Thèse : Zoologie : Paris, Faculté des sciences : 1850. / Titre provenant de l'écran-titre.

Lau Suk Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 128-150). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Table of content --- p.ii / List of Figures --- p.x / List of Tables --- p.xiv / Abbreviation --- p.xv / Abstract --- p.xviii / 論文摘要 --- p.xx / Chapter 1 --- Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Tetraodon nigroviridis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Genomic Sequencing Project --- p.3 / Chapter 1.1.3 --- Tetraodon nigroviridis as Study Model --- p.4 / Chapter 1.1.3.1 --- Genomic Comparison --- p.4 / Chapter 1.1.3.2 --- Gene Order and Structural Studies --- p.5 / Chapter 1.1.3.3 --- Genomic Evolution --- p.6 / Chapter 1.2 --- Transcriptional Regulation and Transcription Factors Binding Sites Prediction --- p.7 / Chapter 1.2.1 --- Transcriptional Regulation --- p.7 / Chapter 1.2.1.1 --- Chromatin Remodeling --- p.7 / Chapter 1.2.1.2 --- Locus Control Regions (LCR) and Boundary Elements --- p.8 / Chapter 1.2.1.3 --- Promoter Structure --- p.9 / Chapter 1.2.1.4 --- Transcriptional Machinery Assembly --- p.10 / Chapter 1.2.2 --- Transcription Factors and Their Binding Sites --- p.11 / Chapter 1.2.3 --- Transcription Factor Binding Site Prediction --- p.12 / Chapter 1.3 --- Acetylcholinesterase --- p.15 / Chapter 1.3.1 --- Background --- p.15 / Chapter 1.3.2 --- Regulation ofAChE --- p.17 / Chapter 1.3.2.1 --- Transcriptional Level --- p.17 / Chapter 1.3.2.2 --- Post-transcriptional Level --- p.19 / Chapter 1.3.2.3 --- Post-translational Level --- p.20 / Chapter 1.3.2.3.1 --- Oligomerization --- p.20 / Chapter 1.3.2.3.2 --- Glycosylation --- p.21 / Chapter 1.3.2.3.3 --- Phosphroylation --- p.22 / Chapter 1.3.3 --- Functions of AChE --- p.23 / Chapter 1.3.3.1 --- Hydrolysis Acetylcholine --- p.23 / Chapter 1.3.3.2 --- Embryonic Development --- p.23 / Chapter 1.3.3.3 --- Haemotopotesis and Thrombopsiesis --- p.24 / Chapter 1.3.3.4 --- Neuritogensis --- p.24 / Chapter 1.3.3.5 --- Amyloid Fibre Assembly --- p.24 / Chapter 1.3.3.6 --- Apoptosis --- p.25 / Chapter 1.3.4 --- AChE and Alzheimer's disease --- p.25 / Chapter 1.3.4.1 --- Treatment for AD Patients --- p.27 / Chapter 1.4 --- Inducible Cell Expression Systems --- p.28 / Chapter 1.5 --- Objectives --- p.32 / Chapter 2 --- Chapter 2 Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Primer Design --- p.34 / Chapter 2.2.2 --- Cell Culture --- p.34 / Chapter 2.2.3 --- Transformation --- p.35 / Chapter 2.2.4 --- Plasmids Preparation --- p.35 / Chapter 2.2.5 --- Plasmids Screening --- p.36 / Chapter 2.2.6 --- RNA Extraction --- p.36 / Chapter 2.2.7 --- Reverse Transcriptase Polymerase Chain Reaction and Construction tnAChE/pCR4 vector --- p.37 / Chapter 2.2.8 --- Genomic Analysis --- p.37 / Chapter 2.2.9 --- Protein Sequence Analysis --- p.38 / Chapter 2.2.10 --- Genomic DNA Extraction --- p.39 / Chapter 2.2.11 --- Construction of Reporter Vectors ptnAChE_565/pGL3 and ptnAChK1143/pGL3 --- p.39 / Chapter 2.2.12 --- Luciferase Assay --- p.40 / Chapter 2.2.13 --- Transcription Factors and Promoter Prediction --- p.40 / Chapter 2.2.14 --- Protein Assay --- p.41 / Chapter 2.2.15 --- AChE Activity Determined by Ellman's Method --- p.41 / Chapter 2.2.16 --- Histochemistry --- p.42 / Chapter 2.2.17 --- Protein Extraction from Tissues --- p.42 / Chapter 2.2.18 --- Construction of Bacterial Expression Vector His-MBP-tnAChEAC/pHISMAL --- p.43 / Chapter 2.2.19 --- Protein Expression in Bacterial Expression System --- p.43 / Chapter 2.2.20 --- Purification and Thrombin Cleavage of His-MBP- tnAChEAC --- p.44 / Chapter 2.2.21 --- SDS Electrophoresis --- p.44 / Chapter 2.2.22 --- Western Blotting --- p.45 / Chapter 2.2.23 --- Construction of Tet-Off Expression Vector --- p.45 / Chapter 2.2.24 --- Transient Expression of tnAChEAC --- p.46 / Chapter 2.2.25 --- Establishment of Stable Tet-Off CHO Cell Lines Overexpressing tnAChEAC --- p.47 / Chapter 2.2.26 --- MTT Assay --- p.47 / Chapter 2.2.27 --- Partial Purification of tnAChEΔC --- p.48 / Chapter 3 --- Chapter 3 Sequence Analysis of AChE Gene of Tetraodon nigroviridis --- p.49 / Chapter 3.1 --- Results --- p.49 / Chapter 3.1.1 --- Cloning of tnAChE from Tetraodon nigroviridis Brain --- p.49 / Chapter 3.1.2 --- "Comparative genomic analysis of tnAChE with Human, Rat, Mouse, Takifugu rubripes, ZebrafishAChE" --- p.49 / Chapter 3.1.3 --- Primary Sequence Analysis --- p.52 / Chapter 3.1.4 --- Promoter and Transcriptional Factors Predictedin tnAChE Promoter Region --- p.60 / Chapter 3.1.4.1 --- Promoter Region Analysis In Silico --- p.60 / Chapter 3.1.4.2 --- Promoter Activity Analysis --- p.76 / Chapter 3.2 --- Discussion --- p.78 / Chapter 4 --- Characterization of tnAChE in Prokaryotic and Eukaryotic Tet-Off Inducible Expression System --- p.91 / Chapter 4.1 --- Results --- p.91 / Chapter 4.1.1 --- AChE Expresses in Tetraodon nigroviridis --- p.91 / Chapter 4.1.2 --- Expression of recombinant tnAChE in Bacterial Expression System --- p.94 / Chapter 4.1.2.1 --- Construction of His-MBP-tnAChEΔC/pHISMAL Construct --- p.94 / Chapter 4.1.2.2 --- His-MBP-tnAChEAC Expression in E. coli Strains BL21 (DE) and C41 --- p.94 / Chapter 4.1.3 --- Expression of tnAChEAC in Mammalian Expression System --- p.99 / Chapter 4.1.3.1 --- Construction of tnAChEAC/pTRE2hgyo Mammalian Expression Vector --- p.99 / Chapter 4.1.3.2 --- Transient Expression of tnAChEAC --- p.99 / Chapter 4.1.3.3 --- Establishment of Tet-Off CHO Cells Stably Expressing the Inducible tnAChEAC --- p.101 / Chapter 4.1.3.4 --- Characterization of Tet-Off tnAChEAC Stably Transfected Cell Clones --- p.103 / Chapter 4.1.3.5 --- Effect of Over Expressed tnAChEAC on cell viability --- p.103 / Chapter 4.1.3.6 --- Partial Purification of tnAChEAC from Stably Transfected Cells --- p.107 / Chapter 4.1.3.7 --- tnAChE and tnAChEAC in Different pH Values --- p.112 / Chapter 4.1.3.8 --- Kinetic Study of tnAChEAC --- p.112 / Chapter 4.1.3.9 --- Inhibition of AChE Activity of Partial Purified tnAChEAC by Huperzine --- p.112 / Chapter 4.2 --- Discussion --- p.116 / Chapter 4.2.1 --- Bacterial Expression System --- p.116 / Chapter 4.2.2 --- Expression of tnAChEΔC in Mammalian System --- p.119 / Chapter 5 --- General Discussion --- p.124 / Chapter 5.1 --- Summaries --- p.124 / Chapter 5.2 --- Further works --- p.126 / Chapter 6 --- References --- p.128 / Appendix 1 internet software and database used in this project --- p.151 / Appendix 2 tnAChE mRNA sequence --- p.152 / Appendix 3 ptnAChE-1143 sequence --- p.154 / Appendix 4 Six open reading frame translation of ptnAChE-1143 --- p.156

