Cell culture systems have provided many insights into eukaryotic gene expression and other biochemical mechanisms. Since the cell represents the smallest living unit of any organism it provides a desirable in vitro system, allowing biochemical studies without the complex physiology of an entire animal. However, processes involving intracellular mechanisms, such as development, aging or carcinogenis, eventually require the analysis of the intact organism. Transgenic animals are a very promising tool to approach questions of this magnitude. Fish in general and the zebrafish (Brachydanio rerio) in particular are an excellent model system for transgenic research, mainly due to their extramaternal fertilization and development and their short generation cycle throughout the year. The recent derivation of zebrafish cell lines has opened up possibilities for in vitro analysis of this popular model species, and expression of heterologous genes under the influence of promoter and other regulatory nucleic acid aequences. In contrast to mammalian expression systems, little nucleic acid sequences controlling gene expression in fish are known. Therefore we examined mammalian expression systems in fish cells in order to determine their efficiency quantitatively. Emphasis was given to zebrafish cultures with the goal of eventually injecting in vitro manipulated embryo cells into host embryos and thereby creating transgenic chimera. / Graduation date: 1993

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Alzheimer’s disease (AD) is the most prevalent form of dementia. There is considerable evidence that AD is caused by accumulating amyloid beta peptides in the brain, as a result of amyloid precursor protein (APP) cleavage by secretase enzymes. The presenilin proteins are central to the gamma-secretase cleavage of the intramembrane domain of APP. Aberrant splicing and point mutations in the human presenilin genes, PSEN1 and PSEN2, have been linked to familial forms of AD, through aberrant APP cleavage resulting in irregular amyloid beta formation. Paper 1 gives a review of the literature on AD research and how animal models are used to elucidate mechanisms of AD pathogenesis. The zebrafish model is used in this thesis to investigate genes with potential relevance to AD initiation and pathogenesis. Paper 2 demonstrates that lowlevel aberrant splicing of exon 8 in psen1 transcripts in zebrafish embryos produces potent dominant negative effects that increased psen1 transcription, cause a dramatic hydrocephalus phenotype, decreased pigmentation and other developmental defects. Similar effects are also observed after low-level interference with splicing of exon 8 in psen2 transcripts. In paper 3, a microarray analysis was performed to analyse global gene expression changes to illuminate the molecular aetiology of the phenotypic effects described in paper 2. Of the 100 genes that showed greatest dysregulation after psen1 or psen2 manipulation, 12 genes were common to both treatments. Five of these have known function and showed increased expression. Cyclin G1 (ccng1) was of particular interest as the human CCNG1 protein shows increased immunoreactivity in the cytoplasm of neurons in human AD brains. Phylogenetic and conserved synteny analysis confirmed the orthology of zebrafish ccng1 with human CCNG1. Expression of zebrafish ccng1 in developing embryos at 24 hours post fertilization (hpf) was observed in the eye, tectum and somites. Decreased Ccng1 expression does not lead to any developmental defects and also cannot rescue the hydrocephalus or pigmentation phenotypes of embryos with aberrant splicing of psen1 exon 8. An analysis of zebrafish ccng1 function in paper 4 (thesis chapter in the form of a manuscript) indicates that truncation of Ccng1 appears to cause developmental defects in the brain, notochord and somites, however, it does not decrease the level of normal ccng1 transcript. The CCNG1 paralogue, Cyclin G2, (CCNG2), is also expressed in zebrafiish (ccng2). Decreasing the expression of Ccng2 results in similar effects on embryo development as truncating Ccng1. Therefore, the truncated forms of Ccng1 potentially interfere with Ccng2 function in a dominant negative manner. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342482 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 58-59). Also available on the World Wide Web.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology and Genetics, Department of Biological Sciences. Title from pdf file main screen (viewed on April 28, 2010). Accompanied by four supplementary video recording files. Includes bibliographical references.

