Forkhead domain基因家族編碼了很多對於胚胎發育至關重要的轉錄因子，而Foxp4則屬於p-subtype forkhead轉錄因子其中一員。Foxp4在胚胎發育期間的表達十分活躍，在發育中的腦部的不同地方表達，但其於中樞神經系統發育中的調控角色並不清楚。Foxp4基因剔除小鼠在出生前死於心臟的缺陷表型(心二分支) ，在此時間段，腦部的發育才剛剛開始，因此我們無法利用Foxp4基因剔除小鼠作為研究中樞神經系統發育的動物模型。最近，我們的團隊利用小腦組織培養技術及siRNA發佈的研究顯示，Foxp4在小鼠小腦中的蒲金氏細胞(Purkinje cell)中擔當著重要的維持作用。這項研究結果加深了我們對研究Foxp4在中樞神經系統發育中的調控角色的決心。 / 本論文旨在利用斑馬魚作為實驗模型，研究foxp4在斑馬魚中樞神經系統發育中的表達及調控角色。RT-PCR結果顯示foxp4在斑馬魚發育中的bud stage開始表達，並在及後的階段維持其表達水平。利用原位雜交技術 (whole mount in-situ hybridization)，我們發現foxp4表達的地區主要集中於發育中的腦部。在成年斑馬魚中，foxp4表達在不同組織和器官，包括腦部，眼睛和心臟。成年斑馬魚腦部切片原位雜交 (sectioned in-situ hybridization)則顯示，foxp4在小腦的蒲金氏細胞和視頂蓋(optic tectum)的periventricular gray zone表達。 / 為了進一步探究foxp4對於胚胎發育過程中的功能，我們利用微注射技術，把反義嗎啉 (morpholino) MO1注射到斑馬魚胚胎中，大幅度抑制foxp4的表達水平。胚胎受精後48小時，MO1注入的胚胎顯示出第四腦室腦積水的缺陷表型。組織學分析顯示，第四腦室以下的延髓被壓縮致形態異常。此外，利用原位雜交技術及不同的分子標記，我們發現胚胎的中後腦邊界也會出現輕度畸形，而後腦的神經元數量及排列亦受到影響。 / 本項研究展示foxp4在胚胎中樞神經系統的發展的重要性，亦提供了新的見解。我們認為foxp4可能是調控腦室發育的重要成員，但在此方面與foxp4相關的分子機制仍須作更深入的研究。 / The forkhead domain gene family encodes a large group of transcription factors that play essential roles in development. Foxp4 is one of the members in the Foxp subfamily that expressed in different parts of developing central nervous system (CNS) and its function is less characterized. Previous study on Foxp4-knockout mice resulted in early embryonic lethality due to defective heart tube development that hindered the functional study of Foxp4 in CNS development. Recently, our laboratory reported that Foxp4 functions as a maintenance role in the Purkinje cell in the mouse cerebellum. Nevertheless, the role of foxp4 in CNS development was still unclear. / In this study, we used zebrafish as a model to study the expression pattern and functional study of foxp4 in the developing CNS. RT-PCR analysis showed that foxp4 transcript was expressed at the bud stage and maintained in the later embryonic stages. Whole-mount in-situ hybridization showed that foxp4 expressed in the cephalic region during embryonic development. In adult zebrafish, foxp4 expresses in different tissues and organs including brain, eye and heart. Sectioned in-situ hybridization of the adult zebrafish brain showed that foxp4 was specifically expressed in the Purkinje cell and the periventricular gray zone of optic tectum. / To further investigate the function of foxp4 during embryonic development, we injected antisense morpholino, MO1 into the zebrafish embryo to knockdown foxp4. By 48 hour post fertilization (hpf), MO1-injected embryos displayed hydrocephalus in the 4th ventricle. Histological analysis revealed that the medulla oblongata below the 4th ventricle was compressed by the edema resulting in abnormal morphology of medulla oblongata in the MO1-injected morphant. In addition, a mild malformation of the mid-hindbrain boundary, disrupted hindbrain patterning was observed in MO1-injected morphant. / Our findings provide new insight into the function of foxp4 in embryonic CNS development. We suggested that foxp4 may be essential in regulating the brain ventricle development while the molecular mechanism underlying the functional role of foxp4 requires further investigation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Wai Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 92-102). / Abstracts also in Chinese. / Abstract --- p.iii / 摘要 --- p.v / Acknowledgement --- p.vii / Figure and table list --- p.xi / Abbreviation --- p.xii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Zebrafish as a developmental model --- p.1 / Chapter 1.2 --- Zebrafish development with highlights --- p.3 / Chapter 1.2.1 --- CNS development --- p.3 / Chapter 1.3 --- Forkhead domain gene in development --- p.5 / Chapter 1.3.1 --- History of forkhead domain gene --- p.5 / Chapter 1.3.2 --- Functional roles of forkhead domain genes in development --- p.6 / Chapter 1.4 --- Foxp subfamily --- p.8 / Chapter 1.4.1 --- Diverse functions of Foxp1, 2, 3 and 4 --- p.8 / Chapter 1.4.2 --- Relationship between Foxp subfamily members --- p.10 / Chapter 1.5 --- Foxp4 --- p.11 / Chapter 1.5.1 --- Genomic organization and protein structure of mFoxp4 --- p.11 / Chapter 1.5.2 --- Previous studies of mFoxp4 --- p.14 / Chapter 1.5.3 --- Foxp4 studies in other model organisms --- p.14 / Chapter 1.5.3.1 --- Rat --- p.15 / Chapter 1.5.3.2 --- Xenopus --- p.16 / Chapter 1.5.3.3 --- C. elegans --- p.16 / Chapter 1.5.4 --- Zebrafish foxp4 --- p.17 / Chapter 1.5.5.1 --- Genomic organization and protein structure of foxp4 --- p.17 / Chapter 1.5.5.2 --- Sequence alignment of foxp4 with other models --- p.19 / Chapter 1.6 --- Hypothesis, aim and strategy of the study --- p.22 / Chapter Chapter 2 --- Expression of foxp4 in zebrafish embryo and adult zebrafish brain / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and methods --- p.25 / Chapter 2.2.1 --- Animals --- p.25 / Chapter 2.2.2 --- Materials --- p.26 / Chapter 2.2.3 --- Semi-quantitative PCR --- p.35 / Chapter 2.2.3.1 --- cDNA of zebrafish embryo --- p.35 / Chapter 2.2.3.2 --- Isolation of adult zebrafish organs --- p.36 / Chapter 2.2.3.3 --- RNA extraction and reverse transcription --- p.36 / Chapter 2.2.3.4 --- Polymerase chain reaction --- p.37 / Chapter 2.2.4 --- Subcloning of DNA fragment / Chapter 2.2.4.1 --- Preparation of cloning vectors --- p.40 / Chapter 2.2.4.2 --- Subcloning of DNA fragments --- p.40 / Chapter 2.2.4.3 --- Transformation of DNA into competent cells --- p.40 / Chapter 2.2.4.4 --- Preparation of recombinant plasmid DNA --- p.41 / Chapter 2.2.5 --- Whole mount in-situ hybridization of zebrafish embryo --- p.45 / Chapter 2.2.5.1 --- Preparation of equipment --- p.45 / Chapter 2.2.5.2 --- Preparation of zebrafish embryos --- p.45 / Chapter 2.2.5.3 --- Preparation of RNA probe --- p.46 / Chapter 2.2.5.4 --- Whole-mount in-situ hybridization --- p.48 / Chapter 2.2.6 --- Sectioned in-situ hybridization of adult zebrafish brain --- p.49 / Chapter 2.2.6.1 --- Histology of adult zebrafish brain --- p.49 / Chapter 2.2.6.2 --- Sectioned in-situ hybridization --- p.50 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Expression profile of foxp4 in different stages of zebrafish embryo --- p.51 / Chapter 2.3.2 --- Expression pattern of foxp4 in different stages of zebrafish embryo --- p.54 / Chapter 2.3.3 --- Expression profile of foxp4 in different zebrafish organs and tissues --- p.57 / Chapter 2.3.4 --- Expression pattern of foxp4 in adult zebrafish brain --- p.59 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 3 --- Functional analysis of foxp4 in zebrafish embryonic development / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Materials and methods --- p.64 / Chapter 3.2.1 --- Materials --- p.64 / Chapter 3.2.2 --- Design of morpholino --- p.68 / Chapter 3.2.3 --- Sequencing of morpholino target regions of foxp4 --- p.70 / Chapter 3.2.4 --- Microinjection --- p.70 / Chapter 3.2.4.1 --- Preparation of materials and equipment --- p.70 / Chapter 3.2.4.2 --- Preparation of injection needle --- p.70 / Chapter 3.2.4.3 --- Preparation of morpholinos --- p.70 / Chapter 3.2.4.4 --- Calibration of injection volume --- p.71 / Chapter 3.2.4.5 --- Microinjection of zebrafish embryo --- p.71 / Chapter 3.2.5 --- Western blotting to assay foxp4 translation inhibition --- p.72 / Chapter 3.2.5.1 --- Preparation of protein extracts --- p.72 / Chapter 3.2.5.2 --- Coomassie blue staining --- p.73 / Chapter 3.2.5.3 --- Western blotting --- p.74 / Chapter 3.2.6 --- Whole mount in-situ hybridization --- p.74 / Chapter 3.3 --- Results --- p.75 / Chapter 3.3.1 --- MO1 knockdown efficiency assayed by Western blotting --- p.75 / Chapter 3.3.2 --- General morphology of morphants --- p.77 / Chapter 3.3.3 --- Histology at the hindbrain region showing the phenotype --- p.79 / Chapter 3.3.4 --- Whole mount in-situ hybridization of different molecular markers --- p.81 / Chapter 3.4 --- Discussion --- p.85 / Chapter Chapter 4 --- Future directions and conclusion / Chapter 4.1 --- Future directions --- p.89 / Chapter 4.2 --- Conclusion --- p.91 / Reference --- p.92