Made available in DSpace on 2014-12-17T14:33:04Z (GMT). No. of bitstreams: 1 PabloAM.pdf: 4013213 bytes, checksum: 97a52b7c01ae798889ede43cc9641282 (MD5) Previous issue date: 2010-02-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Given the great diversity of fishes, the Order Tetraodontiformes stands to show genetic and morphological characteristics enough singular. The fishes of this order have a compact DNA which favors molecular studies, as well as comparisons with more basal species. Model of genome evolution, there are still many gaps in knowledge about their chromosomal patterns and how evolutionary rearrangements influence the marked variation in DNA content of this order. In view of this, we present cytogenetic analyzes of the species Acanthostracion quadricornis (Ostraciidae), A. polygonius (Ostraciidae) Melichthys niger (Balistidae) Cantherhines macrocerus (Monacanthidae) and C. pullus (Monacanthidae), Lagocephalus laevigatus, Colomesus psittacus and Canthigaster figueiredoi (Tetraodontidae), to contribute with cytogenetic data for this group. The analysis was performed by C-banding, Ag-RONs, coloring with base-specific fluorochromes DAPI-CMA3, restriction enzymes AluI, EcoRI, TaqI, PstI and HinfI and in situ hybridization with probes for ribosomal DNA 18S and 5S. The heterochromatic ultrastructure of A. quadricornis and A. polygonius revealed a outstanding heterochromatin content, which may indicate that the accumulation or loss of extensive heterochromatin content could be responsible for large variations in genomic content displayed in different Tetraodontiformes families. The species Cantherhines macrocerus, C. pullus (Monacanthidae) and Melichthys niger (Balistidae) shows a huge karyotypic similarity both numerically and structural. L. laevigatus showed similar cytogenetic features (2n = 44 and single RONs) to the species of the genus Takifugu, which reinforces the idea of their phylogenetic relationships. C. psittacus presented the highest diploid number described for the family (2n = 56) and large amount of HC, features that related with its sister family Diodontidae. Cytogenetic analysis in C. figueiredoi revealed heterochromatic polymorphisms, RONs multiple and Bs chromosomes. These events are rare in marine fishes, and are possibly associated with the strong restructuring and genomic reduction that this family has been suffered. These features, plus the morphological and molecular data suggests that these species share the same ancestral branch, with a possible monophyletic origin. In this study, new contributions to the knowledge of evolutionary patterns facing by Tetraodontiformes are provided and discussed under cytotaxonomyc, genomic and evolutionary perspectives. / Frente ? grande diversidade de peixes, a Ordem Tetraodontiformes se destaca por exibir caracter?sticas gen?ticas e morfol?gicas bastantes singulares. Os peixes desta Ordem apresentam um DNA compacto o que favorece estudos moleculares, assim como compara??es com esp?cies mais basais. Modelo de evolu??o gen?mica, ainda existem v?rias lacunas de conhecimento sobre seus padr?es cromoss?micos e como os rearranjos evolutivos influenciaram na marcante varia??o no conte?do de DNA desta Ordem. Diante disto o presente estudo apresenta an?lises citogen?ticas das esp?cies, Acanthostracion quadricornis (Ostraciidae), A. polygonius (Ostraciidae), Melichthys niger (Balistidae), Cantherhines macrocerus (Monacanthidae), C. pullus (Monacanthidae), Lagocephalus laevigatus, Colomesus psittacus e Canthigaster figueiredoi (Tetraodontidae) visando contribuir com mais dados citogen?ticos para o grupo. As an?lises foram realizadas atrav?s do bandamento C, Ag-RONs, colora??o com fluorocromos base-espec?ficos DAPICMA3, enzimas de restri??o AluI, EcoRI, TaqI, PstI e HinfI e pela hibrida??o in situ com sondas de DNA ribossomal 18S e 5S. A ultra-estrutura heterocromat?nica de A. quadricornis e A. polygonius, revelaram um marcante conte?do heterocrom?tico, situa??o que pode indicar que o ac?mulo ou perda de extenso conte?do de heterocromatinas poderiam