, e a publica??o est? cadastrada como "tese" on 2017-10-06T13:39:56Z (GMT) / Submitted by PPG Biotecnologia Farmac?utica (mpbf@pucrs.br) on 2017-10-18T11:48:05Z No. of bitstreams: 1 DAIANE_BRIDI-DIS.pdf: 2205656 bytes, checksum: 0d6301199a5443bf6ca3d070c2d27d6e (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-10-20T12:39:52Z (GMT) No. of bitstreams: 1 DAIANE_BRIDI-DIS.pdf: 2205656 bytes, checksum: 0d6301199a5443bf6ca3d070c2d27d6e (MD5) / Made available in DSpace on 2017-10-20T12:43:25Z (GMT). No. of bitstreams: 1 DAIANE_BRIDI-DIS.pdf: 2205656 bytes, checksum: 0d6301199a5443bf6ca3d070c2d27d6e (MD5) Previous issue date: 2017-08-11 / Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of resistant crops, after its introduction in 1996. Glyphosate can be used alone, but is commonly used as an active ingredient of the Roundup? herbicide. This herbicide contains several adjuvants in addition to glyphosate, which may promote an unknown e.g. toxicity Polioxietilenamida (POEA). Zebrafish is gaining popularity in behavioral research, because of its physiological similarity to mammals, ease of manipulation, robust performance, low cost, external fertilization, transparency of embryos larval stages and rapid development. The aim of this study was to evaluate the effects of glyphosate and Roundup? on behavioral and morphological parameters in zebrafish larvae and adult. Zebrafish larvae at 3 days post-fertilization (dpf) and adults were exposed to glyphosate (0.01, 0.065, and 0.5 mg/L) and Roundup? (0.01, 0.065, and 0.5 mg/L) for 96 hours. Immediately after the treatment, behavioral parameters such as locomotor activity and aversive behavior and morphology for the larvae and locomotion, agressive behavior and memory for the adults were analyzed. Zebrafish larvae, the results indicated that there were significant differences in the locomotor activity and aversive behavior by glyphosate and Roundup? when compared to the control. However, there was a decrease in distance traveled and the time spent in zone without stimulation for exposed larvae at doses of glyphosate and Roundup?. A significant decrease in body lenght was observed for larvae exposed to Roundup? in all concentrations tested. Our findings demonstrated that glyphosate and Roundup? exposure reduced the distance traveled, the mean speed and the line crossings in the highest concentration of glyphosate (0.5 mg / L) and 0.065 and 0.5mg/L Roundup? in animals adults. We verified that Roundup?-treated adult zebrafish showed a significant impairment in memory. Our results showed that glyphosate and Roundup? had an effect on agressive behavior. Our findings demonstrated that the effects of isolated and commercial forms of glyphosate promoted differences on locomotion, behavior and morphology of the treated animal, suggesting similar mechanisms of toxicity and cellular response. / O glifosato tornou-se o herbicida mais utilizado no mundo, devido ? ado??o ampla de culturas resistentes, ap?s sua introdu??o em 1996. O glifosato pode ser usado sozinho, mas ? comumente utilizado como ingrediente ativo do herbicida Roundup?. Este herbicida cont?m v?rios adjuvantes, tal como a Polioxietilenamida (POEA), que podem promover uma toxicidade desconhecida. O peixe-zebra est? ganhando popularidade na pesquisa comportamental, devido ? similaridade fisiol?gica com os mam?feros, facilidade de manipula??o, baixo custo, fertiliza??o externa, transpar?ncia de embri?es nos est?gios larvais