Wang Yajun. / "April 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 212-248). / 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.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2012-09-19T19:02:38Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ValidacaoFarmagologicaPreferencia.pdf: 809036 bytes, checksum: ad66113d2aa6556dde18a768e6d045c5 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2012-09-28T12:38:04Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ValidacaoFarmagologicaPreferencia.pdf: 809036 bytes, checksum: ad66113d2aa6556dde18a768e6d045c5 (MD5) / Made available in DSpace on 2012-09-28T12:38:05Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_ValidacaoFarmagologicaPreferencia.pdf: 809036 bytes, checksum: ad66113d2aa6556dde18a768e6d045c5 (MD5) Previous issue date: 2012 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A ansiedade é uma desordem complexa e com grande relevância clínica, cujo estudo com modelos animais é importante para pesquisar sobre seus mecanismos e drogas para o seu tratamento. O zebrafish figura como um potencial modelo animal para pesquisas farmacológicas da ansiedade. Um modelo de ansiedade é a preferência claro-escuro, que já foi validado comportamentalmente em zebrafish, contudo necessita de uma validação farmacológica. Objetiva-se descrever a sensibilidade da preferência claro-escuro em zebrafish adultos para as drogas mais utilizadas na clínica da ansiedade, foram administradas pela imersão do animal na solução: Benzodiazepínicos (Clonazepam); Agonistas parciais 5-HT1A (Buspirona); Antidepressivo tricíclico (Imipramina); Antidepressivo ISRS (Fluoxetina e Paroxetina); Antipsicóticos (Haloperidol e Risperidona); Psicostimulante (Dietilpropiona); Beta bloqueadores (Propranolol) e Depressores do SNC (Etanol). Os parâmetros analisados foram o tempo despendido pelo animal no ambiente escuro, o tempo da primeira latência e número de alternâncias. O clonazepam administrado por 300s aumentou o tempo no escuro na menor concentração e reduziu a atividade locomotora, a administração durante 600s da concentração intermediária diminuiu o tempo no escuro e da primeira latência, assim como aumentou a atividade locomotora, indicando efeito ansiolítico. A buspirona aumentou o tempo de permanência no escuro provavelmente devido a redução da atividade motora. A imipramina e a fluoxetina aumentaram o tempo no escuro e da primeira latência e diminuíram o número de alternâncias, indicando ação ansiogênica. A paroxetina não alterou o tempo no escuro, entretanto aumentou o tempo da primeira latência e diminuiu a atividade locomotora. O haloperidol diminuiu a ansiedade na menor concentração, curiosamente aumentou a atividade motora na maior concentração, ao contrário da risperidona que diminuiu a atividade na maior concentração. A dietilpropriona não modificou o tempo no escuro, mas aumentou o tempo da primeira latência e diminuiu a atividade motora apenas na menor concentração. O propranolol reduziu somente o tempo no escuro. O etanol foi efetivo na redução da ansiedade com a concentração intermediária e diminuiu a atividade locomotora em uma concentração menor Os dados corroboram com relatos da literatura em Danio rerio tanto neste modelo em administração intraperitoneal como em outros modelos por administração hídrica e em roedores, quando foi possível a comparação. / Anxiety is a complex disorder with large clinical relevance, whose study with animal models is important for research about their mechanisms and drugs for their treatment. The zebrafish appears as a potential animal model for pharmacological research in anxiety. A model of anxiety is the light-dark preference, which has been validated behaviorally