Thesis (M.Sc.)--York University, 2009. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 53-57). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR51603

Thesis (Ph. D.)--Miami University, Dept. of Zoology, 2004. / Title from second page of PDF document. Includes bibliographical references (p. 116-127).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Currently, the zebrafish (Danio rerio) is widely used in research related to anxiolytic properties and pharmacological mechanisms of alcoholism. Previous research has shown its effects on several behavioral parameters, similar to that already observed in exposure to anxiolytic drugs, as well as attenuation of fear responses against the predator. The detection of the presence of predator can occur in a visual way, and threatened fish has the ability to communicate about risk to other individuals through the release of a chemical called disturbance substance. The predatory threat causes physiological adjustments as a result of the stress response, which aims to maintain the homeostasis of the animal, and the secretion of the hormone cortisol, the main endocrine indicator of this response. As ethanol can affect the reactions of fear, we evaluated its possible effects on cortisol levels in zebrafish as a result of exposure to the visual perception of predator and disturbance substance. Finally, our study demonstrated for the first time the interference of low doses of ethanol (0.25%, 0.50%, and 1%) for acute exposure on the stress response triggered by the presence of the predator, blocking cortisol secretion in both fish: donors and receivers. In the absence of ethanol, we detected stress response with an increase in cortisol levels in fish tissue disturbance substance of donor fish due to the presence of predators, as well as in fish receptors due to the perception of chemical information. Therefore, we postulate that ethanol may interfere with the visual perception of both predator and/or secretion of disturbance substance, as well as the ability to detect this substance. / Recentemente o peixe zebra (Danio rerio) tem sido amplamente utilizado em pesquisas ligadas as propriedades ansiolíticas e mecanismos farmacológicos do alcoolismo. Pesquisas anteriores tem demonstrado seus efeitos em diversos parâmetros comportamentais, similares aos já verificados na exposição a drogas ansiolíticas. Atenuação das reações de medo, ocasionadas pela presença do predador também tem sido demonstradas, em decorrência da exposição ao etanol. A detecção da presença do predador pode ocorrer de maneira visual, onde o peixe ameaçado tem a capacidade de comunicar demais indivíduos sobre o risco, através da liberação de substância química, denominada substância distúrbio. A ameaça predatória ocasiona ajustes fisiológicos em consequência da resposta de estresse, que tem por objetivo manter a homeostase do animal, sendo a secreção do hormônio cortisol o principal indicador desta resposta. Uma vez que o etanol pode afetar as reações de medo, avaliamos seus possíveis efeitos no aumento dos níveis de cortisol de peixes zebra, em consequência da exposição visual ao predador e percepção da substância distúrbio. Finalmente, nosso trabalho demonstrou por primeira vez a interferência de baixas doses de etanol (0,25%, 0,50% e 1%) em exposição aguda, na resposta de estresse desencadeada pela presença do predador, bloqueando a secreção de cortisol tanto em peixes zebra doadores como peixes zebra receptores da substância distúrbio. Na ausência de etanol, detectamos resposta de estresse com aumento dos níveis de cortisol tecidual em peixes doadores de substância distúrbio devido a presença do predador, assim como em peixes receptores devido a percepção da informação química. Tendo isto em conta, postulamos que o etanol pode interferir na percepção visual do predador e/ou a secreção de substância distúrbio, assim como na capacidade de detecção desta substância por peixes receptores.

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Cardiac, metabolic and growth response of early-stage chicken embryos to perturbations in yolk environment was investigated. Also, effects of parental hypoxia exposure on hypoxia resistance, thermal tolerance and body length of zebrafish larvae were investigated. In the first study, thyroxine, triiodothyronine and testosterone produced differential effects on heart rate and development rate of chicken embryos during the first 4 days of development. Triiodothyronine caused a dose-dependent increase in heart rate when applied at 40 or 70 hours of age, while thyroxine caused a dose-dependent increase in heart rate when applied at 40 hours only. Testosterone and propyl-thiouracil (deiodinase antagonist) did not have an effect on heart rate. Development rate was not changed by thyroxine, triiodothyronine, testosterone or propyl-thiouracil, which suggested that heart rate changes did not result from changes in embryo maturity. In the second study, chicken embryos exposed to yolks of different bird species during early-stage embryonic development showed changes in heart rate, mass-specific oxygen consumption and body mass that scaled with the egg mass, incubation period length, and yolk triiodothyronine and testosterone levels of the species from which yolk was derived. In the third study, this phenomenon was investigated between layer and broiler chickens. Heart rate, oxygen consumption and body mass of broiler and layer embryos were significantly changed by a breed-specific change in yolk environment. Yolk triiodothyronine and testosterone concentrations of broiler and layer eggs did not suggest that these hormones were responsible for physiological and morphological changes observed. The final study demonstrated that hypoxia resistance and body lengths, but not thermal tolerance of zebrafish larvae was increased by parental hypoxia exposure.