e desenvolvimento r?pido. O objetivo deste estudo foi avaliar os efeitos do glifosato e do Roundup? sobre par?metros comportamentais e morfol?gicos em peixe-zebra no est?gio larval e adulto. As larvas com 3 dias p?s-fertiliza??o (dpf) e adultos foram expostos ao glifosato (0,01, 0,065 e 0,5 mg/L) e Roundup? (0,01, 0,065 e 0,5 mg/L) por 96 horas. Imediatamente ap?s o tratamento, realizamos a an?lise de par?metros comportamentais, como atividade locomotora, comportamento aversivo e morfologia para larvas e locomo??o, comportamento agressivo e mem?ria aversiva para adultos. Nas larvas houveram diferen?as significativas na atividade locomotora e comportamento aversivo nos animais tratados com glifosato e Roundup? quando comparado ao controle. Foi observada uma diminui??o na dist?ncia percorrida e na resposta aversiva nas larvas expostas ao glifosato e Roundup?. Observou-se uma diminui??o significativa no comprimento corporal das larvas expostas ao Roundup? em todas as concentra??es testadas. Nossos resultados demonstraram que a exposi??o ao glifosato ou Roundup? reduziu a dist?ncia percorrida, a velocidade m?dia e o n?mero de cruzamentos na maior concentra??o de glifosato (0,5mg/L) e 0,065 e 0,5mg/L de Roundup? em animais adultos. Verificamos que peixe-zebra adulto tratado com Roundup? apresentou um comprometimento significativo na mem?ria. Nossos resultados demostraram que o glifosato e o Roundup? tiveram efeito sobre o comportamento agressivo. Assim, nossos achados demonstraram que os efeitos das formas isoladas e comerciais de glifosato promoveram diferen?as na locomo??o, comportamento e morfologia do animal tratado, sugerindo mecanismos semelhantes de toxicidade e resposta celular.

by Henry, Kam Yin Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 148-160). / ZEBRAFISH (BRACHYDANIO RERIO) / ACKNOWLEDGEMENTS / LIST OF CONTENTS / ABSTRACT / ABBREVIATION / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Transgenic fish --- p.1 / Chapter 1.2 --- Zebrafish --- p.4 / Chapter 1.3 --- The grass carp GH gene and protein / Chapter 1.3.1 --- The genomic sequence --- p.5 / Chapter 1.3.2 --- The cDNA sequence --- p.7 / Chapter 1.3.3 --- The grass carp GH protein --- p.7 / Chapter 1.4 --- Functional aspects of promoter regions / Chapter 1.4.1 --- PEPCK --- p.9 / Chapter 1.4.2 --- RSV-LTR --- p.10 / Chapter 1.4.3 --- hMT-IIA --- p.10 / Chapter 1.4.4 --- MMTV-LTR --- p.11 / Chapter 1.5 --- Eukaryotic gene expression in cultured cells / Chapter 1.5.1 --- COS-7 and HepG2 cells --- p.11 / Chapter 1.5.2 --- Transfection system --- p.12 / Chapter 1.5.3 --- Fate of DNA after transfection --- p.13 / Chapter 1.6 --- Electroporation and microinjection as tools for gene transfer / Chapter 1.6.1 --- Electroporation: Theory and operation --- p.13 / Chapter 1.6.2 --- Microinjection: Design of microinjector --- p.16 / Chapter 1.6.3 --- Fate of DNA after gene transfer in embryos / Transient expression --- p.16 / Stable transformation --- p.17 / Inheredity of transgene --- p.17 / Chapter 1.7 --- The aims of the present study --- p.18 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Electrophoresis of DNA / Chapter 2.1.1.1 --- Agarose gel electrophoresis --- p.19 / Chapter 2.1.1.2 --- PAGE --- p.20 / Chapter 2.1.2 --- Purification of DNA --- p.21 / Chapter 2.1.3 --- Recovery of DNA fragments / Chapter 2.1.3.1 --- Electroelution --- p.22 / Chapter 2.1.3.2 --- Geneclean kit --- p.23 / Chapter 2.1.4 --- Standard recombinant DNA