ser respons?veis pelas extensas varia??es no conte?do gen?mico exibidas nas diferentes fam?lias dos Tetraodontiformes. As esp?cies Cantherhines macrocerus, C. pullus (Monacanthidae) e Melichthys niger (Balistidae) apresentam uma grande similaridade cariot?pica, tanto num?rica, como estruturalmente. Lagocephaluslaevigatus mostrou caracter?sticas citogeneticas similares (2n=44 e RONs simples) as esp?cies do g?nero Takifugu, o que refor?a a id?ia de seu relacionamento filogen?tico, e Colomesus psittacus apresentou o maior n?mero dipl?ide descrito para a fam?lia (2n=56) e grande quantidade de HC, caracter?sticas que o relacionariam com a fam?lia irm? Diodontidae. An?lises citogen?ticas em C. figueiredoi revelaram polimorfismos heterocrom?ticos, RONs m?ltiplas e cromossomos Bs, sendo estes eventos raros para peixes marinhos, estando possivelmente associados ? marcante reestrutura??o e redu??o gen?mica que esta fam?lia sofreu. Estas caracter?sticas, somadas aos dados morfol?gicos e moleculares sugerem que estas esp?cies compartilham de um mesmo ramo ancestral, com poss?vel origem monofil?tica. Neste trabalho novas contribui??es ao conhecimento dos padr?es evolutivos enfrentados pelos Tetraodontiformes s?o fornecidas e discutidas sob perspectivas citotax?nomicas, gen?micas e evolutivas.

Made available in DSpace on 2014-12-17T15:18:12Z (GMT). No. of bitstreams: 1 LorenaCBL.pdf: 1131500 bytes, checksum: f844b916fb8784c601514bd7da1743b9 (MD5) Previous issue date: 2007-02-26 / The Tetraodontiformes order is composed for about 400 species of fish, distributed in ten families, with circuntropical distribution. The morphologic diversity of each family reflects in, part, the different levels of specialization. This group represents an ancestry after-Perciformes and constitutes the last branch of the diffusion of the Tele?steos, occupying a position of prominence. The phylogenetics relationships of the Tetraodontiformes exist diverse works examining and, in all, these families are recognized as groups brothers, being Diodontidae next to Tetraodontidae and Balistidae next to Monacanthidae. Although it possesss a representative number of species, the works involving of the families Balistidae and Monacanthidae are few exemplary, especially species of oceanic islands. In this work cytogenetic studies in five species had been analyzed Cantherhines macrocerus, Cantherhines pullus (Monacanthidae), Melichthys niger (Balistidae), Sphoeroides testud?neus (Tetraodontidae) and Chilomycterus antennatus (Diodontidae); through conventional coloration, Ag-NORs and C banding. Ahead of the different karyological trends of evolution presented by the Tetraodontiformes, the present work also searched to verify the relation existence enters the total size of the chromosomes with the amount of DNA in these groups of Tetraodontiformes. For such, they had been correlated the total size of the chromosomes of these species, with values of content of available DNA in literature. The cytogenetics analyses for the species C.macrocerusJ C.pullus (Monacanthidae) and M.niger (Balistidae), had disclosed 40 chromosomes, all acrocentrics. All possess only one pair of NORs and pericentromeric heterochromatin. For S.testud?neus the found dyploid number was equal 2n=46, with NF=78 (16m+18sm+8st+4a), while that for C.antennatus it possesss 2n=50, with NF=76 (4m+22st+24a). Both species possess simple NORs and pericentromeric heterochromatin blocks. In M.niger, the presence of positive marking (heterochromatin and NOR) in the secondary constriction in the second chromosomic