Yu Chi Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 124-139). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abbreviations --- p.iv / Abstract --- p.v / 論文摘要 --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction / Chapter 1.2 --- HBV and its potential oncogenic properties / Chapter 1.3 --- Aim of the present study / Chapter 1.4 --- Expressed sequence tag (EST) analysis: an approach to reveal gene expression pattern in a specific tissue / Chapter 1.5 --- cDNA subtraction / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- Plating out the adult human normal liver cDNA library / Chapter 2.2 --- PCR amplification of cloned human normal liver cDNA inserts / Chapter 2.3 --- Cycle sequencing of cloned human normal liver cDNA inserts / Chapter 2.4 --- mRNA preparation from the HCC tissue and its surrounding normal counterpart / Chapter 2.5 --- PCR-Select cDNA subtraction / Chapter 2.6 --- Construction of HCC subtracted cDNA library by T/A cloning method / Chapter 2.7 --- PCR amplification of cloned subtracted cDNA / Chapter 2.8 --- Cycle sequencing of cloned subtracted cDNA / Chapter 2.9 --- Sequence analysis / Chapter 2.10 --- Differential hybridization of HCC subtracted clones / Chapter Chapter 3 --- Results --- p.46 / Chapter 3.1 --- The sequencing results of adult human normal liver cDNA clones / Chapter 3.2 --- Categorization of ESTs sequenced from the adult normal liver / Chapter 3.3 --- Adaptor ligation efficiency analysis / Chapter 3.4 --- Primary and secondary PCR Amplification / Chapter 3.5 --- PCR analysis of subtraction efficiency / Chapter 3.6 --- The sequencing results of subtracted HCC cDNA clones / Chapter 3.7 --- Categorization of ESTs sequenced from the subtracted HCC cDNA library / Chapter 3.8 --- Differential hybridization of subtracted cDNA clones / Chapter Chapter 4 --- Discussions --- p.90 / Chapter 4.1 --- Characterization of the ESTs generated from human normal liver cDNA library / Chapter 4.2 --- EST analysis on subtracted HCC cDNA clones / Chapter 4.3 --- Candidate genes differentially expressed in HCC / Appendix A The coordinates of dot blots (in numerical order according to clone numbers) / Appendix B The coordinates of dot blots (in alphabetical order according to putative identity) / References --- p.124

Au Chi Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 157-169). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Table of Contents --- p.ii / Abstract --- p.v / 論文摘要 --- p.vii / Abbreviations --- p.viii / List of Figures --- p.ix / List of Tables --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Hepatitis B virus and Hepatocellular carcinoma --- p.3 / Chapter 1.3 --- Pathogenesis of HBV related HCC --- p.6 / Chapter 1.4 --- Current screening test and tumor markers --- p.10 / Chapter 1.5 --- Expressed sequence tag (EST) sequencing --- p.13 / Chapter 1.6 --- Aim of the present study --- p.15 / Chapter 1.7 --- Characterization of KIAA0022 --- p.16 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of liver HCC and normal counterpart libraries --- p.19 / Chapter 2.2 --- Plating out the human liver cDNA libraries --- p.19 / Chapter 2.3 --- PCR amplification of clones human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4 --- Cycle sequencing of cloned human liver cancer and the normal counterpart cDNA libraries --- p.21 / Chapter 2.4.1 --- Dye-primer cycle sequencing (Pharmacia) --- p.21 / Chapter 2.4.1.1 --- Using Pharmacia LBKA.L.F. DNA sequencer --- p.21 / Chapter 2.4.1.2 --- Using Li-Cor 4200 Automated DNA sequencer --- p.22 / Chapter 2.4.2 --- Dye-terminator cycle sequencing (Pharmacia) --- p.22 / Chapter 2.5 --- Sequences analysis --- p.23 / Chapter 2.6 --- Cloning of full-length cDNA of KIAA0022 --- p.24 / Chapter 2.6.1 --- Amplification of KIAA0022 gene using PCR --- p.24 / Chapter 2.6.2 --- Purification of the PCR product --- p.25 / Chapter 2.6.3 --- Ligation --- p.25 / Chapter 2.6.4 --- One Shot® TOP 10 Chemical Transformation --- p.25 / Chapter 2.6.5 --- Small-scale preparation of the plasmid DNA --- p.26 / Chapter 2.6.6 --- Large-scale preparation of the plasmid DNA Table of Contents (continued) --- p.26 / Chapter 2.6.7 --- DNA sequencing of the full-length cDNA of KIAA0022 --- p.28 / Chapter 2.7 --- Northern Hybridization --- p.29 / Chapter 2.7.1 --- The Human multiple tissue Northern Blot --- p.29 / Chapter 2.7.2 --- Synthesis of the radiolabeled DNA probe --- p.29 / Chapter 2.7.3 --- Hybridization of the Northern blot --- p.30 / Chapter 2.8 --- Subcellular localization of KIAA0022 by tagging with green fluorescence protein (GFP) --- p.30 / Chapter 2.8.1 --- Amplification and purification of the KIAA0022 gene product --- p.30 / Chapter 2.8.2 --- Restriction enzymes digestion --- p.31 / Chapter 2.8.3 --- DNA ligation --- p.31 / Chapter 2.8.4 --- Preparation of the Escherichia coli competent cells for transformation --- p.31 / Chapter 2.8.5 --- Transformation of the plasmid DNA into competent Escherichia coli cells --- p.32 / Chapter 2.8.6 --- Small-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.7 --- Large-scale preparation of the plasmid DNA --- p.32 / Chapter 2.8.8 --- DNA sequencing of the cloned plasmid DNA --- p.33 / Chapter 2.8.9 --- Transfection --- p.33 / Chapter 2.8.10 --- Fluorescence microscopy examination --- p.33 / Chapter 2.9 --- Yeast two-hybrid screening assay --- p.34 / Chapter 2.9.1 --- "Cloning of the KIAA0022 gene into the yeast two-hybrid DNA-BD vector, pGBKT7" --- p.34 / Chapter 2.9.2 --- Small-scale transformation of pGBKT7-KIAA0022 plasmid --- p.34 / Chapter 2.9.2.1 --- Preparation of yeast competent cells --- p.34 / Chapter 2.9.2.2 --- Transformation of the pGBKT7-KIAA 0022 plasmid into the yeast strain PJ69-2A --- p.35 / Chapter 2.9.3 --- Screening a pretransformed library by yeast mating --- p.35 / Chapter 2.9.4 --- β -Galactosidase analysis - colony lift filter assay --- p.36 / Chapter 2.9.5 --- Analysis of yeast plasmid inserts using PCR and DNA sequencing --- p.37 / Chapter 2.9.5.1 --- PCR --- p.37 / Chapter 2.9.5.2 --- DNA sequencing --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Results of ESTs sequencing in normal counterpart and HCC libraries --- p.38 / Chapter 3.1.1 --- The sequencing results of the normal counterpart cDNA clones --- p.38 / Chapter 3.1.2 --- Sequencing results of the human liver cancer cDNA clones --- p.41 / Chapter 3.1.3 --- The accuracy of the automated sequencing technique --- p.41 / Chapter 3.1.4 --- Catalogue of normal counterpart ESTs --- p.45 / Chapter 3.1.5 --- Catalogue of liver cancer ESTs --- p.47 / Chapter 3.2 --- Identification of genes differentially expressed in HCC using in silico method --- p.115 / Chapter 3.3 --- Sequence analysis of KIAA0022 --- p.121 / Chapter 3.3.1 --- Structural analysis of KIAA0022 --- p.121 / Chapter 3.3.2 --- Homology alignment --- p.122 / Chapter 3.4 --- Tissue distribution and expression profile of KIAA0022 using Northern blot analysis --- p.132 / Chapter 3.5 --- Subcellular localization of the KIAA0022 tagging by green fluorescence protein --- p.134 / Chapter 3.6 --- Yeast two-hybrid screening assay --- p.136 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Large-scale partial cDNA sequencing --- p.138 / Chapter 4.2 --- Characterization of ESTs --- p.139 / Chapter 4.3 --- Identification of genes differentially expressed in liver cancer using Poisson probability --- p.143 / Chapter 4.4 --- Characterization of KIAA0022 --- p.154 / Reference --- p.157 / Appendix --- p.170