techniques / Chapter 2.1.4.1 --- Dephosphorylation --- p.24 / Chapter 2.1.4.2 --- Kinasing --- p.24 / Chapter 2.1.4.3 --- Ligation --- p.24 / Chapter 2.1.4.4 --- Filling in reaction --- p.25 / Chapter 2.1.4.5 --- Transformation --- p.25 / Chapter 2.1.5 --- Minipreparation of plasmids --- p.26 / Chapter 2.1.6 --- Large preparation of plasmids / Chapter 2.1.6.1 --- Qiagene kit --- p.27 / Chapter 2.1.6.2 --- CsCl density gradient centrifugation --- p.27 / Chapter 2.1.7 --- DNA sequencing --- p.29 / Chapter 2.1.8 --- "Extraction of DNA from embryos, fry and fish" / Method 1 --- p.32 / Method 2 --- p.32 / Chapter 2.1.9 --- Probe labelling / Chapter 2.1.9.1 --- End-labelling --- p.33 / Chapter 2.1.9.2 --- Random priming --- p.33 / Chapter 2.1.10 --- CAT assay --- p.33 / Chapter 2.1.11 --- Polymerase chain reaction(PCR) --- p.35 / Chapter 2.1.12 --- Radioimmunassay(RIA) of FGH --- p.36 / Chapter 2.1.13 --- Dot blotting --- p.38 / Chapter 2.1.14 --- Southern blotting --- p.39 / Chapter 2.2 --- "Linkers, primers and probes" / Chapter 2.2.1 --- Primers --- p.41 / Chapter 2.2.2 --- Linkers --- p.45 / Chapter 2.2.3 --- Probes --- p.47 / Chapter 2.3 --- Construction of chimeric growth hormone genes / Chapter 2.3.1 --- Sources of plasmids --- p.50 / Chapter 2.3.2 --- General principles --- p.50 / Chapter 2.3.3 --- PEPCKgcGHcDNA --- p.51 / Chapter 2.3.4 --- RSVgcGHcDNA --- p.54 / Chapter 2.3.5 --- hMTgcGHcDNAcDNA --- p.56 / Chapter 2.3.6 --- MMTVgcGHcDNA --- p.58 / Chapter 2.3.7 --- "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.60 / Chapter 2.4 --- Expression of chimeric genes in cultured cells / Chapter 2.4.1 --- Culturing of COS-7 and HepG2 cells --- p.66 / Chapter 2.4.2 --- Expression of chimeric genes in COS-7 and HepG2 cells --- p.67 / Chapter 2.5 --- Zebrafish / Chapter 2.5.1 --- "Culturing, Spawning and hatching" --- p.67 / Chapter 2.6 --- Electroporation and microinjection for gene transfer / Chapter 2.6.1 --- Electroporation / Chapter 2.6.1.1 --- Tuning up electroporation --- p.69 / Chapter 2.6.1.2 --- Evidence of gene transfer by electroporation / Chapter 2.6.1.2.1 --- CAT assay --- p.71 / Chapter 2.6.1.2.2 --- Dot blot --- p.71 / Chapter 2.6.1.2.3 --- PCR and Southern blotting of PCR products --- p.72 / Chapter 2.6.1.2.4 --- Southern blotting of fish total DNA --- p.73 / Chapter 2.6.2 --- Microinjection / Chapter 2.6.2.1 --- Handling of microinjection --- p.74 / Chapter 2.6.2.2 --- Evidence of gene transfer by microinjection / Chapter 2.6.2.2.1 --- CAT assay --- p.75 / Chapter 2.6.2.2.2 --- PCR and Southern blotting of PCR products --- p.75 / Chapter 2.7 --- Phenotypic alteration of fish generated from electroporated eggs / Chapter 2.7.1 --- Electroporation and handling of fish generated from electroporation --- p.75 / Chapter 2.7.2 --- Measurement of phenotypic change in fish generated from electroporation --- p.77 / Chapter 2.8 --- Detection of transgene and expression of exogenous DNA / Chapter 2.8.1 --- Transgene detection --- p.78 / Chapter 2.8.2 --- Expression of exogenous DNA --- p.79 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Construction of Chimeric growth hormone genes / Chapter 3.1.1 --- Confirmation of integrity of chimeric genes / PEPCKgcGHcDNA --- p.80 / RSVgcGHcDNA --- p.81 / hMTgcGHcDNA --- p.81 / MMTVgcGHcDNA --- p.81 / "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.82 / Chapter 3.1.2 --- Yield of chimeric genes from CsCl density gradient centrifugation --- p.82 / Chapter 3.2 --- Chimeric gene expression in COS-7 and HepG2 cells / Chapter 3.2.1 --- Expression of chimeric genes in COS-7 cells --- p.89 / Chapter 3.2.2 --- Expression of chimeric genes in HepG2 cells --- p.93 / Chapter 3.3 --- Transfer of chimeric genes into embryos / Chapter 3.3.1 --- Electroporation / Chapter 3.3.1.1 --- Monitoring of electroporation --- p.94 / Chapter 3.3.1.2 --- Evidence for gene transfer / Chapter 3.3.1.2.1 --- CAT assay --- p.98 / Chapter 3.3.1.2.2 --- Dot blotting --- p.98 / Chapter 3.3.1.2.3 --- PCR and Southern blotting of PCR product --- p.101 / Chapter 3.3.1.2.4 --- Southern blotting of DNA from fish generated from electroporation --- p.106 / Chapter 3.3.2 --- Microinjection / Chapter 3.3.2.1 --- CAT assay --- p.109 / Chapter 3.3.2.2 --- PCR --- p.109 / Chapter 3.4 --- Phenotypic alterations of fish / The first experiment --- p.112 / The second experiment --- p.113 / The third experiment --- p.113 / The fourth experiment --- p.122 / Chapter 3.5 --- Detection of transgene and expression of exogenous DNA / Chapter 3.5.1 --- Transgene --- p.128 / Chapter 3.5.2 --- Possible expression of exogenous DNA --- p.129 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Chimeric growth hormone genes --- p.132 / Chapter 4.2 --- Expression of chimeric growth hormone genes in COS-7 and HepG2 cells --- p.134 / Chapter 4.3 --- Transfer of exogenous DNA into embyros --- p.136 / Chapter 4.4 --- Phenotypic alteration of fish developed from electroporated eggs --- p.139 / Chapter 4.5 --- The possible integration and expression of exogenous DNA --- p.143 / Chapter 4.6 --- Conclusions --- p.145 / Chapter 4.7 --- Suggestions for further studies --- p.146 / REFERENCES --- p.148 / Chapter APPENDIX I --- Restriction maps / PEPCKgcGH / PEPCKgcGHcDNA / RSVgcGH / RSVgcGHcDNA / hMTgcGH / hMTgcGHcDNA / MMTVgcGHcDNA / pBH1.2 / pMSG-CAT / pUC19 / hMT-IIA / PBC12BI / RSVCAT / pUC101 / pSEl/S2 / PUCSE2/S1 / pUCS2

Kwok Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 84-100). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.vii / List of figures --- p.xi / List of tables --- p.xiv / Symbols and abbreviations --- p.xv / List of fish names mentioned in the thesis --- p.xviii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Gonadotropins / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.4 / Chapter 1.2 --- Gonadotropin receptor / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.2 --- Expression --- p.7 / Chapter 1.2.3 --- Signaling / Chapter 1.2.3.1 --- cAMP-mediated pathway --- p.7 / Chapter 1.2.3.2 --- Phospholipase C-mediated pathway --- p.9 / Chapter 1.2.4 --- Regulation of expression --- p.12 / Chapter 1.2.5 --- Desensitization of receptors / Chapter 1.2.5.1 --- Uncoupling --- p.13 / Chapter 1.2.5.2 --- Internalization --- p.13 / Chapter 1.3 --- Structure of ovarian follicles --- p.14 / Chapter 1.4 --- The project objectives and long-term significance --- p.16 / Chapter Chapter 2 --- Cloning and Characterization of Zebrafish Follicle-stimulating Hormone (FSH) and Luteinizing Hormone (LH) Receptors ´ؤ Evidence for Distinct Functions of FSH and LH in Follicle Development / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Animals and chemicals --- p.22 / Chapter 2.2.2 --- Isolation of total RNA --- p.22 / Chapter 2.2.3 --- Cloning of zebrafish FSHR (zfFSHR) and LHR (zfLHR) cDNA fragments from the zebrafish ovary --- p.23 / Chapter 2.2.4 --- Rapid amplification of 5´ةcDNA ends (5'-RACE) and full-length