Orientador: Sonia Marli Zingaretti / Banca: janete Apparecida Desiderio / Banca: Juliana da Silva Coppede / Resumo: A grande expansão da cultura de cana-de-açúcar pelo território brasileiro leva à necessidade do desenvolvimento de cultivares melhoradas e adaptadas às diferentes condições de clima a que são submetidas. Os estresses bióticos e abióticos são fatores que afetam a produtividade de uma cultura e entre esses, o estresse hídrico assume grande importância em função do regime de chuvas e do aumento de temperatura iminente nos próximos anos. Analisar a expressão de genes em plantas submetidas a estresses pode contribuir de forma expressiva para elucidar as rotas de defesa das plantas, contribuindo sobremaneira para o melhoramento da cultura e o desenvolvimento de novas variedades. O projeto teve como objetivo obter transformantes de Nicotiana tabacum in vitro com o gene LTP (Lipid Transfer Protein) e avaliar sua funcionalidade em relação ao estresse por deficiência hídrica, em casa de vegetação por hidroponia. Feita a seleção da EST com base em resultados anteriores obtidos pelo grupo de pesquisa, posterior análise em bancos de dados, realizou-se a aquisição do clone no Centro de Estocagem de Genes (BCCCenter) e o re-sequenciamento para comprovar sua identidade. Estudos in silico foram realizados através da utilização de softwares de bioinformática e a análise da função do gene foi realizada a partir da transformação genética de Nicotiana tabacum via Agrobacterium tumefaciens. Seis transformantes de N. tabacum com o inserto de interesse LTP foram obtidos e testados quanto à tolerânci... (Resumo completo, clicar acesso eletrônico abaixo) / The great expansion of sugarcane cultivation across Brazilian territory leads to the need to develop better cultivars and adapted to the different climatic conditions that are submitted. The biotic and abiotic stresses are factors that affect the productivity of a crop and among them, the water stress will assume great importance due to the rainfall regime and the increase of the imminent temperature in the next years. Analyzing the expression of genes in stress - stressed plants can contribute in an expressive way to elucidate as plant defense routes, contribute to the improvement of the culture and the development of new varieties. The objective of this project was to obtain transformers of Nicotiana tabacum in vitro with the LTP (Lipid Transfer Protein) gene and to evaluate its functionality in relation to stress due to water deficiency in a greenhouse by hydroponics. We made EST selection based on previous results obtained by a research group, later analysis in databases, performing a clone acquisition in the Gene Storage Center (BCCCenter) and resequencing to prove its identity. Silicon studies were carried out through the application of bioinformatics software and an analysis of the genetic function was performed from the genetic transformation of Nicotiana tabacum via Agrobacterium tumefaciens. Six N. tabacum transformants with the LTP insert of interest were obtained and tested for tolerance to water deficit by induction of different concentrations of mannitol. Transformer tobacco plants showed better phenotypic performance compared to untransformed plant and good readaptation after stress. The T1 generation these plants will be used in studies for the biological and functional verification of the action of the inserted gene, through the Real-Time qPCR technique. / Mestre

by Chan Ying Chi, Jessica. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 113-127). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iv / Contents --- p.vi / Abbreviations --- p.xi / Single Letter Amino Acid Code --- p.xvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Glycogen Synthase (EC 2.4.1.11) --- p.1 / Chapter 1.2 --- Glycogen Synthase Kinase-3 --- p.4 / Chapter 1.3 --- Structure of Glycogen Synthase Kinase-3 --- p.5 / Chapter 1.4 --- Functions of Glycogen Synthase Kinase-3 --- p.8 / Chapter 1.4.1 --- Substrate Recognition --- p.8 / Chapter 1.4.2 --- Glycogen Synthase Kinase-3 Homologs --- p.10 / Chapter 1.4.2.1 --- Drosophila --- p.10 / Chapter 1.4.2.2 --- Xenopus --- p.11 / Chapter 1.4.2.3 --- Dictyostelium and Others --- p.12 / Chapter 1.4.3 --- Regulation of Glycogen Synthase-3 in Mammalian Systems --- p.13 / Chapter 1.4.4 --- The role of Glycogen Synthase Kinase-3in Mammalian Brain --- p.16 / Chapter 1.4.4.1 --- Glycogen Synthase Kinase-3β --- p.18 / Chapter 1.4.4.2 --- Glycogen Synthase Kinase-3α --- p.21 / Chapter 1.4.5 --- Glycogen Synthase Kinase-3α in Certain Tumor Cells --- p.23 / Chapter 1.5 --- Objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General Techniques / Chapter 2.1.1 --- Plasmid Minipreparation --- p.26 / Chapter 2.1.2 --- Large Scale of Plasmid DNA Purification Using QIAGEN-tip500 --- p.28 / Chapter 2.1.3 --- Extraction of Human Blood Genomic DNA --- p.30 / Chapter 2.1.4 --- UV Spectroscopy for determining DNA/RNA