Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 54-56). / vii, 56 leaves, bound ill. 29 cm

Made available in DSpace on 2014-06-11T19:30:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-29Bitstream added on 2014-06-13T20:39:50Z : No. of bitstreams: 1 sene_vf_me_botib.pdf: 1250166 bytes, checksum: ef91b80ce3068f6731dda41593edae4e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram analisadas seis espécies/citótipos de peixes do gênero Eigenmannia, Eigenmannia sp1, Eigenmannia sp2, E. cf. trilineata, Eigenmannia sp e dois citótipos de E. virescens de diferentes bacias hidrográficas brasileiras, com o uso de técnicas citogenéticas básicas (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e moleculares (hibridação fluorescente in situ com sondas de DNAr 18S e 5S, com sondas teloméricas (TTAGGG)n, com sondas para elementos retrotransponíveis Rex 1 e Rex 3 e também por microdissecção, amplificação e hibridação in situ fluorescente com sonda produzida a partir do cromossomo sexual Y de Eigenmannia sp2). As espécies/citótipos analisados apresentaram intensa variação em seus números diploides, de 2n=28 cromossomos em Eigenmannia sp1, 2n=31/32 em Eigenmannia sp2, 2n=34 em E. cf. trilineata, 2n=36 em Eigenmannia sp e 2n=38 em E. virescens, além da ocorrência de sistema sexual XX-XY no citótipo de E. virescens do rio Ribeirão Claro (chamado de E. virescens-XY) e ausência desse sistema no citótipo do rio Mogi-Guaçu (chamado de E. virescens), bem como a ocorrência de sistema múltiplo do tipo X1X1X2X2-X1X2Y em Eigenmannia sp2 do rio Araquá. O DNAr 5S está organizado em duas classes distintas e foi localizado em diferentes cromossomos entre estas espécies/citótipos, mas sempre em posição terminal dos cromossomos, com exceção apenas do par cromossômico 7 de Eigenmannia sp1, que possui DNAr 5S em posição intersticial. Ainda, sequências de DNAr 5S foram localizadas no par sexual XY do citótipo de E. virescens-XY, evidenciando uma nova característica dos cromossomos sexuais deste grupo. As RONs, identificadas pelo tratamento com nitrato de Prata e pela sonda de DNAr 18S, foram sempre localizadas em compartimentos cromossômicos distintos do DNAr 5S e, apesar de serem localizadas... / Conventional (Giemsa, Ag-NOR, C-banding) and molecular (Fluorescent in situ hybridization with 18S and 5S rDNA probes, telomeric repeats (TTAGGG)n, Rex1 and Rex3 retrotransposable elements and microdissection, amplification and fluorescent in situ hybridization with probes produced from the Y sex chromosome of Eigenmannia sp2.) cytogenetic studies were carried out in six fish species/cytotypes of the genus Eigenmannia from different Brazilian hydrographic basins. The analyzed species/cytotypes presented an intense variation in diploid number, ranging from 2n=28 chromosomes in Eigenmannia sp1, 2n=31/32 in Eigenmannia sp2, 2n=34 in Eigenmannia cf. trilineata, 2n=36 in Eigenmannia sp to 2n=38 in E. virescens, besides the occurrence of a sex chromosome system XX-XY in the cytotypes of E. virescens from Ribeirão Claro river (named as E. virescens-XY) and absence of this sex chromosome system in the cytotypes of Mogi-Guaçu river (named E. virescens), as well as the occurrence of a multiple sex chromosome system X1X1X2X2-X1X2Y in Eigenmannia sp2 from Araquá river. The 5S rDNA is organized in two distinct classes and was located in different chromosomes between these species/cytotypes; on the other hand, the location in the terminal position of chromosomes was a conserved feature, with exception of chromosome pair 7 in Eigenmannia sp1, which had 5S rDNA sites in an interstitial position. Yet, 5S rDNA signals were detected on the XY sex chromosome of E. virescens-XY, showing some new characteristics of sex chromosomes in this group. The NORs, identified by silver nitrate staining and 18S rDNA probes, were always located in distinct chromosome compartments of 5S rDNA and besides located in different chromosomes in all analyzed samples, they remained conserved through the karyotypic differentiation process in this group. The analysis of constitutive heterochromatin... (Complete abstract click electronic access below)