in zebrafish, however, requires a pharmacological validation. The objective is to describe the sensitivity of the light-dark preference in zebrafish adults for the most common drugs in clinical anxiety, were administered by immersing the animal in the solution: Benzodiazepines (Clonazepam), 5-HT1A partial agonists (Buspirone), Tricyclic Antidepressant (Imipramine), Antidepressant SSRIs (Fluoxetine and Paroxetine), Antipsychotics (Haloperidol and Risperidone); Psychostimulant (Diethylpropion), Beta blockers (Propranolol) and CNS depressants (Ethanol). The parameters analyzed were the time spent by the animal in a dark environment, the time of the first latency and number of midline crossings. Clonazepam administered 300 s increased the time in the dark at lower concentrations and reduced locomotor activity, administration during 600 s of the intermediate concentration decreased over time in the dark and the first latency, and increased locomotor activity, indicating anxiolytic effect. Buspirone raised the time spent in the dark, probably due to reduction of motor activity. Imipramine and fluoxetine increased time in the dark and the first latency and decreased the number of alternations, indicating anxiogenic action. Paroxetine did not alter the time in the dark, however the first time increased latency and decreased locomotor activity. Haloperidol decreased anxiety in the lowest concentration, curiously raised motor activity at the highest concentration, instead of risperidone, which decreased the activity at the highest concentration. Diethylpropion did not change over time in the dark but increased the time of the first latency and decreased motor activity only at lower concentrations. Propranolol reduced only time in the dark. Ethanol was effective in reducing anxiety with the intermediate concentration and decreased locomotor activity in a lower concentration. Data corroborate with the literature in Danio rerio both intraperitoneal administration in this model as in other models for water delivery and in rodents, when it was possible to compare.

A critical step in mammalian ovarian follicle development is the transition of gonadotropin-independent preantral follicles to the gonadotropin-dependent antral follicles. However, the molecular mechanisms underlying the transition or early follicle activation are largely unknown. Using zebrafish as the model, we have recently identified Y-box binding protein 1 (YB-1, Ybx1/ybx1), a transcription factor and mRNA binding protein, in early developing oocytes whose expression level was very high in the gonadotropin-independent primary growth (PG) stage but drastically diminished at the beginning of the gonadotropin-dependent secondary growth (SG) stage, i.e., previtellogenic (PV) stage. This has raised interesting questions on the role of Ybx1 in follicle activation as well as how it is controlled. To provide clues to these issues, we first analyzed the regulation of Ybx1 during PG-to-PV transition under IGF-I treatment and the associated signaling pathways. IGF-I, an endocrine/paracrine factor in the growth axis, stimulats Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway in PG follicles. Interestingly, the phosphorylation correlated well with the decline of Ybx1 protein level and the activation of the follicle from the PG follicle pool. This, together with our finding that zebrafish Ybx1 is exclusively produced in PG oocytes in large amount but suddenly disappears during PG-to-PV transition, has prompted us to wonder what the relationship between Ybx1 phosphorylation and degradation. Further experiments showed that Akt directly binds and phosphorylates Ybx1, leading the regulation of Ybx1, including its phosphorylation, cleavage, translocation and degradation, which in turn regulates gene expression and protein synthesis. / In summary, as a multifunctional protein that may play a critical role