Concentration --- p.31 / Chapter 2.1.5 --- Agarose Gel Electrophoresis of DNA --- p.31 / Chapter 2.1.6 --- Purification of DNA Fragment from Agarose Gel using GeneClean III ® (BIO 101 Inc.) Kit --- p.32 / Chapter 2.1.7 --- Restriction Digestion of DNA --- p.32 / Chapter 2.1.8 --- Southern Blot --- p.33 / Chapter 2.1.9 --- Probe Labelling --- p.33 / Chapter 2.1.10 --- Hybridization by Radio-labelling --- p.34 / Chapter 2.1.11 --- DNA Sequencing Reaction --- p.35 / Chapter 2.1.12 --- "Preparation of 6% Polyacrylamide, 8M Urea Denaturing Gel for DNA Sequencing Analysis" --- p.37 / Chapter 2.1.13 --- Preparation of Escherichia coli DH5α Competent Cells --- p.38 / Chapter 2.1.14 --- Modification of 5'Protruding end with T4DNA Polymerase --- p.39 / Chapter 2.1.15 --- Ligation and Transformation of Foregin DNA --- p.39 / Chapter 2.1.16 --- Rapid Screening for the Presence of Plasmid --- p.40 / Chapter 2.2 --- Expression of Glycogen Synthase Kinase-3 / Chapter 2.2.1 --- Preparation of Mammalian cells in Culture --- p.41 / Chapter 2.2.2 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.2.3 --- Western Blot Detection of Glycogen Synthase Kianse-3 --- p.43 / Chapter 2.3 --- Assay of Glycogen Synthase Kinase Promoter Activity / Chapter 2.3.1 --- Preparation of SHSY5Y in Culture --- p.45 / Chapter 2.3.2 --- Trypsinization for Removing Adherent Cells --- p.45 / Chapter 2.3.3 --- Transfection of Mammalian Cells by Calcium Phosphate Precipitation --- p.46 / Chapter 2.3.4 --- Stimulation of Transfection Cells by different Chemicals and Preparation of Cell Extract --- p.47 / Chapter 2.3.5 --- CAT-ELISA and β-Gal ELISA Assay --- p.47 / Chapter 2.4 --- Isolation of Glycogen Synthase Kinase-3α 5，Promoter Region / Chapter 2.4.1 --- 5'Rapid Amplification of cDNA End (5'RACE) --- p.48 / Chapter 2.4.2 --- PromoterFinder DNA Walking --- p.49 / Chapter 2.4.3 --- YAC Clone Genomic Construction --- p.50 / Chapter 2.5 --- Construction of Plasmid for Assay of Glycogen Synthase Kinase-3α Promoter Activity --- p.53 / Chapter 2.6 --- Genomic Organization of Glycogen Synthase Kinase-3α --- p.53 / Chapter 2.7 --- Primer Extension Assay / Chapter 2.7.1 --- Isolation of Total RNA by TRIZOL Reagent --- p.57 / Chapter 2.7.2 --- Primer Extension by SuperScript II --- p.57 / Chapter 2.8 --- Reagents and Buffers / Chapter 2.8.1 --- Nucleic Acid Electrophoresis Buffers --- p.59 / Chapter 2.8.2 --- Reagents for Preparation of Plasmid DNA --- p.59 / Chapter 2.8.3 --- Media for Bacterial Culture --- p.60 / Chapter 2.8.4 --- Reagents for Southern Blot --- p.60 / Chapter 2.8.5 --- Reagents for SDS-PAGE --- p.61 / Chapter 2.8.6 --- Reagents for Western Blot --- p.62 / Chapter 2.8.7 --- Reagents for DNA Sequencing --- p.62 / Chapter Chapter 3 --- Isolation of 5´ة Glycogen Synthase Kinase-3α Promoter Region / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results --- p.66 / Chapter 3.2.1 --- 5' Rapid Amplification of cDNA End (5'RACE) --- p.66 / Chapter 3.2.2 --- PromoterFinder DNA Walking --- p.68 / Chapter 3.2.3 --- YAC Clone Library Construction --- p.71 / Chapter 3.2.3.1 --- Southern Blotting --- p.71 / Chapter 3.2.3.2 --- Isolation of Sequence Upstream of Glycogen Synthase Kinase-3α region from YAC Clone Using PromoterFider DNA Walking --- p.71 / Chapter 3.2.3.3 --- Sequences of 5，Glycogen Synthase Kinase -3α Promoter --- p.73 / Chapter 3.2.4 --- Primer Extension Assay --- p.78 / Chapter 3.2.5 --- Assay of Glycogen Synthase Kinase-3α Promoter Activity using CAT-ELISA --- p.78 / Chapter 3.2.6 --- Genomic Structure of Glycogen Synthase Kinase-3α --- p.84 / Chapter 3.3 --- Discussion --- p.90 / Chapter 3.3.1 --- Glycogen Synthase Kinase-3a Promoter --- p.90 / Chapter 3.3.2 --- Glycogen Synthase Kianse-3a Promoter Activity --- p.92 / Chapter 3.3.3 --- Prospective and Future Studies --- p.94 / Chapter Chapter 4 --- Expression of Glycogen Synthase Kinase-3 / Chapter 4.1 --- Introduction --- p.96 / Chapter 4.2 --- Results Expression of GSK-3 under Stresses --- p.97 / Chapter 4.3 --- Discussion --- p.105 / Chapter 4.3.1 --- Post-translation regulation of Glycogen Synthase Kinase-3 --- p.105 / Chapter 4.3.2 --- Prospective and Future Studies --- p.107 / Chapter Chapter 5 --- Conclusion --- p.109 / Chapter 5.1 --- Promoter study --- p.110 / Chapter 5.2 --- Genomic organization study --- p.111 / Chapter 5.3 --- Expression study --- p.112 / Reference --- p.113 / Appendices / Appendix I G/C contents of GSK-3α Promoter Region --- p.128 / Appendix II Restriction sites of GSK-3α Promoter Region --- p.134 / Appendix III Primers designed on GSK-3α Promoter Region --- p.139 / Appendix IV Restriction sites of GSK-3α cDNA --- p.142 / Appendix V Vectors --- p.150 / Appendix VI Adaptors Sequences --- p.152 / Appendix VII Anti-GSK-3 Antibody --- p.153 / Appendix VIII Raw data of GSK-3α promoter activity assay --- p.154

"November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 131-152). / 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.

