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

Découvert en 1964, le virus d'Epstein-Barr appartient à la famille des gamma-herpèsvirus. Ce virus à ADN présente une forte prévalence (90% de la population adulte est infectée). Ce fut le premier virus identifié comme associé à des cancers (lymphome de Burkitt et d'Hodgkin, carcinome gastrique et de l'oropharynx). Ce virus a pour spécificité de posséder deux cycles distincts : latent et lytique (production de particules virales). Le facteur de transcription viral EB1 (ou Zebra) est un élément clé lors de l'initiation du cycle lytique et semble une cible importante pour l'élaboration de nouveaux traitements. Une première partie de ce travail concerne la caractérisation d'une protéine cellulaire (l'Ubinucléine) qui interagit et inhibe l'activité de EB1. Cette protéine voyage entre noyaux et jonctions serrées. Elle appartient à la famille des « NACos » (nuclear and adhesion complex components). La fonction de l'Ubinucléine n'est pas connue et sa protéomique quand elle est localisée dans les jonctions serrées, a été réalisée. Des études fonctionnelles montrent que l'Ubinucléine interagit avec plusieurs partenaires cellulaires, emprunte la voie d'endocytose dépendante de la clathrine et que sa localisation cellulaire (nucléaire ou dans les jonctions serrées) est affectée par la PKA. Dans une seconde partie, nous nous sommes intéressés à des molécules inhibitrices du facteur de transcription viral EB1. Après criblage à haut débit de composés chimiques (EMBL-Heidelberg), des tests in-vitro ont permis d'en sélectionner un pour des essais in-vivo. Ce composé chimique inhibe l'activité du facteur de transcription EB1 et bloque précocement la mise en place du cycle lytique dans des cellules de lymphome de Burkitt. Il semble donc intéressant d'améliorer l'efficacité et la spécificité de cette molécule. / Discovered in 1964, Epstein-Barr virus belongs to the Gamma-herpesvirus family. This DNA virus shows an important prevalence (90% of the adult population is infected). It was the first virus identified as associated with cancers (Hodgkin and Burkitt lymphomas, Gastric and oropharyngal carcinomas). It presents two distinct cycles: Latency and Lytic cycle (viral particle production). Viral transcription EB1 (or Zebra) is a key element for lytic cycle initiation and seems to be an important target for future treatment development. A first part of this work concerned the characterization of the cellular protein Ubinuclein that inhibits EB1 activity. This protein travels between nucleus and tight junctions. It belongs to the “NACos” (nuclear and adhesion complex components) protein family. Ubinuclein function is not known and its proteomic was performed when it was localized at tight junction. Next, functional studies showed that Ubinuclein interacts with various cellular partners and goes though the clathrin dependent endocytosis pathway. It's localisation (nuclear or at tight junction) changes with PKA activity. In the second part of this work, we focus on viral transcription factor EB1 inhibitors. After high-throughput screening of compounds (EMBL-Heidelberg), in-vitro assays allowed to select one molecule for in-vivo experiments. This compound inhibits the activity of the transcription factor EB1 and stops early lytic cycle establishment in Burkitt lymphoma cells. Further work needs to be done to increase efficacy and specificity of this molecule.