pair suggesting the occurrence of a rearrangement, possibly a fusing involving these homologous ones, indicating that these events had been important for the establishment of the karyological history of this group. A maintenance of the chromosomic constancy found in the populations of C.macrocerus (Monacanthidae) and S.testudineus (Tetraodontidae) perhaps if must for the aiding of the gene flow through oceanic chains. These data contrast with the differentiated kinds of chromosomes of C.antennatus between the Northeast coast and Southeastern, suggesting that the ecological standards of each species, added to the conditions of the marine environment, can be responsible for the karyological delineation of each species. The found characteristics for the species C.macrocerus, C.pullus, M.niger, S.testudineus and C.antennatus add it the available data for other species of Tetraodontiformes. From the data gotten in the present study, it can be inferred that the DNA content possesss direct relation with the total length of the chromosomes / RESUMO A ordem Tetraodontiformes ? composta por cerca de 400 esp?cies de peixes, distribu?das em dez fam?lias, com distribui??o circuntropical. A diversidade morfol?gica de cada fam?lia reflete em, parte, os diferentes n?veis de especializa??o. Este grupo representa uma linhagem p?s-Perciformes e constitui o ?ltimo ramo da difus?o dos Tele?steos, ocupando uma posi??o filogen?tica de destaque. Nesta ordem encontram-se algumas das esp?cies de peixes mais conhecidas popularmente, os baiacus, fam?lias Diodontidae e Tetraodontidae; e os cangulos, fam?lias Balistidae e Monacanthidae. Existem diversos trabalhos examinando as rela??es filogen?ticas dos Tetraodontiformes e, em todos, estas fam?lias s?o reconhecidas como grupos irm?os, sendo Diodontidae mais pr?ximo de Tetraodontidae e Balistidae mais pr?ximo de Monacanthidae. Embora possua um n?mero representativo de esp?cies, s?o poucos os trabalhos citogen?ticos envolvendo exemplares das fam?lias Balistidae e Monacanthidae, especialmente esp?cies insulares. Neste trabalho foram analisadas citogeneticamente as esp?cies Cantherhines macrocerus, Cantherhines pullus (Monacanthidae), Melichthys niger (Balistidae), Sphoeroides testudineus (Tetraodontidae) e Chilomycterus antennatus (Diodontidae); atrav?s de colora??o convencional, Ag-RONs e bandamento C. Diante das diferentes tend?ncias de evolu??o cariot?picas apresentadas pelos Tetraodontiformes, o presente trabalho tamb?m buscou verificar a exist?ncia de rela??o entre o tamanho total dos cromossomos com a quantidade de DNA nestes grupos de Tetraodontiformes. Para tal, foram correlacionados o tamanho total do complemento hapl?ide destas esp?cies, com valores de conte?do de DNA dispon?veis na literatura. As an?lises citogen?ticas para as esp?cies C.macrocerus, C.pullus (Monacanthidae) e M.niger (Balistidae), revelaram um cari?tipo composto de 40 cromossomos, todos acroc?ntricos. Todas possuem apenas um par de RONs e heterocromatina pericentrom?rica. Para S.testudineus o n?mero dipl?ide encontrado foi igual a 2n=46, com NF=78 (16m+18sm+8st+4a), enquanto que para C.antennatus possui 2n=50, com NF=76 (4m+22st+24a). Ambas esp?cies possuem RONs simples e blocos heterocrom?ticos centrom?ricos. Em M. niger, a presen?a de marca??o positiva (heterocromatina e RON) na constri??o secund?ria no 2?par cromoss?mico em M. niger sugerindo a ocorr?ncia de um rearranjo, possivelmente uma fus?o envolvendo estes hom?logos, indicando que estes eventos foram importantes para o estabelecimento dos cari?tipos deste grupo.A