Thesis (Ph. D. in Biological Sciences)--Vanderbilt University, Aug. 2009. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--University of Alabama at Birmingham, 2009. / Title from PDF t.p. (viewed July 19, 2010). Includes bibliographical references.

A busca pelo homem por uma vida saudável tem impulsionado pesquisas por novas substâncias capazes de atender tal desejo. O composto fenólico resveratrol (3, 4\', 5- trihidroxiestilbeno) é uma dessas substâncias que apresenta uma variedade de ações farmacológicas, como potencial antioxidante, capacidade antiinflamatória, proteção contra doenças cardíacas e câncer. Apesar dos inúmeros estudos sobre os benefícios do resveratrol à saúde, há poucos dados na literatura sobre sua toxicidade em organismos aquáticos, e principalmente sua concentração no ambiente, tornando o presente estudo fundamental para a contribuição de informações sobre a ecotoxicidade do resveratrol no ambiente aquático. O presente estudo avaliou a toxicidade do resveratrol em embriões e larvas de Danio rerio (zebrafish). Para isso foi realizado o ensaio in vitro de citotoxicidade do resveratrol, ensaios de ecotoxicidade e ensaio de biomarcadores enzimáticos. A avaliação do resveratrol por cromatografia líquida de alta pressão (HPLC) também foi realizada. De acordo com os resultados obtidos, o índice de citotoxicidade (IC50), concentração do resveratrol que causou a morte de 50% das células da linhagem NCTC-L929 foi de 39 mg L-1. A concentração de resveratrol que causa mortalidade em 50% dos organismos expostos (CL50), nos ensaios de ecotoxicidade crônica de curta duração com larvas do peixe Danio rerio foi de 51,37 mg L-1. A CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 96 h de duração foi de 75,33 mg L-1 e a CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 168 h de duração foi de 50,87 mg L-1. Nas concentrações mais elevadas de resveratrol foram observadas deformidades em embriões e larvas. O resveratrol alterou as atividades das enzimas LDH e ChE no estágio embriolarval de Danio rerio. Na análise do resveratrol por HPLC não foi observado degradação do composto. / The concern about human being healthy life has driven researchers to study new compounds capable of reaching that desire. Resveratrol (3, 4\', 5 trihydroxystilbene) a phenolic compound, is one of these substances which presents a variety of pharmacological actions, as antioxidant potential, antiinflammatory capacity, protection against heart and cancer diseases. Despite the numerous studies on the benefits of resveratrol to health, there is little evidence in the literature of its toxicity to aquatic organisms, and especially its concentration in the environment, making the present study fundamental for the contribution of information on the ecotoxicity of resveratrol in the aquatic environment. The present study evaluated the toxicity of resveratrol in embryos and larvae of Danio rerio (zebrafish). For this purpose the in vitro cytotoxicity of resveratrol assay, ecotoxicity assays and enzyme biomarker assay were performed. The evaluation of resveratrol by high pressure liquid chromatography (HPLC) was also performed. According to the results, the cytotoxicity index (IC50), concentration of resveratrol that caused the death of 50% of the cells of the NCTC-L929 lineage was 39 mg L-1. The concentration of resveratrol that causes mortality in 50% of exposed organisms (LC50) in the short-lived chronic ecotoxicity assays with larvae of the Danio rerio fish was 51.37 mg L-1. The LC50 obtained in the embryo-active acute ecotoxicity test of the Danio rerio fish with 96 h duration was 75.33 mg L-1 and the LC50 obtained in the embryo-active acute ecotoxicity assay of the Danio rerio fish with 168 h duration was 50.87 mg L-1. At higher concentrations of resveratrol deformities were observed in embryos and larvae. Resveratrol altered the activities of LDH and ChE enzymes in the embryonal stage of Danio rerio. No degradation of resveratrol was observed in the HPLC analysis of compound.