by Patrick, Tik-wan Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-155). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.vi / Abbreviations --- p.viii / List of figures --- p.ix / List of tables --- p.xiv / Chapter Chapter One: --- Introduction --- p.1-17 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- The Human genome project --- p.2 / Chapter 1.3 --- The EST approach --- p.3 / Chapter 1.4 --- Significance of the EST approach --- p.3 / Chapter 1.5 --- Human heart cDNA sequencing --- p.5 / Chapter 1.6 --- Significance of the human adult heart EST project --- p.7 / Chapter 1.7 --- Ribosomal proteins --- p.8 / Chapter 1.7.1 --- The ribosomal constituents --- p.8 / Chapter 1.7.2 --- Eukaiyotic ribosomal proteins --- p.10 / Chapter 1.8 --- Mammalian ribosomal proteins --- p.11 / Chapter 1.8.1 --- Evolution of mammalian ribosomal proteins --- p.11 / Chapter 1.8.2 --- Significance of mammalian ribosomal proteins --- p.12 / Chapter 1.9 --- Possible functional roles of ribosomal protein --- p.14 / Chapter 1.10 --- Nomenclature of ribosomal proteins --- p.16 / Chapter 1.11 --- The theme of the thesis --- p.17 / Chapter Chapter Two: --- Materials and Methods --- p.18-49 / Chapter 2.1 --- Cycle sequencing --- p.18 / Chapter 2.1.1 --- Plating out the cDNA library --- p.18 / Chapter 2.1.2 --- Amplification of the cDNA clones by PCR --- p.19 / Chapter 2.1.3 --- Purification and quantitation of the PCR product --- p.20 / Chapter 2.1.4 --- Cycle DNA sequencing --- p.20 / Chapter 2.2 --- Cloning of hrpL29 in pUC 18 cloning vector --- p.21 / Chapter 2.2.1 --- Amplification of the phage by plate lysate --- p.21 / Chapter 2.2.2 --- Amplification of the insert by PCR --- p.22 / Chapter 2.3 --- Screening for hrpL29 transformant --- p.22 / Chapter 2.3.1 --- Mini-preparation of plasmid DNA (Sambrook et al，1989) --- p.22 / Chapter 2.3.2 --- Large scale preparation of plasmid DNA --- p.24 / Chapter 2.4 --- Primer design for cloning of an intron of hrpL29 --- p.26 / Chapter 2.5 --- Isolation of the intron of hrpL29 by PCR --- p.26 / Chapter 2.6 --- Restricted endonuclease digestion --- p.27 / Chapter 2.7 --- Purification of DNA from the agarose gel --- p.27 / Chapter 2.8 --- Dephosphorylation of linearized plasmid DNA --- p.29 / Chapter 2.9 --- DNA ligation --- p.29 / Chapter 2.10 --- "Preparation of competent bacterial cells for transformation (Hanahan,1985)" --- p.30 / Chapter 2.11 --- Plasmid DNA Transformation --- p.31 / Chapter 2.12 --- Unicycle DNA sequencing by T7 polymerase (Pharmacia) --- p.32 / Chapter 2.13 --- Synthesis of radiolabelled DNA probe --- p.33 / Chapter 2.14 --- "Oligonucleotide synthesis, deprotection and purification" --- p.34 / Chapter 2.14.1 --- Oligonucleotide synthesis --- p.34 / Chapter 2.14.2 --- Deprotection and purification of oligonucleotides --- p.35 / Chapter 2.15 --- Southern analysis --- p.36 / Chapter 2.15.1 --- "Isolation of genomic DNA from leukocytes (Ciulla et al,1988)" --- p.36 / Chapter 2.15.2 --- Restricted digestion and fractionation of genomic DNA --- p.37 / Chapter 2.15.3 --- Southern transfer of DNA onto a membrane support --- p.37 / Chapter 2.15.4 --- Prehybridization of the Southern blot --- p.40 / Chapter 2.15.5 --- Hybridization of the Southern blot --- p.40 / Chapter 2.16 --- Northern analysis --- p.41 / Chapter 2.16.1 --- "Isolation of total RNA by using the AGPC-RNA method (Chomczynski and Sacchi,1987, modified)" --- p.41 / Chapter 2.16.2 --- Separation of total RNA by electrophoresis and transfer onto a membrane support --- p.43 / Chapter 2.16.3 --- Prehybridization of the Northern blot --- p.46 / Chapter 2.16.4 --- Hybridization of the Northern blot --- p.47 / Chapter 2.17 --- First strand cDNA synthesis (Pharmacia) --- p.48 / Chapter 2.18 --- PCR of the first strand cDNA --- p.48 / Chapter Chapter Three: --- Results --- p.50-113 / Chapter 3.1 --- Partial sequencing of adult human heart cDNA clones --- p.50 / Chapter 3.2 --- DNA homology searching by using the program BLASTN --- p.52 / Chapter 3.2.1 --- Catalogue of the 502 ESTs of the cardiovascular system --- p.54 / Chapter 3.2.2 --- Classification and frequency of the human adult heart cDNA clones --- p.63 / Chapter 3.3 --- Submission of the cDNA sequences to NCBI --- p.64 / Chapter 3.4 --- Pattern of gene expression in the human adult cardiovascular system --- p.66 / Chapter 3.5 --- "Sequence determination of hrpL29 (Law et. al., 1996)" --- p.72 / Chapter 3.5.1 --- Cycle Taq sequencing of hrpL29 --- p.72 / Chapter 3.5.2 --- Subcloning of the hrpL29 cDNA insert into the pUC18 DNA cloning vector --- p.75 / Chapter 3.5.3 --- Unicycle T7 sequencing of hrpL29 --- p.77 / Chapter 3.6 --- Sequence alignment and comparison of hrpL29 with other known sequences in the databases --- p.79 / Chapter 3.7 --- The primary structure of hrpL29 --- p.83 / Chapter 3.8 --- Results of RT-PCR and PCR --- p.88 / Chapter 3.9 --- Genomic analysis of hrpL29 --- p.92 / Chapter 3.9.1 --- Isolation of the first intron of hrpL29 --- p.92 / Chapter 3.9.2 --- Southern analysis of hrpL29 --- p.97 / Chapter 3.10 --- Northern analysis of hrpL29 --- p.103 / Chapter 3.10.1 --- Tissue distribution of hrpL29 mRNA in rat tissues --- p.103 / Chapter 3.10.2 --- Time course of hRPL29 expression in mouse heart --- p.106 / Chapter 3.10.3 --- Time course of hRPL29 expression in mouse brain --- p.110 / Chapter Chapter Four: --- Discussion --- p.114-139 / Chapter 4.1 --- Characterization of the ESTs --- p.114 / Chapter 4.2 --- Significance of the heart EST project --- p.116 / Chapter 4.3 --- Redundancy of the EST sequencing --- p.118 / Chapter 4.4 --- The importance of frequent database searching --- p.119 / Chapter 4.5 --- The importance of an efficient comparison algorithm --- p.120 / Chapter 4.6 --- Human ribosomal protein L29 (hRPL29) --- p.122 / Chapter 4.7 --- Internal duplication in hRPL29 --- p.124 / Chapter 4.8 --- Primary structure analysis of hRPL29 --- p.126 / Chapter 4.9 --- RT-PCR and PCR of the first strand cDNA with primers using the C095-ATG and dT primer --- p.128 / Chapter 4.10 --- Southern analysis of hrpL29 --- p.128 / Chapter 4.11 --- Northern analysis of hrpL29 --- p.133 / Chapter 4.11.1 --- Tissue distribution of the mRNA species of hrpL29 --- p.133 / Chapter 4.11.2 --- Time course of hRPL29 expression in mouse heart and brain --- p.134 / Chapter 4.12 --- Possible functional role of hRPL29 --- p.135 / Chapter 4.13 --- Further aspects --- p.137 / Appendix --- p.140-143 / References --- p.144-155