manuten??o da const?ncia cariot?pica encontrada nas popula??es de C. macrocerus (Monacanthidae) e Sphoeroides testudineus (Tetraodontidae) talvez se deva pelo favorecimento do fluxo g?nico atrav?s das correntes oce?nicas. Estes dados contrastam com os cit?tipos diferenciados de C.antennatus entre a costa Nordeste e Sudeste, sugerindo que os padr?es ecol?gicos de cada esp?cie, somados ?s condi??es do meio ambiente marinho, possam ser respons?veis pela delinea??o cariot?opica de cada esp?cie. As caracter?sticas citogen?ticas encontradas para as esp?cies C.macrocerus, C.pullus, M.niger, S.testudineus e C.antennatus somam-se aos dados dispon?veis para outras esp?cies de Tetraodontiformes. A partir dos dados obtidos no presente estudo, pode-se inferir que o conte?do de DNA possui rela??o direta com o comprimento total do genoma

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-02-22T19:07:45Z No. of bitstreams: 1 JulianaGalvaoBezerra_DISSERT.pdf: 1935169 bytes, checksum: ce2361b269051f4b199f9f2d9f4568f5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-03-06T23:22:30Z (GMT) No. of bitstreams: 1 JulianaGalvaoBezerra_DISSERT.pdf: 1935169 bytes, checksum: ce2361b269051f4b199f9f2d9f4568f5 (MD5) / Made available in DSpace on 2017-03-06T23:22:30Z (GMT). No. of bitstreams: 1 JulianaGalvaoBezerra_DISSERT.pdf: 1935169 bytes, checksum: ce2361b269051f4b199f9f2d9f4568f5 (MD5) Previous issue date: 2016-03-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A ordem Tetraodontiformes se destaca por exibir caracter?sticas morfol?gicas e gen?ticas bastante singulares, representando um dos principais ramos derivados da diversifica??o dos tele?steos. Alguns dos seus grupos constituem os vertebrados com os genomas mais compactos, qualificando-os como modelo de estudo da evolu??o do genoma. Esta caracter?stica gen?mica parece ser o resultado de perdas evolutivas de DNA. Com vistas a realizar compara??es citogen?micas entre esp?cies de alguns grupos de Tetraodontiformes foram realizadas an?lises citogen?ticas nas esp?cies Cantherhines pullus e Monacanthus chinensis (Monacanthidae), Sphoeroides testudineus (Tetraodontidae) e Melichthys N?ger (Balistidae). As an?lises foram ralizadas utilizando as metodologias cl?ssicas (colora??o pelo Giemsa, bandamento C, Ag-RONs), colora??o com fluorocromos base-espec?ficos e mapeamento cromoss?mico atrav?s da hibrida??o in situ fluorescente (FISH) de sequ?ncias ribossomais 18S e 5S e telom?ricas. As esp?cies C. pullus e M. niger revelaram cari?tipos compostos de 40 cromossomos, todos acroc?ntricos. Ambas possuem apenas um par de RONs e heterocromatinas, em maior parte, pericentrom?ricas, contudo, o mapeamento de sequ?ncias telom?ricas em C. pullus mostrou marca??es telom?ricas intersticiais, resultado da din?mica de rearranjos cromoss?micos que ocorre no grupo. Compara??es citogen?ticas entre as esp?cies S. testudineus (2n=46; NF=74) e M. chinensis (2n=34; NF=34) revelaram cari?tipos d?spares em rela??o ao n?mero diploide e de bra?os cromoss?micos, bem como quanto ao diminuto tamanho dos cromossomos de S. testudineus, em rela??o ao grandes cromossomos acroc?ntricos presentes em M. chinensis. A marcante diverg?ncia no tamanho dos cromossomos, estrutura cariot?pica e distribui??o de heterocromatina evidencia a elevada din?mica cromoss?mica e as m?ltiplas tend?ncias carioevolutivas presentes em Tetraodontiformes. Em vista do interesse sobre a evolu??o gen?mica na ordem, novas contribui??es ao conhecimento dos seus genomas e cari?tipos s?o fornecidos e discutidos sob perspectivas citogen?micas e evolutivas.