Our initial studies reported on the cloning and characterization of FoxP3 (T regulatory cell specific) for the first time in any teleost species along with T-bet and STAT6 in zebrafish, which are specific to Th1 and Th2 cells respectively and looked at their expression in the zebrafish.  Our next studies focused on CD4 expression by T cells and addresses the question whether teleost Th cells are functionally similar to their mammalian counterpart.  A CD4 specific antibody was developed and using molecular techniques have been able to characterise for the first time CD4+ cells within the zebrafish. In addition, to the CD4L gene, three other novel CD4 related genes have also been discovered which need to be investigated, which could mean in fish different T helper cell populations exist expressing CD4 related molecules.  Another FoxP3 gene was discovered, a factor important in controlling the differentiation and function of T regulatory (Treg) cells, and so we shifted our investigations to studying these FoxP3 molecules within the zebrafish model.  These two orthologs of mammalian FoxP3 were only found in zebrafish and it appears that higher vertebrates including modern fish only have one copy of the FoxP3 gene.  Through the development of an antibody we were also able to look at the expression of the zebrafish genes within cells and determine where they were being expressed.  Lastly, having all the molecular tools that were developed during the course of this thesis, allowed us to use <i>in vivo </i>zebrafish models of <i>Mycobacterium marinum </i>and <i>Salmonella typhimurium </i>infection, and attempt to determine the types of adaptive immune responses that were being developed. Understanding the regulatory mechanisms behind the teleost adaptive immune response will help to establish the zebrafish model for vertebrate immune function.

CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...

Shi, Yujian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 83-101). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

在哺乳動物中，表皮生長因子(EGF)家族及其同源受體(EGFR)家族是卵泡生成的重要調節者。表皮生長因子受體信號轉導不但在促黃體激素誘導的卵母細胞成熟和排卵中是必不可少的，而且也通過與促性腺激素互相作用而影響卵泡的類固醇生產和生存。表皮生長因子對卵母細胞成熟的促進作用，也保存在金魚和斑馬魚等硬骨魚類。然而，在硬骨魚類中，有關表皮生長因子家族功能的知識仍然是非常有限的。在本研究中，我們的目的如下: (1)試圖找出那些表皮生長因子受體信號轉導路徑傳達表皮生長因子對卵母細胞成熟; (2)表皮生長因子受體和促黃體激素的潛在關係; (3)表皮生長因子和其他身體激素之間的相互作用。所有發現都可以幫助我們更系統地更了解表皮生長因子在斑馬魚卵泡發育中的作用。 / 我們實驗室先前的研究表明，表皮生長因子促進斑馬魚卵母細胞的成熟是通過激活素系統，但不知道其通過什麼細胞內信號轉導路徑調節激活素系統的表達。在這裡，我們使用斑馬魚卵泡培養和藥理抑製劑來研究幾個熟知的表皮生長因子受體轉導信號在表皮生長因子刺激激活素亞基表達的作用。我們發現，有絲分裂原活化蛋白質激酶3/1(MAPK3/1)，p38有絲分裂原活化蛋白質激酶 (p38MAPK)，蛋白激酶C(PKC)和蛋白激酶A(PKA)在抑制素βAA(inhbaa)和抑制素βB(inhbb)亞基表達中起到刺激作用，但對抑制素βAB(inhbab)的表達起抑制作用。另一方面，只有磷脂酰肌醇3激酶(PI3K)調節表皮生長因子對抑制素βB的表達，而它沒有對抑制素βAA和抑制素βB亞基的表達起任何淨影響。 / 除了表皮生長因子在斑馬魚卵巢自己產生的作用， 探討表皮生長因子和其他因素之間的相互作用也是很重要的，因為卵泡是由多種激素和彼此互動的因素控制。 / 在雌性哺乳動物的生殖中，表皮生長因子受體轉導促黃體激素的作用是十分重要的，這使我們很感興趣研究這種情況是否在斑馬魚一樣發生。 和哺乳動物不同，人绒毛膜促性腺激素(hCG)沒有激活斑馬魚表皮生長因子受體。另一方面，和哺乳動物相似，人绒毛膜促性腺激素誘導有絲分裂原活化蛋白質激酶3 / 1在斑馬魚卵泡細胞中的磷酸化，但它是通過蛋白激酶A不是表皮生長因子受體的。此外，在雌性哺乳動物中，人绒毛膜促性腺激素對有絲分裂原活化蛋白質激酶3 / 1的作用是使卵母細胞成熟, 但這作用可能不在斑馬魚中發生，因為它負調控抑制素βAB的表達。 / 表皮生長因子在斑馬魚卵巢的另一個有趣地方是胰島素的存在改變它對調節芳香化酶表達中的作用。表皮生長因子對芳香化酶表達起輕微的抑制效果，但它提高了胰島素對芳香化酶表達的刺激作用。此外，胰島素，促性腺激素和表皮生長因子一起的時候，更加增加了芳香化酶的表達。然而，表皮生長因子抑制佛司可林(FK)對芳香化酶和抑制素αmRNA表達的刺激作用。 / 兩者合計，我們提供了一些基本但重要的信息有關表皮生長因子及其受體在斑馬魚卵巢的功能，包括每個表皮生長因子受體信號轉導信號路徑的分子機制及它們參與調節激活素亞基的表達；表皮生長因子，促黃體激素和胰島素之間的相互作用。這些信息可以讓我們進一步探討表皮生長因子在斑馬魚卵泡以及其他脊椎動物的重要角色。 / In mammals, epidermal growth factor (EGF) family and its cognate receptor (EGFR) family are the key regulators of folliculogenesis. EGFR signaling not only is indispensable in luteinizing hormone-induced oocyte maturation and ovulation, but also influences steroidogenesis and survival of follicles via interacting with gonadotropins. The positive role of EGF on oocyte maturation is shown to conserve in teleosts such as goldfish and zebrafish. However, the knowledge about the functions of EGF family in teleosts is still very limited. In the present study, we sought to find out the EGFR signaling pathways that mediate the action of EGF on oocyte maturation, the potential relationship of EGFR and LH and the interaction between EGF and systemic hormones. All these findings help us to understand more about the role of EGF in zebrafish folliculogenesis in a more systematic manner. / Our lab previously demonstrate that EGF promotes oocyte maturation in zebrafish via activin system but the contribution of the EGFR signaling pathways in regulating the activin system is not known. Here, we investigated the role of several well-known EGFR signaling cascades in EGF-stimulated activin subunit expression using cultured zebrafish follicle and pharmacological inhibitors. We found that MAPK3/1, p38 MAPK, PKC and PKA played stimulatory role in the expression of inhbaa and inhbb but suppressive role in inhbab. On the other hand, only PI3K was found to mediate the action of EGF on the expression of inhbab while it did not have any net effect on the expression of inhbaa and inhbb. / Apart from its own action in zebrafish ovary, it is essential to investigate the interaction or cross-talk between EGF and other factors because folliculogenesis is controlled by many hormones and factors interacting with each other. / The vital mediatory action of EGFR on LH in female reproduction of mammals was led us to ask whether the situation is the same as in zebrafish. However, unlike in mammals, hCG did not transactivate zebrafish EGFR. On the other hand, similar to mammals, hCG did induce MAPK3/1 phosphorylation in zebrafish follicle cells but it was via PKA dependent but not EGFR dependent manner. Moreover, hCG-induced MAPK3/1 activation is involved in oocyte maturation in mammals while in zebrafish, the function of this activation might not be the same as in mammals because it negatively regulated the expression of inhbab. / Another interesting issue about EGF in zebrafish ovary is that its role in regulating the expression of aromatase in response to insulin. EGF alone has slight suppressive effect on aromatase expression but it boosted the stimulatory effect of insulin on aromatase expression. Furthermore, insulin, hCG and EGF together even increased the expression of aromatase. However, EGF always suppressed the stimulatory effect of forskolin on aromatase and inhibin alpha mRNA expression. / Taken together, we provided some fundamental but important information about the function of EGF and its receptor in zebrafish ovary by elucidating the molecular mechanism of each EGFR signaling pathways involved in regulating the expression of activins subunits, the interaction between EGF, LH and insulin. All these studies not only allow us to further explore the elegant roles of EGF in zebrafish folliculogenesis, but also in other vertebrates in the future. / 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. / Chung, Chi Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 116-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract in English --- p.I / Abstract in Chinese --- p.III / Acknowledge --- p.V / Table of contents --- p.VI / List of figures and tables --- p.XI / Symbols and abbreviations --- p.XVI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Folliculogenesis --- p.1 / Chapter 1.1.1 --- In mammals --- p.1 / Chapter 1.1.2 --- In teleosts --- p.2 / Chapter 1.1.2.1 --- Vitellogenesis --- p.3 / Chapter 1.1.2.2 --- Oocyte maturation --- p.3 / Chapter 1.2 --- Epidermal growth factor receptor family --- p.4 / Chapter 1.2.1 --- Structure --- p.4 / Chapter 1.2.2 --- Signal transduction pathways --- p.5 / Chapter 