in early follicle development, Ybx1 is subject to regulation by external factors such as IGF-I, which stimulated Ybx1 phosphorylation via PI3K/Akt but not MAPK pathway. Once Ybx1 is phosphoylated by Akt in the cytoplasm of PG follicle, on one hand, it will be cleaved and translocated to the nucleus to regulate gene expression. On the other hand, the phosphor-Ybx1 can also be degraded through the Ub-proteasome pathway, leading the release of free mRNA to further translation. All these promote the synthesis of many growth- and differentiation-related proteins, which will facilitate early follicle activation. Our findings suggest that the oocyte may serve as the headquarter to programme follicle activation and that the oocyte Ybx1 protein may play a critical role in this event. The delineation of the signaling pathways involved in IGF-I-induced Ybx1 phosphorylation and the regulation of Ybx1 as well as its function in gene transcription and protein synthesis during PG-to-PV transition will provide insight into the mechanism of early follicle activation and puberty initiation. / 哺乳动物卵巢卵泡发育的一个关键步骤是从促性腺激素非依赖的窦前卵泡向促性腺依赖的窦状卵泡的转变过程。但是这一早期卵泡激活的分子机制却不是非常清楚。利用斑马鱼为模型，我们在早期发育的卵母细胞中发现一种名叫Y-box结合蛋白1 （YB-1, Ybx1/ybx1）的转录因子和mRNA 结合蛋白，它在促性腺激素不依赖的初级生长期卵泡（PG）中大量表达，但是在促性腺激素依赖的第二生长期卵泡（SG）,也叫卵黄发生前期(PV)中表达量大大降低。这引发我们猜想YB-1 可能在早期卵泡激活（PG-to-PV 转变）中发挥着重要作用，并且想知道它的这一表达量的巨变是如何被调控的。为了弄清楚这些问题，我们首先分析了IGF-I 处理下Ybx1 在PG-to-PV 的转变中是怎样被调控的，以及相关的信号通路。我们发现在PG 阶段，IGF-I 这种存在于生长轴中的内分泌／旁分泌因子，通过PI3K/Akt 而不是MAPK 通路促进Ybx1 的磷酸化。有趣的是，这种磷酸化的升高正好伴随着Ybx1 蛋白水平的下降以及PG 卵泡的激活。结合我们之前的发现：斑马鱼Ybx1 只在PG 卵母细胞中大量表达但在PG-to-PV 的转变过程中突然消失，促使我们猜想Ybx1 磷酸化和它的降解之间应该存在一定的关系。进一步的实验表明Akt 激酶直接结合并磷酸化Ybx1,导致一系列对Ybx1 调控，包括它的磷酸化，切割，转位以及降解，所有这些又将促进基因的表达调控及蛋白的合成。 / 总之， 多功能蛋白Ybx1 可能在早期卵泡发育过程中发挥着至关重要的作用。外界刺激因子，如IGF-I，通过PI3K/Akt 而非MAPK 途径促进Ybx1 磷酸化。一旦Ybx1 在PG 卵泡细胞质中被Akt 磷酸化，一方面Ybx1 将会被切割并且转位到细胞核中去调节基因表达，另一方面，磷酸化的Ybx1 还会通过泛素蛋白酶途径被降解，从而释放出mRNA 去进一步的翻译。所有这些将促进许多生长和分化相关的蛋白合成，从而促进早期卵泡的激活。我们的研究结果表明，卵母细胞很可能是程序性卵泡激活的核心部分，存在于卵母细胞中的Ybx1 蛋白在这一过程中起着关键作用。研究IGF-I 参与诱导的Ybx1 磷酸化的信号通路以及在PGto-PV 转变过程中对Ybx1 蛋白的调控和它在基因表达及蛋白合成中的作用，将有力的帮助我们弄清早期卵母细胞激活及青春期的启动机制。 / Zhang, Lingling. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 104-127). / Abstracts also in Chinese. / Title from PDF title page (viewed on 06, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Yu, Man Ying Susana. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-117). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of contents --- p.viii / Symbols and abbreviations --- p.xii / Scientific names --- p.xiv / List of figures --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Regulation of ovarian follicle development --- p.3 / Chapter 1.2.1 --- Endocrine regulation --- p.3 / Chapter 1.2.1.1 --- Gonadotropins- FSH and LH --- p.3 / Chapter 1.2.1.2 --- Co-gonadotropin- growth hormone --- p.5 / Chapter 1.2.2. --- Paracrine regulation --- p.6 / Chapter 1.2.2.1 --- Activin --- p.6 / Chapter 1.2.2.2 --- Insulin-like growth factor I (IGF-I) --- p.7 / Chapter 1.3 --- The GH-IGF-I axis --- p.7 / Chapter 1.3.1 --- The somatomedin hypothesis --- p.8 / Chapter 1.3.2 --- "Structure and signaling of GH, GHR" --- p.8 / Chapter 1.3.3 --- Structure and signaling of IGF system --- p.9 / Chapter 1.3.4 --- Role of GH-IGF system in reproduction --- p.11 / Chapter 1.3.5 --- GH action in ovarian functions --- p.12 / Chapter 1.3.6 --- IGF-I action in ovarian functions --- p.13 / Chapter 1.3.7 --- The mini GH-IGF axis within the ovary --- p.14 / Chapter 1.4 --- Objectives of present study --- p.14 / Chapter Chapter 2 --- "Expression Profiles of the GH-IGF System in the Ovary of Zebrafish, Danio rerio" --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Material and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of tissues and different stages of follicles from the zebrafish --- p.22 / Chapter 2.2.3 --- Separation of somatic follicle layers and oocytes --- p.22 / Chapter 2.2.4 --- Primary