Resumo: Foram analisadas seis espécies/citótipos de peixes do gênero Eigenmannia, Eigenmannia sp1, Eigenmannia sp2, E. cf. trilineata, Eigenmannia sp e dois citótipos de E. virescens de diferentes bacias hidrográficas brasileiras, com o uso de técnicas citogenéticas básicas (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e moleculares (hibridação fluorescente in situ com sondas de DNAr 18S e 5S, com sondas teloméricas (TTAGGG)n, com sondas para elementos retrotransponíveis Rex 1 e Rex 3 e também por microdissecção, amplificação e hibridação in situ fluorescente com sonda produzida a partir do cromossomo sexual Y de Eigenmannia sp2). As espécies/citótipos analisados apresentaram intensa variação em seus números diploides, de 2n=28 cromossomos em Eigenmannia sp1, 2n=31/32 em Eigenmannia sp2, 2n=34 em E. cf. trilineata, 2n=36 em Eigenmannia sp e 2n=38 em E. virescens, além da ocorrência de sistema sexual XX-XY no citótipo de E. virescens do rio Ribeirão Claro (chamado de E. virescens-XY) e ausência desse sistema no citótipo do rio Mogi-Guaçu (chamado de E. virescens), bem como a ocorrência de sistema múltiplo do tipo X1X1X2X2-X1X2Y em Eigenmannia sp2 do rio Araquá. O DNAr 5S está organizado em duas classes distintas e foi localizado em diferentes cromossomos entre estas espécies/citótipos, mas sempre em posição terminal dos cromossomos, com exceção apenas do par cromossômico 7 de Eigenmannia sp1, que possui DNAr 5S em posição intersticial. Ainda, sequências de DNAr 5S foram localizadas no par sexual XY do citótipo de E. virescens-XY, evidenciando uma nova característica dos cromossomos sexuais deste grupo. As RONs, identificadas pelo tratamento com nitrato de Prata e pela sonda de DNAr 18S, foram sempre localizadas em compartimentos cromossômicos distintos do DNAr 5S e, apesar de serem localizadas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Conventional (Giemsa, Ag-NOR, C-banding) and molecular (Fluorescent in situ hybridization with 18S and 5S rDNA probes, telomeric repeats (TTAGGG)n, Rex1 and Rex3 retrotransposable elements and microdissection, amplification and fluorescent in situ hybridization with probes produced from the Y sex chromosome of Eigenmannia sp2.) cytogenetic studies were carried out in six fish species/cytotypes of the genus Eigenmannia from different Brazilian hydrographic basins. The analyzed species/cytotypes presented an intense variation in diploid number, ranging from 2n=28 chromosomes in Eigenmannia sp1, 2n=31/32 in Eigenmannia sp2, 2n=34 in Eigenmannia cf. trilineata, 2n=36 in Eigenmannia sp to 2n=38 in E. virescens, besides the occurrence of a sex chromosome system XX-XY in the cytotypes of E. virescens from Ribeirão Claro river (named as E. virescens-XY) and absence of this sex chromosome system in the cytotypes of Mogi-Guaçu river (named E. virescens), as well as the occurrence of a multiple sex chromosome system X1X1X2X2-X1X2Y in Eigenmannia sp2 from Araquá river. The 5S rDNA is organized in two distinct classes and was located in different chromosomes between these species/cytotypes; on the other hand, the location in the terminal position of chromosomes was a conserved feature, with exception of chromosome pair 7 in Eigenmannia sp1, which had 5S rDNA sites in an interstitial position. Yet, 5S rDNA signals were detected on the XY sex chromosome of E. virescens-XY, showing some new characteristics of sex chromosomes in this group. The NORs, identified by silver nitrate staining and 18S rDNA probes, were always located in distinct chromosome compartments of 5S rDNA and besides located in different chromosomes in all analyzed samples, they remained conserved through the karyotypic differentiation process in this group. The analysis of constitutive heterochromatin... (Complete abstract click electronic access below) / Orientador: Fausto Foresti / Coorientador: José Carlos Pansonato Alves / Banca: Daniela Cristina Ferreira / Banca: Orlando Moreira Filho / Mestre