by Yam Kwok Fai. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-149). / Acknowledgments --- p.i / List of Contents --- p.ii / List of Figures --- p.viii / List of Tables --- p.xii / List of Primers --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth Hormone (GH) --- p.1 / Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3 / Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4 / Chapter 1.2.2 --- Biosynthesis and Degradation of GHR --- p.6 / Chapter 1.2.3 --- Regulation of GHR Level --- p.7 / Chapter 1.2.4 --- The Structure of GHR --- p.9 / Chapter 1.2.5 --- The Structure of GHR Gene --- p.13 / Chapter 1.2.6 --- Growth Hormone Binding Protein (GHBP) --- p.14 / Chapter 1.2.7 --- The GH/Prolactin/Cytokine/Erythropoietin Receptor Superfamily --- p.15 / Chapter 1.2.8 --- Proposed Signal Transduction Pathway --- p.17 / Chapter 1.2.9 --- GHR Related Dwarfism --- p.22 / Chapter i). --- Substitution of certain amino acid residues in the extracellular domain --- p.22 / Chapter ii). --- Deletion of the extracellular domain --- p.23 / Chapter a). --- deletion of a small portion of the binding protein / Chapter b). --- deletion of a large portion of the binding protein / Chapter c). --- deletion of a large portion of the binding domain and the whole transmembrane domain / Chapter iii). --- Associated with normal GHBP --- p.24 / Chapter 1.3 --- Objectives of Cloning Vertebrate GHR cDNAs --- p.24 / Chapter Chapter 2 --- General Experimental Methods / Chapter 2.1 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.26 / Chapter 2.2 --- Isolation of Total RNA --- p.26 / Chapter 2.3 --- Isolation of mRNA --- p.26 / Chapter a). --- directly from tissue / Chapter b). --- from isolated total RNA / Chapter 2.4 --- Spectrophotometric Quantification and Qualification of DNA and RNA --- p.29 / Chapter 2.5 --- First Strand cDNA Synthesis --- p.29 / Chapter 2.6 --- Polymerase Chain Reaction (PCR) --- p.30 / Chapter 2.7 --- Agarose Gel Electrophoresis --- p.31 / Chapter 2.8 --- Formaldehyde Agarose Gel Electrophoresis of RNA --- p.31 / Chapter 2.9 --- Capillary Transfer of DNA/RNA to a Nylon Membrane (Southern/Northern Blotting) --- p.32 / Chapter a). --- DNA denaturing / Chapter b). --- Capillary transfer / Chapter 2.10 --- DNA Radiolabelling --- p.33 / Chapter a). --- By random primer translation / Chapter b). --- By nick translation / Chapter 2.11 --- Spuncolumn Chromatography --- p.34 / Chapter 2.12 --- Hybridization of Southern/Northern Blot --- p.35 / Chapter 2.13 --- Autoradiography --- p.35 / Chapter 2.14 --- Linearization and Dephosphorylation of Plasmid DNA --- p.36 / Chapter 2.15 --- Restriction Digestion of DNA --- p.36 / Chapter 2.16 --- Purification of DNA from Agarose Gel using GENECLEAN® Kit --- p.36 / Chapter 2.17 --- 3' End Modification of PCR Amplified DNA --- p.37 / Chapter 2.18 --- Ligation of DNA Fragments to Linearized Vector --- p.37 / Chapter 2.19 --- Preparation of Escherichia coli Competent Cells --- p.38 / Chapter 2.20 --- Transformation of the Escherichia coli Strain DH5a --- p.38 / Chapter 2.21 --- Minipreparation of Plasmid DNA --- p.39 / Chapter 2.22 --- DNA Purification by Phenol/Chloroform Extraction --- p.39 / Chapter 2.23 --- Ethanol Precipitation of DNA and RNA --- p.40 / Chapter 2.24 --- Preparation of Plasmid DNA using Wizard´ёØ Minipreps DNA Purification Kit from Promega --- p.40 / Chapter 2.25 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.41 / Chapter 2.26 --- DNA Sequencing --- p.42 / Chapter 2.26.1 --- DNA Sequencing Reaction / Chapter a). --- T7 sequencing / Chapter b). --- PCR sequencing / Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.44 / Chapter i). --- Preparation of 8% polyacrylamide gel solution / Chapter ii). --- Casting the gel / Chapter iii). --- Electrophoresis / Chapter Chapter 3 --- Molecular Cloning of Golden Hamster (Mesocricetus auratus) GHR cDNA / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Experimental Methods / Chapter 3.2.1 --- Animals and Tissues --- p.47 / Chapter 3.2.2 --- PCR Cloning of GHR cDNA Fragments in the Cytoplasmic Domain --- p.47 / Chapter 3.2.2.1 --- Primer design and PCR strategy --- p.47 / Chapter 3.2.2.2 --- PCR studies on the hamster liver and kidney first strand cDNA --- p.49 / Chapter 3.2.2.3 --- Southern analysis of the PCR products --- p.50 / Chapter 3.2.2.4 --- Subcloning and sequencing of PCR amplified cDNA fragments --- p.50 / Chapter 3.2.3 --- Screening of a Hamster Liver cDNA Library --- p.51 / Chapter 3.2.3.1 --- Preparation of the plating bacteria --- p.51 / Chapter 3.2.3.2 --- Phage titering of the λ ZAP library --- p.51 / Chapter 3.2.3.3 --- Primary screening of the amplified hamster liver cDNA library --- p.52 / Chapter 3.2.3.4 --- Plaque uplifting and hybridization with hamster GHR cDNA fragment --- p.52 / Chapter 3.2.3.5 --- Purification of putative clones from primary screening --- p.53 / Chapter 3.2.3.6 --- Checking the size of the DNA insert --- p.53 / Chapter 3.2.3.7 --- In vitro excision to release phagemid from the phage vector --- p.54 / Chapter 3.2.3.8 --- Plasmid minipreparation of the putative clones --- p.56 / Chapter 3.2.3.9 --- Nucleotide sequencing of the DNA inserts of different clones --- p.56 / Chapter 3.2.4 --- Tissue Distribution of GHR in Hamster Tissues and the Relative Expression Level of GHR mRNAin these tissues --- p.58 / Chapter 3.2.5 --- Cloning of the Full-length GHR cDNA into a Mammalian Vector --- p.59 / Chapter 3.2.5.1 --- PCR amplification of the full-length hamster GHR cDNA --- p.59 / Chapter 3.2.5.2 --- Preparation of the hamster GHR cDNA insert for ligation --- p.60 / Chapter 3.2.5.3 --- Linearization of pRc/CMV expression vector --- p.60 / Chapter 3.2.5.4 --- Ligation of the linearized expression vector with