follicle cell culture --- p.22 / Chapter 2.2.5 --- Total RNA extraction --- p.23 / Chapter 2.2.6 --- Reverse transcription --- p.23 / Chapter 2.2.7 --- "Validation of semi-quantitative RT-PCR assays for GH (gh), GHR (ghr), IGF-I (igf1), IGF-II (igf2), and IGF-I receptor (igf1r)" --- p.24 / Chapter 2.2.8 --- Data analysis --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Validation of semi-quantitative RT-PCR assays --- p.25 / Chapter 2.3.2 --- Spatial expression of GH-IGF in different tissues of zebrafish --- p.26 / Chapter 2.3.3 --- "Localization of gh, ghr, igf1, igf2 and igf1r within the zebrafish follicle" --- p.26 / Chapter 2.3.4 --- Temporal expression profiles of GH-IGF system during folliculogenesis --- p.28 / Chapter 2.4 --- Discussion --- p.28 / Chapter Chapter 3 --- Regulation of the GH-IGF-I System and Its Cross-talk with the Activin System in the Zebrafish Ovary --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Material and methods --- p.45 / Chapter 3.2.1 --- Animals --- p.45 / Chapter 3.2.2 --- Chemicals and hormones --- p.45 / Chapter 3.2.3 --- Primary follicle cell culture --- p.45 / Chapter 3.2.4 --- Preparation of ovarian fragments --- p.45 / Chapter 3.2.5 --- Total RNA extraction --- p.45 / Chapter 3.2.6 --- RT-PCR --- p.47 / Chapter 3.2.7 --- Construction of real-time PCR standards --- p.47 / Chapter 3.2.8 --- Real-time PCR and semi-quantitative RT-PCR --- p.48 / Chapter 3.2.9 --- Data analysis --- p.49 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- "Expression of growth hormone (gh), growth hormone receptors (ghr1 and ghr2\ IGF-I (igf1), IGF-II (igf2), IGF-I receptor (igf1ra and igf1rb), activin subunits (inhba and inhbb) and follistatin (fst) in cultured zebrafish ovarian fragments" --- p.49 / Chapter 3.3.2 --- "Establishment of real-time RT-PCR for zebrafish inhba, inhbb and bactin" --- p.50 / Chapter 3.3.3 --- GH regulation of activin expression in cultured zebrafish follicle cells --- p.50 / Chapter 3.3.4 --- GH regulation of IGF-I in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.5 --- IGF-I regulation of activin expression in cultured zebrafish follicle cells --- p.51 / Chapter 3.3.6 --- Activin regulation of IGF system --- p.52 / Chapter 3.4 --- Discussion --- p.52 / Chapter Chapter 4 --- Production of recombinant zebrafish growth hormone --- p.69 / Chapter 4.1 --- Introduction --- p.69 / Chapter 4.2 --- Material and Methods --- p.71 / Chapter 4.2.1 --- Animals --- p.71 / Chapter 4.2.2 --- Construction of expression plasmids pPIC9K/zfGH --- p.71 / Chapter 4.2.3 --- Production of recombinant zebrafish GH using Pichia pastoris --- p.73 / Chapter 4.2.4 --- SDS-PAGE and silver staining --- p.74 / Chapter 4.2.5 --- Purification --- p.74 / Chapter 4.2.6 --- Primary follicle cell culture --- p.75 / Chapter 4.2.7 --- Zebrafish hepatic cell culture --- p.76 / Chapter 4.2.8 --- RNA extraction and RT-PCR --- p.76 / Chapter 4.2.9 --- Real-time PCR --- p.77 / Chapter 4.2.10 --- Cell culture and transient transfection --- p.78 / Chapter 4.2.11 --- Luciferase reporter gene assay --- p.78 / Chapter 4.2.12 --- Data analysis --- p.79 / Chapter 4.3 --- Results --- p.79 / Chapter 4.3.1 --- Production of recombinant zebrafish GH --- p.79 / Chapter 4.3.2 --- Effect of recombiant zfGH on the expression of activin β Aand βB in cultured zebrafish follicle cells --- p.80 / Chapter 4.3.3 --- Effect of zfGH on the expression of igf1 in cultured zebrafish hepatic cells --- p.80 / Chapter 4.3.4 --- Luciferase reporter gene assay --- p.81 / Chapter 4.4 --- Discussion --- p.81 / Chapter Chapter 5 --- General Discussion --- p.94 / Chapter 5.1 --- Overview --- p.94 / Chapter 5.2 --- Major achievements of the present study --- p.95 / Chapter 5.2.1 --- Demonstration of a local mini-GH-IGF-I axis within the zebrafish ovary --- p.96 / Chapter 5.2.2 --- Differential expression profiles of the GH-IGF system during folliculogenesis --- p.96 / Chapter 5.2.3 --- The inter-relationship of GH-IGF and activin-follistatin systems --- p.96 / Chapter 5.2.4 --- Production of recombinant zebrafish GH --- p.97 / Chapter 5.3 --- Future prospects --- p.97 / References --- p.102 / Symbols and Abbreviations / Symbols / α Alpha / β Beta / Abbreviations / 20β-HSD 20β-hydroxysteroid dehydrogenase / bp Base pair / cAMP Cyclic adenosine monophosphate / cDNA Complementary cDNA / CHO Chinese hamster ovary / "DHP 17α, 20β-dihydroxy-4-prenane-3 -one" / DNA Deoxyribonucleic acid / EGF Epidermal growth factor