1.2.2.1 --- MEK1/2-MAPK3/1 pathway --- p.6 / Chapter 1.2.2.2 --- p38 MAPK pathway --- p.6 / Chapter 1.2.2.3 --- PI3K pathway --- p.7 / Chapter 1.2.2.4 --- PKC pathway --- p.7 / Chapter 1.2.3 --- Reproductive function of EGFR signaling --- p.8 / Chapter 1.2.3.1 --- In mammals - mediator of luteinizing hormone --- p.9 / Chapter 1.2.3.2 --- In teleosts --- p.10 / Chapter 1.3 --- Interaction of EGF family with other hormones --- p.11 / Chapter 1.4 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- The Involvement of Diverse EGFR Signaling Pathways in Regulating the Expression of Activin Subunits in Zebrafish / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Materials and methods --- p.16 / Chapter 2.2.1 --- Animals and chemicals --- p.16 / Chapter 2.2.2 --- Primary zebrafish follicle cell culture --- p.17 / Chapter 2.2.3 --- Follicle incubation --- p.18 / Chapter 2.2.4 --- Immunoblotting --- p.18 / Chapter 2.2.5 --- Immunohistochemistry --- p.18 / Chapter 2.2.6 --- RNA extraction and reverse transcription --- p.19 / Chapter 2.2.7 --- Real-time quantitative polymerase chain reaction (qPCR) --- p.20 / Chapter 2.2.8 --- Data analysis --- p.20 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Recombinant human EGF up-regulated the expression of all known activin subunits in zebrafish ovary via specific activation of zebrafish EGFR --- p.21 / Chapter 2.3.2 --- MAPK3/1, p38 MAPK, PKC and PKA played similar roles in regulating EGF-stimulated expression of activin subunits --- p.22 / Chapter 2.3.3 --- PI3K pathway was involved in EGF-stuimulated expression of inhbab but not inhbaa and inhbb in zebrafish follicle cells --- p.24 / Chapter 2.3.4 --- MAPK3/1, p38 MAPK and PKC all together were sufficient to mediate the action of EGF on the expression of all activin subunits in zebrafish follicle cells --- p.24 / Chapter 2.3.5 --- Signaling cross-talk between PKA, MAPK3/1 and PI3K in zebrafish follicle cells --- p.25 / Chapter 2.3.6 --- EGFR signaling in the intact zebrafish follicles --- p.25 / Chapter 2.3.7 --- EGF and hCG togher up-regulatory the expression of inhbab synergistically in zebrafish follicle cells --- p.25 / Chapter 2.4 --- Discussion --- p.26 / Chapter Chapter 3 --- hCG-induced MAPK3/1 Phosphorylation in the Zebrafish Follicle Cells is Independent of EGFR Activation / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and methods --- p.54 / Chapter 3.2.1 --- Animals and chemicals --- p.54 / Chapter 3.2.2 --- Primary zebrafish follicle cell culture --- p.54 / Chapter 3.2.3 --- Immunoblotting --- p.54 / Chapter 3.2.4 --- RNA extraction and reverse transcription --- p.54 / Chapter 3.2.5 --- Real-time quantitative polymerase chain reaction (qPCR) --- p.54 / Chapter 3.2.6 --- Data analysis --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- hCG stimulated MAPK3/1 phosphorylation in zebrafish follicle cells --- p.54 / Chapter 3.3.2 --- hCG-induced MAPK3/1 phosphorylation was PKA-dependent but EGFR-independent --- p.55 / Chapter 3.3.3 --- Role of hCG-induced MAPK3/1 activation in regulating the expression of activin subunits --- p.55 / Chapter 3.4 --- Discussion --- p.56 / Chapter Chapter 4 --- Interaction between EGF, hCG and insulin on the Expression of inha and cyp19a1a in Zebrafish Follicles - a Potential Link between Nutrition and Reproduction / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and chemicals --- p.69 / Chapter 4.2.1 --- Animals and chemicals --- p.69 / Chapter 4.2.2 --- Primary zebrafish follicle cell culture --- p.69 / Chapter 4.2.3 --- Follicle incubation --- p.69 / Chapter 4.2.4 --- Immunhistochemistry --- p.69 / Chapter 4.2.5 --- RNA extraction and reverse transcription --- p.70 / Chapter 4.2.6 --- Real-time quantitative polymerase chain reaction (qPCR) --- p.70 / Chapter 4.2.7 --- Data analysis --- p.70 / Chapter 4.3 --- Results --- p.70 / Chapter 4.3.1 --- Recombinant human insulin up-regulated the expression of inha and cyp19a1a in zebrafish follicles --- p.70 / Chapter 4.3.2 --- Interaction between insulin and hCG on the expression of cyp19a1a and inha in intact zebrafish follicles and follicle cells --- p.71 / Chapter 4.3.3 --- Interaction of cAMP pathway and insulin in the regulation of cyp19a1a and inha in zebrafish follicle and zebrafish follicle cells --- p.72 / Chapter 4.3.4 --- Differential effect of EGF on insulin, hCG and FK-regulated expression of cyp19a1a and inha in zebrafish follicles and follicle cells --- p.72 / Chapter 4.3.5 --- Insulin activated Akt pathway in both follicle cells and oocyte in zebrafish follicles --- p.73 / Chapter 4.4 --- Discussion --- p.73 / Chapter Chapter 5 --- General Discussion --- p.96 / Chapter Appendix --- Chapter Phosphorylation of MAPK3/1 in the Follicle in Vivo Evidence for Roles of MAPK3/1 in the Two Compartments of Follicles during Final Maturation and Ovulation / Introduction --- p.104 / Materials and methods --- p.105 / Animals and chemicals --- p.105 / Immunohistochemistry --- p.105 / Results --- p.105 / Discussion --- p.106 / References --- p.116

Chan Chung Yiu Patrick. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-151). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Abbreviations --- p.xv / Chapter CHAPTER 1 --- Literature review --- p.1 / Chapter 1.1 --- Aquatic heavy metal contaminations --- p.1 / Chapter 1.1.1 --- Biology of heavy metals --- p.1 / Chapter 1.1.2 --- Mechanism of heavy metal toxicity --- p.2 / Chapter 1.2 --- Environmental monitoring of aquatic heavy metal contaminations --- p.4 / Chapter 1.2.1 --- Bioconcentration effects of heavy metals --- p.4 / Chapter 1.2.2. --- The concept of bioindicator in pollution assessment --- p.5 / Chapter 1.3 --- Metallothioneins --- p.7 / Chapter 1.3.1 --- Biological functions of MT and its metal inducibility --- p.7 / Chapter 1.4 --- Zebrafish (Daino reio) as an animal model --- p.11 / Chapter 1.4.1 --- Biology of zebrafish --- p.11 / Chapter 1.4.2 --- Current applications of transgenic zebrafish --- p.12 / Chapter 1.5 --- The use of cell culture systems in toxicology research --- p.13 / Chapter 1.6 --- Project aims --- p.16 / Chapter CHAPTER 2 --- Quantification of zMT mRNA levels using real-time PCR --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.1.1 --- The use of zebrafish embryos in toxicity assessment --- p.17 / Chapter 2.1.2 --- MT mRNA as a bioindicator of metal exposure --- p.18 / Chapter 2.1.3 --- Quantification of gene transcripts by RT-PCR --- p.19 / Chapter 2.1.4 --- Specific aims of this chapter --- p.27 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- Animal --- p.28 / Chapter 2.2.2 --- Cell culture --- p.31 / Chapter 2.2.3 --- General molecular biology techniques --- p.33 / Chapter 2.2.4 --- mRNA quantification by Real-time PCR --- p.35 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Heavy metal toxicity --- p.42 / Chapter 2.3.1.1 --- In vivo metal toxicity in zebrafish adult --- p.43 / Chapter 2.3.1.2 --- In vivo metal toxicity in zebrafish embryos at late epiboly --- p.44 / Chapter 2.3.1.3 --- In vivo metal toxicity in zebrafish eleutheroembryo --- p.46 / Chapter 2.3.1.4 --- In vitro metal toxicity on ZFL cell line --- p.48 / Chapter 2.3.2 --- Optimization of real-time PCR conditions --- p.50 / Chapter 2.3.2.1 --- Construction of relative standard curve --- p.50 / Chapter 2.3.2.2 --- Optimization of primer concentration --- p.52 / Chapter 2.3.2.3 --- Melting curve analysis for PCR specificity --- p.53 / Chapter 2.2.3 --- Quantification of zMT mRNA by Real-time PCR --- p.54 / Chapter 2.2.3.1 --- Relative