the full-length hamster GHR cDNA --- p.61 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PCR Amplification of Hamster GHR cDNA Fragments --- p.61 / Chapter 3.3.1.1 --- RT-PCR --- p.61 / Chapter 3.3.1.2 --- Southern blot analysis --- p.62 / Chapter 3.3.1.3 --- Subcloning and nucleotide sequencing of PCR amplified hamster GHR cDNA fragments --- p.64 / Chapter 3.3.2 --- Screening of an Amplified λZAP Hamster Liver cDNA Library --- p.70 / Chapter 3.3.2.1 --- Preparation of the cDNA probe and phage titering --- p.70 / Chapter 3.3.2.2 --- Screening of the cDNA library --- p.70 / Chapter 3.3.2.3 --- PCR study of the 5' and 3' regions of the DNA insert of the clones selected for secondary screening --- p.72 / Chapter 3.2.3.4 --- Nucleotide sequencing of the full-length hamster GHR cDNA --- p.73 / Chapter 3.2.3.5 --- Tissue distribution of GHR in hamster and the relative expression level of the GHR mRNA in these tissues --- p.73 / Chapter 3.2.3.6 --- Cloning of the full-length hamster GHR cDNA into a mammalian expression vector --- p.79 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Cloning of the Full-length hamster GHR cDNA --- p.81 / Chapter 3.4.2 --- Comparison of the Nucleotide and the Predicted Amino Acid Sequences of the Hamster GHR with other Cloned GHRs --- p.82 / Chapter 3.4.3 --- Tissue Distribution of GHR in Hamster and the Relative Expression Level of the GHR mRNA in these Tissues --- p.89 / Chapter 3.4.4 --- Further Studies on Hamster GHR --- p.90 / Chapter Chapter 4 --- Molecular Cloning of Chinese Bullfrog (Rana tigria rigulosa) GHR cDNA from Adult Frog Liver / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Experimental Methods / Chapter 4.2.1 --- Animal and Tissues --- p.93 / Chapter 4.2.2 --- Cloning of the Cytoplasmic Domain of Frog GHR cDNA by PCR --- p.93 / Chapter 4.2.2.1 --- RT-PCR --- p.93 / Chapter 4.2.2.2 --- Southern blot analysis of PCR amplified products --- p.95 / Chapter 4.2.2.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.95 / Chapter 4.2.2.4 --- Restriction analysis of GHR cDNA fragment between GHR p1 and GHR p2 --- p.95 / Chapter 4.2.2.5 --- PCR cloning of other portions of frog GHR cDNA --- p.96 / Chapter 4.2.2.6 --- Subcloning and sequencing of PCR amplified GHR cDNA fragment using primers other than GHR p1 and GHR p2 --- p.97 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Cloning of the Intracellular Domain of Frog GHR cDNA by RT-PCR --- p.97 / Chapter 4.3.1.1 --- RT-PCR --- p.97 / Chapter 4.3.1.2 --- Southern blot analysis --- p.98 / Chapter 4.3.1.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.98 / Chapter 4.3.1.4 --- Restriction enzyme analysis of GHR cDNA fragments --- p.102 / Chapter 4.3.1.5 --- PCR cloning of other portions of frog GHR cDNA --- p.103 / Chapter 4.3.1.6 --- Subcloning and sequencing of PCR products from other portions of frog GHR cDNA --- p.103 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Cloning of the Full-length frog GHR cDNA --- p.109 / Chapter 4.4.2 --- Further Studies on Frog GHR --- p.117 / Chapter Chapter 5 --- Attempts on the Molecular Cloning of Teleost GHR cDNA / Chapter 5.1 --- Introduction --- p.119 / Chapter 5.2 --- Experimental Methods / Chapter 5.2.1 --- Animals and Tissues --- p.120 / Chapter 5.2.2 --- PCR Cloning of Teleost GHR cDNA fragments --- p.120 / Chapter 5.2.2.1 --- Design of PCR primers --- p.120 / Chapter 5.2.2.2 --- Preparation of mRNA and synthesis of first strand cDNA --- p.122 / Chapter 5.2.2.3 --- PCR studies on dace and snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.2.3.1 --- PCR studies on dace liver first strand cDNA --- p.122 / Chapter 5.2.2.3.2 --- PCR studies on snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.3 --- "Northern Analysis on Dace, Snakehead fish and Eel mRNA" --- p.123 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Molecular Studies on Dace GHR cDNA --- p.123 / Chapter 5.3.1.1 --- PCR studies on dace first strand cDNA --- p.123 / Chapter 5.3.2 --- PCR Studies on Teleost First Strand cDNA --- p.128 / Chapter 5.3.3 --- Northern Analysis on Teleost mRNA --- p.128 / Chapter 5.4 --- Discussion --- p.130 / Chapter 5.4.1 --- PCR Studies on Teleost GHR cDNA --- p.130 / Chapter 5.4.2 --- Northern Analysis on Teleost mRNA --- p.131 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Achievement of this Project --- p.134 / Chapter 6.1.1 --- Hamster GHR --- p.134 / Chapter 6.1.2 --- Frog GHR --- p.135 / Chapter 6.1.3 --- Teleost GHR --- p.136 / Chapter 6.2 --- Postulation on Cloned GHRs at the Molecular Level --- p.136 / Bibliography --- p.141 / Appendices --- p.150

by Ng Yuen Keng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-148). / Table of contents --- p.1 / List of figures --- p.5 / List of tables --- p.12 / List of abbreviations --- p.13 / Abbreviation table for amino acids --- p.16 / Chapter Chapter 1 --- Literature Review --- p.17 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.2 --- The Hematopoietin/cytokine receptor superfamily --- p.20 / Chapter 1.3 --- The PRLR protein --- p.22 / Chapter 1.3.1 --- The receptor size --- p.22 / Chapter 1.3.2 --- Primary structure --- p.22 / Chapter 1.3.3 --- Structure of the extracellular domain --- p.26 / Chapter 1.3.4 --- Structure of the cytoplasmic domain --- p.30 / Chapter 1.3.5 --- Characteristics of specific PRL binding to PRLR --- p.32 / Chapter 1.5 --- The PRLR gene --- p.33 / Chapter 1.6 --- Heterogeneity of PRLR --- p.33 / Chapter 1.7 --- Signal transduction of PRLR --- p.35 / Chapter 1.7.1 --- JAK: a novel family of cytoplasmic protein tyrosine kinases --- p.35 / Chapter 1.7.2. --- Interaction between JAK2 and PRLR --- p.37 / Chapter 1.7.3 --- STAT proteins: mediators of PRL-dependent gene transcription --- p.37 / Chapter 1.7.4 --- Other signaling pathways of PRLR --- p.38 / Chapter 1.7.5 --- Future prospects on PRLR signaling --- p.38 / Chapter 1.8 --- Regulation of PRLR gene expression --- p.39 / Chapter 1.9 --- Objective of cloning the PRLR cDNA in male Syrian golden hamster --- p.42 / Chapter Chapter 2 --- PCR cloning of hamster PRLR cDNA fragment from adult male hamster liver --- p.44 / Chapter 2.1. --- Introduction --- p.44 / Chapter 2.2. --- Materials and Methods --- p.45 / Chapter 2.2.1 --- Primer design and PCR strategy --- p.45 / Chapter 2.2.2 --- Collection of liver --- p.46 / Chapter 2.2.3 --- Reverse transcription of polyadenylated RNA --- p.46 / Chapter 2.2.4 --- Nested PCR --- p.47 / Chapter 2.2.5 --- Southern analysis of the PCR products --- p.48 / Chapter 2.2.6 --- Subcloning of PCR product --- p.49 / Chapter 2.2.7 --- Sequence determination of the positive recombinant clone --- p.49 / Chapter 2.2.8 --- Sequence alignment and homology comparison --- p.50 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Nucleotide sequence alignment and primer design --- p.55 / Chapter 2.3.2 --- Nested PCR --- p.55 / Chapter 2.3.3 --- Subcloning of the PCR product --- p.56 / Chapter 2.3.4 --- Analysis of nucleotide and predicted amino acid sequences --- p.56 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3 --- Nucleotide sequence determination of the 5' and the 3' ends of hamster PRLR cDNA --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Collection of liver --- p.71 / Chapter 3.2.2 --- Total RNA preparation and poly (A) + RNA isolation --- p.72 / Chapter 3.2.3 --- Double stranded cDNA synthesis --- p.73 / Chapter 3.2.4 --- Adaptor ligation --- p.74 / Chapter 3.2.5 --- 5´ة and 3' RACE PCR --- p.74 / Chapter 3.2.6 --- Cloning of the RACE PCR products --- p.76 / Chapter 3.2.7. --- Sequence determination of the RA CE PCR products --- p.77 / Chapter 3.2.8. --- Sequence analysis of the RACE PCR products --- p.78 / Chapter 3 .2.9 --- Northern blot analysis of hamster PRLR mRNA in male hamster tissues --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.1.1 --- RNA preparation and double stranded cDNA synthesis --- p.79 / Chapter 3.3.2 --- RACE PCRfor the 5' and the 3' ends of hamster PRLR cDNA --- p.84 / Chapter 3.3.3 --- Cloning of the 5' and 3'RACE PCR products --- p.92 / Chapter 3.3.4 --- Sequence determination of the RACE PCR products --- p.92 / Chapter 3.3.5 --- Nucleotide sequence analysis of hamster PRLR full length cDNA --- p.101 / Chapter 3.3.6 --- Northern blot analysis of hamster PRLR --- p.101 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter 4 --- Attempts to study the PRLR gene expression in male hamster tissues --- p.113 / Chapter 4.1 --- Introduction --- p.113 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.2.1 --- Collection of tissues --- p.115 / Chapter 4.2.2 --- Total RNA preparation and poly (A)+ RNA isolation --- p.116 / Chapter 4.2.3 --- Reverse Transcription --- p.116 / Chapter 4.2.4 --- Polymerase chain reaction for detecting the presence of hamster PRLR cDNA in various tissues --- p.117 / Chapter 4.2.5 --- Nested PCR for detecting heterogeneity in PRLR cDNA sizes in various tissues --- p.117 / Chapter 4.2.6 --- Analysis and quantitation of PCR products --- p.118 / Chapter 4.3 --- Results --- p.119 / Chapter 4.4 --- Discussion --- p.134 / Chapter Chapter 5 --- General Discussion --- p.137 / References --- p.141 / Appendices --- p.149 / Chapter I. --- "Stock solution preparation (Sambrook et al., 1989)" --- p.149 / Chapter II. --- List of primers --- p.152 / Primers for sequence determination --- p.152 / "Primer for first strand cDNA synthesis and 3' RACE PCR (Frohman et al., 1988 and Loh et al.,1989)" --- p.152 / "Primers for amplifying the actin cDNA fragment (Chan et al.,1995)" --- p.152 / Primers used for PCR-cloning and semi-quantitative analysis of hamster PRLR cDNA --- p.153 / Chapter III. --- "First strand cDNA synthesis primer, cDNA adaptor and adaptor primers used in the 5' and3' end sequence determinations of hamster PRLR cDNA" --- p.154 / Chapter IV. --- "Multiple cloning sites of the pCRII (Invitorgen), pUC 18 (Pharmacia) and pBluescript SK+ vectors (Clontech)" --- p.155 / Chapter VI. --- Nucleic acid molecular weight size markers --- p.158

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-17T11:43:51Z No. of bitstreams: 1 Francisco de Assis Camara Rabelo Filho.pdf: 1431986 bytes, checksum: a5c762b1d10133b3e27a6ed470fcfa1b (MD5) / Made available in DSpace on 2017-03-17T11:43:51Z (GMT). No. of bitstreams: 1 Francisco de Assis Camara Rabelo Filho.pdf: 1431986 bytes, checksum: a5c762b1d10133b3e27a6ed470fcfa1b (MD5) Previous issue date: 2013-02-28 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The yam (Dioscorea spp.) has an important economic and social role in the Northeast of Brazil, providing food with high value to the human diet. It is also a source of income to poor family, employing large number of manpower. Among the phytosanitary problems of this crop, the fungal and nematode diseases are the most important ones. The virus diseases also have high importance, especially for causing cultivar degeneracy, with reduction of plant vigor and consequent qualitative and quantitative production losses. Three viruses stand out in Brazil with occurrence on main cultivars used in Northeast, Yam mosaic virus (YMV) and Yam mild mosaic virus (YMMV), belonging to the genus Potyvirus, family Potyviridae and one virus of the genus Badnavirus, family Caulimoviridae. Recently, two more viruses from the genus Begomovirus and Curtovirus genus, family Geminiviridae were detected. In the present work, initially, the detection and characterization of an YMMV isolate obtained from D. trifida were undertaken by electronic microscope analysis including visualization of cytoplasmatic inclusions typical of the genus Potyvirus. In addition, molecular studies with specific primers for YMMV in RT–PCR, followed by RT–PCR with degenerate primers to viruses of the Potyvirus genus, cloning the product corresponding to the coat protein and phylogenetic analysis including sequences available at the GenBank were done. Sequencing of the complete genome of a YMMV isolate and a detailed study on the nucleotide sequence of this virus by cDNA amplification and cloning in plasmids pGEM-T Easy and the pCR 4 Topo were also realized, and the data deposited in GenBank. The diagnosis of plant virus diseases can be realized by serological and molecular techniques. In yam