Crosses were made between a stock of summer steelhead (Salmo gairdneri) known to be resistant to infection by Ceratomyxa shasta and stocks of summer steelhead known to be susceptible. Ceratomyxosis, the disease caused by C. shasta was initiated by exposure to Willamette River water. I found that the crosses were intermediate in susceptibility to ceratomyxosis relative to the parental stocks. There was no difference in susceptibility to ceratomyxosis between reciprocal crosses of the same stocks. Persistence of moderate susceptibility in the F₂ generation of experimental stock crosses and examples from both wild and hatchery stocks of mixed ancestry indicate long term disease problems may result from introductions of less adapted, foreign stocks. / Graduation date: 1987

Life history within a single species can vary significantly. Many of these differences are associated with varying environmental conditions. Understanding what environmental conditions cue alternate life histories within a single species has been researched extensively. In salmonid fishes, more than almost any other group, varying environmental conditions give rise to individuals within species that take markedly different life history trajectories. Oncorhynchus mykissis a species of salmonid native to the Pacific Northwest region of North America. This species has two life history forms, anadromous and resident. The anadromous form spends a portion of its life in ocean while the resident life history form completes its entire life history in freshwater. Until the decision to migrate and morphological changes associated with smoltification occur, the two life history variants of this species are indistinguishable from each other. This ambiguity in juvenile O. mykiss morphology presents challenges for conservation managers charged with protecting and increasing threatened O. mykiss populations around the Pacific Northwest because conservation efforts cannot be evaluated until juvenile fish make the decision to migrate. Microarray gene expression analysis was used to profile gene expression in juvenile populations of wild and hatchery O. mykiss to identify gene expression variation associated with alternate life history variants. This analysis identified 8 DNA sequences present in both brain and gill tissues that differ in expression in rainbow trout and steelhead hatchery stocks. Differential expression as quantified by microarrays was validated with quantitative real-time PCR. Lastly, the expression of these putative life history markers was preliminarily evaluated in a wild population of O. mykiss at sample locations in the South Fork John Day River Basin, Oregon with known ratios of juvenile anadromous and resident fish.

Gene expression profiles of tissues and cell-lines can be powerful tools for documenting the genetic response to a particular treatment, such as stressors. However, there is a paucity of information on the genetic stress response in the brain. Therefore, we attempted to profile gene expression in the brain of juvenile steelhead trout (Oncorhynchus mykiss) in response to stressors commonly encountered in aquaculture settings and similar to those encountered in hydropower dam mitigation efforts. We subjected fish to a combined out-of-water and low-water stressor totaling three hours. Plasma stress response factors indicate that fish were undergoing a physiological stress response after 3 hours of continuous stressor. We utilized suppression subtractive hybridization to identify cDNA fragments up- or down-regulated in the brain upon completion of the stressor. Forward and reverse subtractions, and sub-cloning of the purified PCR products yielded 59 clones all of which were sequenced. Sequenced cDNA fragments were subjected to BLASTn and BLASTx searches over the course of one year. Fragments fell into the following functional categories: those associated with ATP generation, signal transduction, ion transport, translational machinery, DNA packaging and mobilization, cell structure, and cDNA fragments with cryptic function. Of the 59, 12 were selected for further analysis, and 5 were confirmed to be differentially expressed by northern hybridization. The differentially expressed genes included cytochrome b, NADH dehydrogenase subunit 2, ATPsynthase subunit 6, a cDNA fragment with unknown function, and neuron specific gene 1. Our results present a first attempt to profile gene expression in the brain of fish and demonstrate the power of molecular tools at capturing large amounts of biological information without having to target any one particular gene. A gene expression profile of the brain consequent to stress provides a catalog of responses at a given time point. This catalog can then be used to isolate full-length cDNAs, localize mRNAs in the brain or other tissue, as probes to determine expression patterns and time courses of gene expression in other tissues, and for the quantification of cDNA molecules with real time PCR. / Graduation date: 2003