Neritina zebra à um gastrÃpode comum em bancos de lama e Ãguas salobras. Este estudo tem como objetivos revisar o conteÃdo da literatura especializada e acrescentar informaÃÃes sobre a anatomia de Neritina zebra. Foram analisados os animais coletados no estuÃrio do rio Cearà - CE, no Parque Estadual do rio Cocà â CE e os espÃcimes tombados na coleÃÃo MalacolÃgica "Prof. Henry Ramos Matthews" (CMPHRM) da Universidade Federal do Cearà (UFC) e na coleÃÃo de Moluscos do Museu de Zoologia da Universidade de SÃo Paulo (MZUSP). A rÃdula foi observada e fotografada atravÃs de microscÃpio Ãptico e microscÃpio eletrÃnico de varredura (MEV). A protoconcha foi observada e fotografada atravÃs do MEV. N. zebra apresenta concha globosa, protoconcha com 1,5 voltas, teleoconcha com 3,5 voltas, lÃbio interno denteado, espira baixa, abertura semilunar. Concha apresentando vÃrias cores e vÃrios padrÃes de âziguezagueâ. A columela forma um septo transversal. O opÃrculo à calcÃrio e liso, com apÃfise bifurcada. O mÃsculo columelar à bem desenvolvido, com duas partes de tamanhos diferentes. BrÃnquia bipectinada. O osfrÃdio encontra-se à frente da membrana suspensÃria da brÃnquia, do lado esquerdo do manto. O coraÃÃo apresenta um Ãtrio e um ventrÃculo. A rÃdula à do tipo rhipidoglossa, apresentando a fÃrmula geral .5.1.5.. EsÃfago e intestino longos. O intestino passa atravÃs da cavidade pericardial, estando envolvido pelo ventrÃculo. O sistema reprodutor masculino apresenta uma abertura genital, ducto espermÃtico longo e enrolado, glÃndula prostÃtica e pÃnis com formato laminar do lado direito da regiÃo cefÃlica. O sistema reprodutor feminino apresenta trÃs poros genitais (um vaginal, um âgonoporo principalâ e um âducto enigmÃticoâ), um saco de reforÃo, saco do espermatÃforo, ovÃrio com vÃrias vilosidades digitiformes paralelas, receptÃculo seminal e glÃndulas de albÃmen e da cÃpsula. / Neritina zebra is a common gastropod on mud bottom and brackish water. This study aims to review the literature and add information about the anatomy of Neritina zebra. We analyzed the animals collected in the estuary of the Cearà river â CE, in âParque Estadual do rio CocÃâ - CE and specimens deposited in âColeÃÃo MalacolÃgica Prof. Henry Ramos Matthews" (CMPHRM) of âUniversidade Federal do CearÃâ (UFC) and âColeÃÃo de Moluscos do Museu de Zoologia da Universidade de SÃo Pauloâ (MZUSP). The radula was observed and photographed through optic and scanning electron microscope (SEM). The protoconch was observed and photographed by SEM. N. zebra presents globose shell, protoconch with 1.5 whorls, teleoconch with 3.5 whorls, small teeth on inner lip, low spire, semilunar aperture. Shells present several different colors and patterns of zigzag. The columella forms a transversal septum. Operculum calcareous, smooth, with bifurcated apophysis. The columellar muscle is well developed, with two parts of different sizes. Bipectinate gill. The osphradium is in front of the suspensory membrane of the gill, on the left side of the mantle. The heart presents one auricle and one ventricle. The radula is rhipidoglossa type, presenting the general formula .5.1.5. . Long esophagus and intestine. The intestine passes through the pericardial cavity, enwrapped by the ventricle. The male reproductive system presents one genital opening, very long and coiled spermatic duct, prostate gland and penis with laminar shape on the right side of the cephalic region. The female reproductive system has three genital pores (vaginal pore, main female gonopore and enigmatic duct), a reinforcement sac, a spermatophore sac, an ovary with many small parallel digitiformes vilosities, a seminal receptacle and the albumen and capsule glands.

O ácido retinóico (AR) é sintetizado a partir de oxidações sucessivas do retinol. A última etapa de oxidação é catalisada pelas enzimas RALDHs. O estabelecimento da polaridade ântero-posterior (AP) do coração é crítico para a demarcação das regiões de efluxo (ventrículos) e de influxo (átrios). Nosso grupo propõe que uma onda caudo-rostral (CR) de RALDH2 seja o mecanismo responsável por esta padronização em vertebrados. Para testar o papel da sinalização pelo AR na padronização AP do peixe zebra, manipulamos sua via com o inibidor das enzimas RALDHs, DEAB, com AR e com um inibidor da enzima CYP26 (IC), que cataboliza o AR. Os tratamentos com DEAB durante o período da onda de RALDH2 produziram átrios reduzidos, enquanto que os tratados com AR e com IC apresentam o domínio atrial expandido. Animais tratados com DEAB e AR em um estágio posterior a onda não mostraram diferença significativa ao controle. Estes dados sugerem uma forte correlação entre o evento da onda e a padronização das câmaras cardíacas, semelhante ao que ocorre nos demais amniotos. / The retinoic acid (RA) is synthetized by successive oxidations of retinol. The last oxidation step is catalyzed by the RALDHs enzymes. The establishment of the anteroposterior (AP) polarity is critical for the demarcation of outflow (ventricle) and inflow (atrium) regions. Our group proposes that a caudorostral (CR) wave of RALDH2 is the mechanism responsible for this patterning in vertebrates. In order to test the role of the RA signaling in zebrafish AP patterning, we manipulated its pathway with a RALDH enzymatic inhibitor, DEAB, with RA and with a CYP26 (IC) enzymatic inhibitor, that catabolises the RA. The DEAB treatments during the manifestation of the RALDH wave produced reduced atriums; meanwhile, the treatments with RA and IC presented an atrium expansion. Animals treated with DEAB and RA during a stage posterior to the wave did not present any significant difference. These data suggest a strong correlation between the wave event and the cardiac chamber patterning, similar to the mechanism observed in others amniotes.