crop, molecular tools normally are used due to difficulties in acquiring specific antisera, making more expense the identification of viral agents. Polyclonal antiserum was produced in rabbit for YMMV, using the coat protein of an isolate of YMMV expressed in vitro using Escherichia coli system. / O inhame (Dioscorea spp.) apresenta importante papel socioeconômico na região Nordeste do Brasil. Além de fornecer alimento de alto valor nutritivo para a dieta humana, é fonte de renda para famílias de baixo poder aquisitivo, empregando grande contingente de mão de obra. Dentre os problemas fitossanitários desta cultura, destacam-se as doenças fúngicas e aquelas causadas por nematoides. As viroses apresentam também elevada importância, principalmente por ocasionarem degenerescência de cultivares, com diminuição do vigor das plantas e, consequentemente perdas qualitativas e quantitativas da produção. Três vírus se destacam, em nível de Brasil, com registros nas principais cultivares empregadas no Nordeste, Yam mosaic virus (YMV) e Yam mild mosaic virus (YMMV), pertencentes ao gênero Potyvirus, família Potyviridae e um vírus do gênero Badnavirus, família Caulimoviridae. Recentemente, foram detectados mais dois vírus, pertencentes aos gêneros Begomovirus e Curtovirus, família Geminiviridae. No presente trabalho, inicialmente foram efetuadas a detecção e caracterização de um isolado de YMMV, obtido de D. trifida, por meio de análise eletromicroscópica, incluindo a visualização de inclusões citoplasmáticas típicas do gênero Potyvirus. Em seguida, foram realizados estudos moleculares com primers específicos para o YMMV em RT–PCR, seguido de RT–PCR com primers degenerados para vírus do gênero Potyvirus, clonagem do produto correspondente à capa proteica e análise filogenética, com os dados de sequências disponíveis no GenBank. Também foi realizado o sequenciamento do genoma completo de um isolado de YMMV e estudo detalhado da sequência de nucleotídeo, mediante amplificação do cDNA e clonagem em plasmídeos pGEM-T Easy e do pCR 4 Topo, cujos dados foram depositados no GenBank. A diagnose das fitoviroses pode ser efetuada por técnicas sorológicas e moleculares. Na cultura do inhame, usa-se, em geral, ferramentas moleculares, pela dificuldade na aquisição de antissoros específicos, o que encarece a identificação dos agentes virais. Antissoro policlonal para YMMV foi desenvolvido em coelho usando a proteína capsidial de um isolado do vírus expressa in vitro no sistema Escherichia coli.

A família das galectinas compreende um grupo de lectinas cujos domínios de reconhecimento de carboidratos (CRDs) possuem afinidade específica para ß-galactosídeos. Estas se encontram amplamente distribuídas em células normais e neoplásicas de diferentes organismos e estão envolvidas em uma grande diversidade de mecanismos celulares. As galectinas têm sido foco de estudos recentes, principalmente pelo seu envolvimento em processos inflamatórios e neoplásicos, entretanto, muitas perguntas sobre as interações com diferentes carboidratos, a especificidade destas interações e o papel específico das galectinas em inflamação, adesão celular, progressão tumoral e metástase permanecem ainda sem resposta. O presente projeto focou os estudos estruturais dos domínios de reconhecimento de carboidratos (CRDs) da galectina-4 humana (HGal-4). Nosso trabalho envolveu a clonagem, expressão, purificação dos domínios de reconhecimento de carboidratos (CRD-I e CRD-II) de forma independente. O domínio CRD-I da HGal-4 foi cristalizado e sua estrutura determinada por técnicas de cristalografia de raios-X a 2 Å de resolução. A estrutura cristalográfica do domínio CRD-I da galectina-4 humana possue duas folhas- compostas de seis fitas (S1- S6) e cinco fitas (F1-F6) enoveladas na forma de um -sanduiche. Uma comparação estrutural entre membros da classe das galectinas mostra que este enovelamento global dos CRDs é conservado e que e a diferença em especificidade pelos carboidratos observado pelas diferentes galectinas é consequência de mutações pontuais de aminoácidos. Os resultados obtidos no desenvolvimento do presente projeto serão utilizados como uma ferramenta importante para o entendimento de processos celulares que envolvem a galectina-4 humana, como inflamação, progressão celular e metástase, e conseqüentemente, contribuir para o planejamento de novas estratégias de diagnóstico e tratamento de neoplasias. / The galectin family comprises a group of lectins where the carbohydrate-recognition domains (CRDs) display specific affinity for ß-galactosides. They are widely distributed in normal and neoplasic cells of different organisms and are involved in a great diversity of cellular mechanisms. The galectins have been focus of recent studies, mainly for their involvement in inflammatory and neoplasic processes, however, many questions about the interactions with different carbohydrates, the specificity of these interactions and the specific role of the galectins in inflammation, cell adhesion, tumor progression and metastasis remain unanswered.The present project focused the strctural studies of human galectin-4 (HGal-4) carbohydrate-recognition domains (CRDs). Our work involved the independent cloning, heterologous expression and purification of both carbohydrate-recognition domains (CRD-I and CRD-II). The HGal-4 CRD-I domain has been successfully crystallized and its structure solved by X-ray crystallography techniques at 2 Å resolution. The crystallographic structure of HGal-4 CRD-I domain comprises two -sheets containing six (S1- S6) and five strands (F1-F6) each packed as a -sandwich domain. A structural comparison among members of galectin class of proteins shows that this folding is highly conserved and that the difference in specificity for carbohydrate molecules is consequence of punctual aminoacid mutations. Our results will be used as an important tool towards a better understanding of cellular processes such as inflammation, cell progression and metastasis, and consequently, contribute for the development of new strategies for diagnosis and treatment of neoplasies.