zMT mRNA induction in zebrafish embryos at late epiboly --- p.54 / Chapter 2.2.3.2 --- Relative zMT mRNA induction in zebrafish eleutherombryos --- p.57 / Chapter 2.2.3.3 --- Relative zMT mRNA induction in SJD. 1 cell line --- p.58 / Chapter 2.2.2.4 --- In vitro zMT mRNA induction in ZFL cell line --- p.62 / Chapter 2.4 --- Discussions --- p.64 / Chapter 2.4.1 --- Change in metal sensitivity during zebrafish embryo development --- p.64 / Chapter 2.4.2 --- Developmental stage-specfic inducibility of zMT gene expression and metal toxicity --- p.65 / Chapter 2.4.3 --- In vitro zMT regulation by heavy metal ions --- p.67 / Chapter 2.4.4 --- The potential use of zMT expression as exposure biomarker --- p.69 / Chapter CHAPTER 3 --- Functional analysis of a cloned zMT-II gene promoter in zebrafish cell-lines: SJD.1 and ZFL --- p.71 / Chapter 3.1 --- Zebrafish MT gene promoter --- p.71 / Chapter 3.1.1 --- The structure of zMT promoter --- p.71 / Chapter 3.1.2 --- Functional analysis of cloned zMT-II promoter region in HepG2 cells --- p.72 / Chapter 3.1.3 --- Specific aims of this chapter --- p.77 / Chapter 3.2 --- Materials and methods --- p.78 / Chapter 3.2.1 --- General molecular biology techniques --- p.78 / Chapter 3.2.2 --- Cell culture --- p.79 / Chapter 3.2.3 --- Transient transfection assay --- p.81 / Chapter 3.3 --- Results --- p.84 / Chapter 3.3.1 --- Metal responsiveness of zMT-II promoter by transient transfection --- p.84 / Chapter 3.3.2 --- Deletion analysis --- p.88 / Chapter 3.3.2.1 --- Deletion analysis of zMT-II gene promoter in SJD. 1 cell line --- p.88 / Chapter 3.3.2.2 --- Deletion analysis of zMT-II gene promoter in ZFL cell line --- p.90 / Chapter 3.4 --- Discussions --- p.91 / Chapter 3.4.1 --- Structure of zMT-II gene promoter --- p.91 / Chapter 3.4.2 --- Metal responsiveness of zMT-II promoter --- p.94 / Chapter 3.4.3 --- Deletion analysis of zMT-IIgene promoter --- p.95 / Chapter CHAPTER 4 --- Transgenic zebrafish model for in vivo zMT gene regulation study --- p.97 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.1.1 --- Development of transgenic fish --- p.97 / Chapter 4.1.2 --- The principle of gene delivery --- p.98 / Chapter 4.1.3 --- The application of transgenic zebrafish model --- p.99 / Chapter 4.1.4 --- Specific aim of this chapter --- p.101 / Chapter 4.2 --- Materials and methods --- p.102 / Chapter 4.2.1 --- General molecular biology techniques --- p.102 / Chapter 4.2.2 --- Sequence of primers used --- p.103 / Chapter 4.2.3 --- Engineering of constructs for transgenic zebrafish study --- p.103 / Chapter 4.2.4 --- In vitro efficacy test of the GFP constructs --- p.105 / Chapter 4.2.5 --- Gene transfer into zebrafish embryos by electroporation --- p.107 / Chapter 4.2.6 --- Screening of transgenic candidates --- p.108 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Engineering of DNA constructs for transgenic zebrafish production --- p.110 / Chapter 4.3.2 --- In vitro efficacy test of the DNA constructs --- p.111 / Chapter 4.3.3 --- Optimization of electroporation voltage --- p.117 / Chapter 4.4.4 --- Screening of transgenic candidates --- p.118 / Chapter 4.4 --- Discussion --- p.120 / Chapter 4.4.1 --- Potential application of the zMT promoter transgenic mode --- p.120 / Chapter 4.4.2 --- The use of GFP transgenic zebrafish model in developmental gene regulation study --- p.121 / Chapter 4.4.3 --- In vitro efficacy of the GFP constructs --- p.122 / Chapter 4.4.4 --- Transgene expression in zebrafish --- p.122 / Chapter CHAPTER 5 --- General discussion --- p.126 / REFERENCES --- p.134