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

by Yan Hiu Mei, Carol. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-126). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xii / Chapter CHAPTER 1 --- TRANSGENIC TECHNOLOGY --- p.1 / Chapter 1.1 --- Transgenesis in animals --- p.1 / Chapter 1.2 --- Transgenic fish in toxicology --- p.4 / Chapter 1.2.1 --- Aquatic metal toxicity --- p.4 / Chapter 1.2.2 --- Environmental monitoring of aquatic metal toxicity --- p.5 / Chapter 1.2.3 --- Biomarkers --- p.6 / Chapter 1.3 --- Transgenics in aquaculture --- p.9 / Chapter 1.3.1 --- Revolution is needed in aquaculture --- p.9 / Chapter 1.3.2 --- Aquaculture potential of tilapia in China --- p.10 / Chapter 1.3.3 --- Endocrinology for fish growth --- p.12 / Chapter 1.3.4 --- Growth promotion by exogenous growth hormone in tilapia --- p.14 / Chapter 1.3.5 --- Accelerated growth in transgenic fish --- p.15 / Chapter 1.4 --- General principle in transgenic fish production --- p.16 / Chapter 1.5 --- Project aim --- p.22 / Chapter CHAPTER 2 --- ISOLATION AND CHARACTERIZATION OF ZEBRAFISH METALLOTHIONEIN GENE PROMOTER --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.1.1 --- Metallothionein --- p.23 / Chapter 2.1.2 --- Biological functions --- p.24 / Chapter 2.1.3 --- Metallothionein gene regulations --- p.25 / Chapter 2.1.4 --- Metallothionein as biomarker for metal pollution --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- General molecular biology techniques --- p.28 / Chapter 2.2.2 --- Sequences of PCR primers used --- p.31 / Chapter 2.2.3 --- Cloning zebrafish MT gene 5-flanking region --- p.31 / Chapter 2.2.4 --- Cloning zebrafish MT gene --- p.32 / Chapter 2.2.5 --- Cloning full length zMT gene --- p.33 / Chapter 2.2.6 --- Cell culture --- p.35 / Chapter 2.2.7 --- Transient transfection assay --- p.37 / Chapter 2.2.8 --- Electrophoretic mobility shift assay --- p.39 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Zebrafish metallothionein gene --- p.42 / Chapter 2.3.2 --- Deletion analysis of zMT promoter by transient transfection assay --- p.48 / Chapter 2.3.3 --- Functional characterization of zebrafish metallothionein promoter --- p.57 / Chapter 2.4 --- Discussions --- p.61 / Chapter 2.4.1 --- Zebrafish MT gene --- p.61 / Chapter 2.4.2 --- Functional characterization of zebrafish MT promoter --- p.61 / Chapter CHAPTER 3 --- PREPARATION OF GENE CONSTRUCTS FOR TRANSFER IN ZEBRAFISH --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.1.1 --- Zebrafish as model in toxicological studies --- p.65 / Chapter 3.1.2 --- Reporter gene system --- p.66 / Chapter 3.1.3 --- Transgenic reporter fish --- p.68 / Chapter 3.1.4 --- Gene transfer by electroporation in zebrafish --- p.68 / Chapter 3.1.5 --- Objective --- p.69 / Chapter 3.2 --- Materials and methods --- p.70 / Chapter 3.2.1 --- Design of gene constructs for ectopic expression in zebrafish --- p.70 / Chapter 3.2.2 --- Testing electroporation conditions for zebrafish --- p.72 / Chapter 3.3 --- Results --- p.73 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Engineering gene constructs --- p.76 / Chapter 3.4.2 --- Applications of transgenic zebrafish --- p.79 / Chapter CHAPTER 4 --- GENE TRANSFER EXPERIMENTS ON TILAPIA --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.85 / Chapter 4.2.1 --- Isolation of O. aureus growth hormone --- p.85 / Chapter 4.2.2 --- Engineering gene constructs for ectopic expression in tilapia --- p.86 / Chapter 4.2.3 --- Gene transfer in tilapia --- p.87 / Chapter 4.2.4 --- Screening transgenic tilapia --- p.89 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Tilapia growth hormone --- p.91 / Chapter 4.3.2 --- Gene constructs for ectopic expression in tilapia --- p.94 / Chapter 4.3.3 --- Testing electroporation conditions --- p.96 / Chapter 4.3.4 --- PCR screening for transgenic fish --- p.97 / Chapter 4.4 --- Discussions --- p.101 / Chapter 4.4.1 --- Tilapia growth hormone --- p.101 / Chapter 4.4.2 --- Electroporation experiments on of tilapia eggs --- p.101 / Chapter 4.4.3 --- Improvements on gene construct design for tilapia --- p.104 / Chapter 4.4.4 --- Ethical and safety considerations --- p.106 / Chapter CHAPTER 5 --- REFERENCES --- p.114 / APPENDIX --- p.127