Over the past twenty years there has been an exponential increase in the investigation of maternal effects. Understanding the adaptive function of maternal allocation strategies is integral to interpreting the evolutionary outcomes of sexual selection. Thus, model animal systems that facilitate experimental manipulation and controlled investigation of the physiological and behavioural mechanisms underlying maternal effects are important to evolutionary biologists. The zebra finch (Taeniopygia guttata) has been used as a model to investigate avian life-history, signalling behaviour, neurophysiology, mate choice, and more recently, maternal effects. However, a potentially influential and rarely addressed problem with this species is the process of domestication. Within this thesis we aimed to both test current predominant maternal allocation hypotheses, but for the first time in both domesticated and wild zebra finches. Chapter 2 develops on earlier work using domesticated zebra finches that has demonstrated differential allocation of maternally derived yolk androgens and antioxidants in eggs dependent on paternal attractiveness. This chapter specifically tests the ratio of these two yolk resources within individual eggs and shows that the balance of androgens to antioxidants varies by offspring sex and paternal attractiveness. Specifically, we found that mothers allocated a smaller androgen to antioxidant ratio to daughters when paired to green ringed (unattractive) males compared to red ringed (attractive) males. This pattern was reversed for sons, where mothers allocated a larger ratio of androgen to antioxidant when paired to red ringed (attractive) compared to green ringed (unattractive) males. We also show that brood sex ratio depended on both female condition and male attractiveness. It is concluded that investigating female allocation of individual resources within egg yolks may lead to incorrect assumptions on offspring fitness consequences, and that individual female state is an important consideration when predicting a resource allocation strategy. Throughout this thesis colour bands are used as a method to manipulate male attractiveness. In chapter 3 the influence of these bands was further tested to elucidate whether they affect male behaviour or quality. Wild birds were used for this chapter as preferences for bands based on colour have only once been demonstrated in wild birds and it was felt this should also be replicated. We confirmed a female preference for males based on colour bands worn in mate choice trials, with red bands preferred over green. Interestingly, we also found that colour of bands worn by males for an extended period in the single sex aviary influenced both their song rate and condition. Males that had worn red bands sang more in mate choice trials than both green banded or un-banded males. In addition red banded males were found to be in significantly better physical condition. These data suggest that earlier experiments in which it has been assumed that colour bands do not manipulate any form of intrinsic male quality should be re-evaluated. The final two data chapters, 4 and 5, return to investigating maternal allocation in response to male attractiveness, but for the first time in wild birds. Chapter 4 presents an experiment that was conducted on a wild, nest box breeding population of birds. Maternal resources allocation was investigated in both an experimental manipulation of male attractiveness, and also by correlating resource allocation with paternal phenotypic traits. A limited sample size meant few conclusions could be drawn from the experimental study, but significant positive correlations were found between both egg size and yolk testosterone (T) concentration and male phenotypic traits. This suggested that wild zebra finches may follow a positive investment strategy but requires further investigation. In chapter 5 experiments were repeated on wild birds that had been brought into captivity, to allow both an improved sample size and further control of influential environmental features. Again, female allocation strategies are tested using colour bands to manipulate male attractiveness, to allow direct comparisons with work on domesticated zebra finches. We found that females laid significantly heavier eggs for attractive compared to unattractive males, supporting the positive investment hypothesis. In addition we found an interaction between offspring size and paternal attractiveness treatment, with daughters of red banded (attractive) males being smaller than sons. This experiment is the first to demonstrate the influence of colour bands on maternal allocation in wild zebra finches and also provides further support for the positive investment hypothesis in this species. The final chapter discusses how overall patterns of female allocation were shown to be similar among wild and domesticated populations. It is concluded that demonstrated variations between populations and/or contexts reported in these studies cannot be explained by inherent differences between wild and domesticated individuals. Thus, the zebra finch remains a robust and reliable model for testing the evolution of avian maternal allocation strategies.

published_or_final_version / abstract / Anatomy / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

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

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