A purificação da proteína VP3 do vírus da anemia das galinhas (CAV), expressada em um sistema de expressão procariótico como uma proteína de fusão com cauda de histidina, está demonstrada neste estudo. Extraiu-se o DNA da partícula do CAV, obtida de fígado de galinha infectado. Amplificou-se o gene da proteína VP3 a partir do DNA extraído pela reação da polimerase em cadeia (PCR), para subseqüentes clonagem e expressão protéica. O produto recombinante da expressão (pTrc-VP3) foi identificado por PCR e sequenciamento. A técnica de Western blotting determinou a expressão da proteína de fusão VP3 com cauda de histidina de peso molecular de aproximadamente 21 KDa. Através da eluição do gel, obteve-se a purificação da proteína VP3 expressada com homogeneidade julgada pelo gel de poliacrilamida e dodecil sulfato de sódio. A proteína VP3 purificada foi reconhecida por anticorpos do CAV no método Western blotting. Este resultado indica que a proteína VP3 recombinante expressada no sistema do vetor pTrcHis2 pode ser utilizada como antígeno para detectar anticorpos contra o CAV. / Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21 KDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies.

As modificações das lipoproteínas de baixa densidade (LDL) são uma etapa essencial na aterogênese pois acarretam a geração de LDL eletronegativa [LDL(-)] que apresenta propriedades quimiotática, citotóxica, imunogênica e pró-inflamatória. O objetivo deste trabalho foi a produção de hibridomas secretores de anticorpos monoclonais anti-LDL(-), a clonagem dos genes que codificam para as cadeias variáveis destes anticorpos, e sua expressão como fragmentos de anticorpo de cadeia única (scFv). A LDL(-) isolada de plasma humano foi utilizada como antígeno para imunização de camundongos BALB/c. Após triagem dos clones, dois anticorpos monoclonais foram obtidos baseados em sua reatividade pela LDL( -) e não pela LDL nativa: 1A3H2 (1A3) e 2C7D5F10 (2C7). Os cDNAs codificante para a cadeia pesada (VH) e cadeia leve (VL), de ambos os anticorpos, foram obtidos por meio de RT-PCR utilizando bibliotecas de oligonucleotídeos que reconhecem todas os genes de domínios variáveis das famílias de VH e VL murinas. Os genes da VH e VL obtidos foram clonados no vetor pGEM-T Easy (Promega®) e suas seqüências determinadas. A VH do anti-LDL(-) 1A3 pertence família J558.84 e fragmento gênico JH2, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A VH do anti¬-LDL(-) 2C7 pertence a família Vmu 3.2 (J558) e fragmento gênico JH4, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A partir disso, oligonucleotídeos sintéticos foram sintetizados a fim de clonar estes segmentos gênicos no vetor pPlgLE de expressão em Pichia pastoris. Foram realizadas três construções: o scFv 1A3, scFV 2C7 e um scFv híbrido (VH do 1A3 e VL do 2C7). Das três construções obtidas, conseguimos expressar o scFv do anti-LDL 2C7D5F10 que demonstrou ser capaz de reconhecer o antígeno. A proteína recombinante expressa tem grande potencial de ser usada no diagnóstico clínico incluindo imunoensaios in vitro e como reagentes para exames que envolvam a obtenção e análise de imagens. / Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating electronegative LDL [LDL(-)], which has chemotactic cytotoxic, immunogenic and proinflammatory properties. The aim of this study was the generation of anti-LDL(-) mAbs, the cloning of the genes that code for their variable domains and their expression as single-chain Fv (scFv). LDL(-) was isolated from human blood plasma and used as an antigen for immunization of Balb/c mice. Upon screening, two different mAbs were selected based on their ability to recognize LDL(-) and not native LDL: 1A3H2 (1A3) e 2C705F10 (2C7). The cDNAs that code for VH and VL were obtained by RT-PCR using specific immunoglobulin primer libraries wich recognize all VH and VL murine families. The VH and VL genes were cloned in pGEM-T Easy (Promega®) and sequenced. The anti-LDL(-) 1A3 uses a VH segment from J558.84 and a JH2 segment, while VL uses a 8.24/Jk5 segments. The anti-LDL(-) 2C7 uses a VH segment from Vmu 3.2 (J558) and a JH4 segment, while VL uses a 8.24/Jk5 segments. Oligonucleotides were synthetized and those gene segments were cloned in pPIGLE a Pichia pastoris immunoglobulin expression vector. We obtained three scFv constructions: scFV 1A3, scFv 2C7 and a husk hybrid, harboring 1A3 VH and 2C7 VL. Among those, we expressed the scFv anti¬-LDL(-) 2C7 that are able to recognize the antigen. The recombinant protein has a great potential for clinicai diagnostic applications, including in vitro immunoassays and as imaging reagents.