Tse, Chung Kwan Anna. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 86-105). / Abstracts in English and Chinese. / Abstract in English --- p.i / Abstract in Chinese --- p.iii / Acknowledgement --- p.v / Table of content --- p.vii / List of figures and tables --- p.xi / Symbols and abbreviation --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Folliculogenesis --- p.1 / Chapter 1.1.1 --- In Mammals --- p.1 / Chapter 1.1.2 --- In Teleosts --- p.2 / Chapter 1.1.2.1 --- Vitellogenesis --- p.3 / Chapter 1.1.2.2 --- Oocyte Maturation --- p.4 / Chapter 1.1.3 --- Oocyte Control of Folliculogenesis --- p.5 / Chapter 1.2 --- Epidermal Growth Factor Family --- p.7 / Chapter 1.2.1 --- Epidermal Growth Factor Ligand Family --- p.7 / Chapter 1.2.1.1 --- Discovery --- p.7 / Chapter 1.2.1.2 --- Structure --- p.8 / Chapter 1.2.1.3 --- Shedding --- p.8 / Chapter 1.2.1.4 --- Functions --- p.9 / Chapter 1.2.2 --- Epidermal Growth Factor Receptor Family --- p.10 / Chapter 1.2.2.1 --- Structure --- p.10 / Chapter 1.2.2.2 --- Ligand Binding and Dimerization --- p.11 / Chapter 1.2.2.3 --- Signaling and Internalization --- p.12 / Chapter 1.2.3 --- EGF Family in Reproduction --- p.13 / Chapter 1.2.3.1 --- Reproductive Function of EGF Family --- p.13 / Chapter 1.2.3.2 --- EGF Network as Downstream Mediator of Gonadotropins --- p.15 / Chapter 1.3 --- Objectives of the Present Study --- p.18 / Chapter Chapter 2 --- Spatiotemporal Expression of the EGF Family in Zebrafish Ovary / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Chemicals --- p.22 / Chapter 2.2.2 --- Animals --- p.22 / Chapter 2.2.3 --- Isolation of Different Tissues and Ovarian Follicles --- p.22 / Chapter 2.2.4 --- Separation of Oocyte and Follicle layers --- p.23 / Chapter 2.2.5 --- Embryo Collection --- p.23 / Chapter 2.2.6 --- Tyrphostin AG 1478 Treatment of Embryos --- p.23 / Chapter 2.2.7 --- Total RNA Isolation and Reverse Transcription --- p.24 / Chapter 2.2.8 --- Semi-quantitative and Real-time Polymerase Chain Reaction (PCR) --- p.24 / Chapter 2.2.9 --- Data Analysis --- p.25 / Chapter 2.3 --- Results --- p.27 / Chapter 2.3.1 --- Validation of Semi-quantitative PCR Quantification --- p.27 / Chapter 2.3.2 --- Tissue Distribution of the EGF Family --- p.27 / Chapter 2.3.3 --- Spatial Expression of the EGF and the Activin Families within the Follicles --- p.27 / Chapter 2.3.4 --- Temporal Expression of the EGF Family in Folliculogenesis --- p.29 / Chapter 2.3.5 --- Temporal Expression of the EGF Family in Embryogenesis --- p.29 / Chapter 2.3.6 --- Effect of Blocking EGFR on Embryonic Development --- p.30 / Chapter 2.4 --- Discussion --- p.31 / Chapter Chapter 3 --- Regulation of the EGF Family in Cultured Follicle Cells and Their Effects on the Expression of Activin Subunits / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Chemicals --- p.58 / Chapter 3.2.2 --- Primary Follicle Culture --- p.58 / Chapter 3.2.3 --- Ovarian Fragment Incubation --- p.58 / Chapter 3.2.4 --- Total RNA Isolation and Reverse Transcription --- p.58 / Chapter 3.2.5 --- Semi-quantitative and Real-time Polymerase Chain Reaction (PCR) --- p.59 / Chapter 3.2.6 --- Microinjection --- p.59 / Chapter 3.2.7 --- Data analysis --- p.59 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Expression of the EGF Family in Cultured Follicle Cells --- p.61 / Chapter 3.3.2 --- Effects of EGF on the EGF Family Expression in Cultured Follicle Cells --- p.61 / Chapter 3.3.3 --- Effects of BTC and HB-EGF on the Expression of EGF Ligands in Cultured Follicle Cells --- p.61 / Chapter 3.3.4 --- Effect of EGF Ligands on the Expression of Activin Subunits in Cultured Follicle Cells --- p.62 / Chapter 3.3.5 --- Effects of Actinomycin D on Ovarian Fragments --- p.62 / Chapter 3.3.6 --- Effects of Microinjecting Anti-EGF Morpholino on Full Grown Follicles --- p.62 / Chapter 3.4 --- Discussion --- p.64 / Chapter Chapter 4 --- General Discussion --- p.81 / References --- p.86