A melanopsina foi descoberta em 1998 por Ignacio Provencio e colaboradores em melanóforos de Xenopus leavis. Desde sua descoberta, esse fotopigmento surgiu como um possível candidato a intermediar os fenômenos de sincronização nos vertebrados. Nos mamíferos, a melanopsina é encontrada num pequeno subgrupo de células ganglionares da retina, conhecido como células ganglionares retinianas intrinsecamente fotossensíveis (ipRGCs) e o seu papel como fotopigmento responsável pela percepção luminosa, que leva à sincronização das espécies dessa classe aos ciclos de claro e escuro, já foi estabelecido. A melanopsina está presente na retina de todas as classes de vertebrados estudadas até o momento, mas, em contraposição a essa afirmação, a sua estrutura tem maior semelhança com opsina de invertebrados do que com opsina de vertebrados, sugerindo que sua fototransdução ocorra através da via dos fosfoinositídeos. Essa hipótese foi confirmada por diversos trabalhos na literatura e estudos posteriores demonstraram que, em vertebrados não mamíferos, a melanopsina é codificada por dois genes: um ortólogo ao de mamíferos, Opn4m, e um ortólogo ao de X. leavis, Opn4x, levantando diversas questões a respeito da funcionalidade dessa opsina. Nosso grupo vem estudando esse fotopigmento nos tecidos periféricos de vertebrados desde 2001, sendo que foi pioneiro em demonstrar, em melanóforos de Xenopus laevis, que a dispersão dos grânulos de melanina se dá através da fotoativação da melanopsina que desencadeia a cascata de fosfoinositídeos. E estudos mais recentes vêm colocando a melanopsina como um dos possíveis fotopigmentos responsáveis pela sincronização de relógios periféricos em organismos como peixes e anfíbios. Nesse sentido, a linhagem de células ZEM-2S do peixe teleósteo Danio rerio é um ótimo modelo para o estudo das vias de fototransdução em relógios periféricos. Já foi demonstrado que essa linhagem de células é responsiva a estímulos luminosos, exibindo uma proliferação diferencial frente a diferentes regimes de claro e escuro, e ativando a expressão de genes de relógio como clock, per1 e cry1b, que conhecidamente são responsáveis por sincronizar os ritmos biológicos ao fotoperíodo ambiental. Nossos experimentos de imunocitoquímica detectaram a presença das duas proteínas codificadas pelos genes opn4m-1 e opn4m-2 da melanopsina, e mostraram uma significativa diferença na distribuição das proteínas Opn4m-1 e Opn4m-2. Análises de PCR quantitativo mostraram que um pulso de luz azul de 10 min é capaz de alterar a expressão dos genes de relógio per1b, per2, cry1a e cry1b, e que essa alteração ocorre através da via dos fosfoinositídeos em células embrionárias ZEM-2S de Danio rerio. Em adição mostramos que para promover a alteração dos genes de relógio, a via dos fosfoinositídeos interage com outras vias de sinalização como a via do óxido nítrico (NO) e a via das proteína quinases ativadas por mitógenos (MAPKs). Esses dados sugerem que a melanopsina seja um dos principais candidatos a intermediar os processos de sincronização nessas células, pois a somatória dos resultados de detecção da melanopsina, estimulação dentro de seu espectro de absorção e ativação da via dos fosfoinositídeos, a coloca a frente de outras opsinas como vertebrate ancient opsin (Va-opsin) e teleost multiple tissue opsin (Tmt-opsin) e de outros