cDNA --- p.24 / Chapter 2.2.5 --- Isolation of ovarian follicles --- p.25 / Chapter 2.2.6 --- Sampling of the ovaries from sexually immature zebrafish --- p.25 / Chapter 2.2.7 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.8 --- Construction of expression plasmids --- p.26 / Chapter 2.2.9 --- Transient transfection and reporter gene assay --- p.27 / Chapter 2.2.10 --- Establishment and characterization of stable zfFSHR or zfLHR-expressing cell lines --- p.28 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Cloning of FSHR and LHR cDNA from the zebrafish ovary --- p.29 / Chapter 2.3.2 --- Functional characterization of zfFSHR and zfLHR --- p.30 / Chapter 2.3.3 --- Expression of zfFSHR and zfLHR during sexual maturation --- p.31 / Chapter 2.3.4 --- Stage-dependent expression of zfFSHR and zfLHR in the ovarian follicles --- p.32 / Chapter 2.4 --- Discussion --- p.33 / Chapter Chapter 3 --- Down-regulation of FSHR and LHR Expression in the Zebrafish Follicle Ceils by Gonadotropin (hCG) and Its Sigaling Mechanism / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Animals --- p.54 / Chapter 3.2.2 --- Chemicals and hormones --- p.54 / Chapter 3.2.3 --- Primary follicle cell culture --- p.55 / Chapter 3.2.4 --- Total RNA isolation --- p.55 / Chapter 3.2.5 --- "Validation of semi-quantitative RT-PCR assays for FSHR, LHR and GAPDH" --- p.56 / Chapter 3.2.6 --- Data analysis --- p.57 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.57 / Chapter 3.3.2 --- Gonadotropin regulation of FSHR and LHR expression in cultured zebrafish ovarian follicle cells --- p.58 / Chapter 3.3.3 --- Effect of db-cAMP and forskolin on FSHR and LHR expression --- p.59 / Chapter 3.3.4 --- Effects of H89 on hCG-induced suppression of FSHR and LHR expression --- p.60 / Chapter 3.4 --- Discussion --- p.60 / Chapter Chapter 4 --- General Discussion --- p.75 / Chapter 4.1 --- Cloning of zebrafish FSHR and LHR cDNAs and demonstration of receptor specificity --- p.77 / Chapter 4.2 --- Evidence for the differential expression of FSHR and LHR in the zebrafish ovarian and follicle development --- p.78 / Chapter 4.3 --- Down-regulation of FSHR and LHR expression in the zebrafish follicle cells by gonadotropin (hCG) --- p.79 / Chapter 4.4 --- Future research direction --- p.80 / References --- p.84

So Wai-Kin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 100-127). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract (in English) --- p.II / Abstract (in Chinese) --- p.IV / Table of contents --- p.VI / List of Figures --- p.X / Symbols and Abbreviations --- p.XII / List of fish names mentioned in the thesis --- p.XIV / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Pituitary --- p.1 / Chapter 1.2 --- Gonadotropins --- p.1 / Chapter 1.2.1 --- Structure --- p.2 / Chapter 1.2.2 --- Signaling --- p.3 / Chapter 1.2.3 --- Expression --- p.5 / Chapter 1.2.4 --- Functions --- p.7 / Chapter 1.2.4.1 --- Gonadotropin actions on gametogenesis --- p.7 / Chapter 1.2.4.2 --- Gonadotropin actions on steroidogenesis --- p.8 / Chapter 1.2.5 --- Regulation --- p.9 / Chapter 1.2.5.1 --- Neuroendocrine control --- p.10 / Chapter 1.2.5.1.1 --- Gonadotropin-releasing hormone (GnRH) --- p.10 / Chapter 1.2.5.1.2 --- Dopamine (DA) --- p.12 / Chapter 1.2.5.2 --- Gonadal steroid feedback --- p.12 / Chapter 1.2.5.2.1 --- Positive feedback --- p.13 / Chapter 1.2.5.2.2 --- Negative feedback --- p.14 / Chapter 1.2.5.3 --- Paracrine regulators within pituitary --- p.15 / Chapter 1.3 --- Objectives of the present study --- p.16 / Chapter Chapter 2 --- "Molecular Cloning and Functional Characterization of Zebrafish FSHβ, LHβ and GTHα subunits" / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and methods --- p.21 / Chapter 2.2.1 --- Chemicals --- p.21 / Chapter 2.2.2 --- Animals --- p.21 / Chapter 2.2.3 --- Genomic DNA isolation --- p.22 / Chapter 2.2.4 --- Total RNA isolation --- p.22 / Chapter 2.2.5 --- Cloning of zebrafish FSHp，LHβ and GTHa fragments --- p.23 / Chapter 2.2.5.1 --- LHβ and GTHα --- p.23 / Chapter 2.2.5.2 --- FSHβ --- p.23 / Chapter 2.2.6 --- "5'- and 3'-RACE of zebrafish FSHp, LHβ and GTHα subunits" --- p.24 / Chapter 2.2.7 --- Construction of expression constructs --- p.25 / Chapter 2.2.8 --- Cell culture and transfection of Flp-In´ёØ CHO cell --- p.26 / Chapter 2.2.9 --- Recombinant production of zebrafish FSH and LH --- p.27 / Chapter 2.2.10 --- Reverse transcription-polymerase chain reaction (RT-PCR) analysis --- p.27 / Chapter 2.2.11 --- Northern blot hybridization --- p.28 / Chapter 2.2.12 --- SEAP reporter gene assay --- p.28 / Chapter 2.2.13 --- Data analysis --- p.29 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- "Cloning of zebrafish FSHβ, LHβ and GTHα subunits" --- p.30 / Chapter 2.3.2 --- "Expression of zebrafish FSHp, LHβ and GTHα in the zebrafish pituitary" --- p.31 / Chapter 2.3.3 --- Recombinant production of zebrafish FSH and LH --- p.32 / Chapter 2.3.4 --- Functional analysis of zebrafish FSH and LH --- p.33 / Chapter 2.4 --- Discussion --- p.34 / Chapter Chapter 3 --- "Spatial Expression Patterns of Zebrafish FSHβ, LHβ and GTHα Subunits in the Pituitary and Their Temporal Expression Profiles during Sexual Maturation and Ovulatory Cycle" / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and methods --- p.61 / Chapter 3.2.1 --- Chemicals --- p.61 / Chapter 3.2.2. --- Animals --- p.62 / Chapter 3.2.3 --- Total RNA isolation from zebrafish pituitaries and reverse transcription --- p.62 / Chapter 3.2.4 --- Validation of RT-PCR on single pituitary --- p.63 / Chapter 3.2.5 --- Real-time PCR --- p.64 / Chapter 3.2.6 --- Tissue preparation for in situ hybridization --- p.64 / Chapter 3.2.7 --- In situ hybridization --- p.65 / Chapter 3.2.8 --- Data analysis --- p.66 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- "PCR amplification of FSHβ, LHβ and GTHα and GAPDH in single zebrafish pituitary" --- p.67 / Chapter 3.3.2 --- "Establishement of real-time RT-PCR for zebrafish FSHβ, LHβ and GTHa and GAPDH" --- p.67 / Chapter 3.3.3 --- "Temporal expression profiles of zebrafish FSHβ, LHβ and GTHα subunits during sexual maturation" --- p.67 / Chapter 3.3.4 --- "Temporal expression profiles of zebrafish FSHp, LHβ and GTHα subunits during ovulatory cycle" --- p.68 / Chapter 3.3.5 --- "In situ hybridization of zebrafish FSHβ, LHβ and GTHα" --- p.69 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- General Discussion / Chapter 4.1 --- Cloning of zebrafish gonadotropin subunit cDNAs --- p.91 / Chapter 4.2 --- Bioactivity and receptor specificity of recombinant zebrafish FSH and LH --- p.91 / Chapter 4.3 --- Expression of gonadotropin subunits during zebrafish sexual maturation and ovulatory cycle --- p.92 / Chapter 4.4 --- "Localization of FSHβ, LHβ and GTHα subunits in zebrafish pituitary" --- p.93 / Chapter 4.5 --- Contributions of the present study --- p.94 / Chapter 4.6 --- Future prospects --- p.95 / References --- p.100