candidatos como Crys fotossensíveis e mecanismos de estresse oxidativo. No curso deste trabalho também conseguimos definir metodologias eficientes de transfecção de RNA de interferência e de DNA plasmidial em células ZEM-2S de D. rerio, que são ferramentas fundamentais nos estudos de expressão gênica nesse modelo / Melanopsin was discovered in 1998 by Ignacio Provencio and colleagues in Xenopus leavis melanophores. Since its discovery, this photopigment has emerged as a possible candidate to mediate synchronization in vertebrates. In mammals the melanopsin is found in a subset of retinal ganglion cells, known as intrinsically photosensitive retinal ganglion cells (ipRGCs) and their role as the photopigment responsible for photoentrainment in mammals has already been established. Melanopsin is present in the retina of all vertebrate classes studied to date, nevertheless, its structure is more similar to invertebrate than to vertebrates opsins, suggesting that their phototransduction pathway occurs through the phosphoinositide pathway. This hypothesis has been confirmed by several studies in the literature. Later studies showed that melanopsin is encoded by two genes in non-mammalian vertebrates, Opn4m orthologous to mammalian and Opn4x orthologous to X. leavis, raising new questions about the functionality of this opsin. Our group has studied this photopigment in vertebrate peripheral tissues since 2001 and, in Xenopus laevis melanophores, we demonstrated that pigment granule dispersion occurs through photoactivation of melanopsin and triggering of phosphoinositide pathway. More recent studies have put melanopsin as a possible photoreceptor responsible for peripheral clocks entrainment in organisms like fish and amphibians. In this context, the ZEM-2S cell line of the teleost fish Danio rerio is a good model to study the mechanism of phototransduction in peripheral clocks. It has been previously demonstrated that this cell line is responsive to light stimuli, exhibiting a differential proliferation when submitted to different light/dark regimes and activating the expression of clock genes such as clock, per1 and cry1b, known to synchronize the biological rhythms to environmental photoperiod. Our immunocytochemistry experiments detected the presence of two proteins encoded by the melanopsin genes opn4m-1 and opn4m-2, and showed a significant difference in the distribution of proteins Opn4m-1 Opn4m-2. Quantitative PCR analyses showed that a 10-min blue light pulse is able to change the expression of the clock genes per1b, per2, cry1b and cry1a, and that this change occurred through the phosphoinositide cascade in embryonic ZEM-2S cells of D. rerio. In addition we showed that, to promote the change in clock gene expression, the phosphoinositide pathway interacts with other signaling pathways such as the nitric oxide (NO) and the mitogen-activated protein kinase (MAPK) pathways. These data suggest that melanopsin is a major candidate to mediate the photoentrainment in these cells, because taken together, the detection of melanopsin, stimulation within its absorption spectrum and activation of the phosphoinositide cascade, puts it ahead of other opsins, as the vertebrate ancient opsin (Va-opsin) and teleost multiple tissue opsin (Tmt-opsin), and other candidates, as photosensitive Crys and mechanisms of oxidative stress. In the course of this work, we could also define efficient methods for transfection of interference RNA and plasmidial DNA in ZEM-2S cells of D. rerio, which are fundamental tools in studies of gene expression in this model

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 35-40). Also available on the World Wide Web.

University of Macau / Institute of Chinese Medical Sciences

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 234-254). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 234-254). Also available on the Internet.