Stephen Siu-chung Chim. / "June 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 152-166). / 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.

Masayo Kotaka. / "May 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 179-190). / 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.

Or Man Wai. / "January 2002." / "glued" in title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / 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.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-119).

Apesar do intenso interesse na regulação metabólica e evolução das vias produtoras de ATP, o porquê de a maioria dos microorganismos multicelulares metabolizarem glicose através de respiração, ao invés da fermentação, ainda permanece sem resposta. Um desses microorganismos é o fungo celulolítico Trichoderma reesei (Hypocrea jecorina. Usando análise EST e microarrays de cDNA, foi estabelecido, em T. reesei, que a expressão dos genes que codificam as enzimas do ciclo de TCA é programada de tal modo a favorecer a oxidação de piruvato pelo ciclo de TCA, ao invés de sua redução a etanol, através da fermentação. Além disso, os resultados indicam que acetaldeído pode ser convertido a acetato, e não a etanol, prevenindo a regeneração de NAD+, um produto chave requerido para o metabolismo anaeróbico. Os estudos também mostram que a maquinaria de controle regulatório por glicose, foi, provavelmente, objeto de pressão evolutiva, a qual dirigiu o fluxo metabólico à respiração, e não à fermentação. / Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long-standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the TCA is programmed in a way that favors the oxidation of pyruvate via the TCA cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD+, a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation.

Orientador: Anete Pereira de Souza. / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:31:52Z (GMT). No. of bitstreams: 1 DaSilva_CarlaCristina_D.pdf: 7310053 bytes, checksum: 1acc7f4e6eb7811b25670f85f9563217 (MD5) Previous issue date: 2014 / Resumo: A seringueira (Hevea brasiliensis), espécie nativa da Amazônia, é a maior fonte de borracha natural do mundo. Programas de melhoramento genético da seringueira têm sido cruciais para a obtenção de caracteres desejáveis. Entretanto, o ciclo de melhoramento da seringueira é muito longo (cerca de 30 anos), tornando-se essencial o desenvolvimento de novas técnicas de avaliação precoce. As bibliotecas de cDNA e Expressed Sequence Tags (ESTs) são ferramentas muito importantes em biologia molecular: possibilitam identificar genes preferencialmente expressos em tecidos ou tipos celulares e também são valiosas fontes de marcadores polimórficos, instrumentos poderosos para genotipagem e mapeamento molecular. O uso de marcadores derivados de ESTs permite construir mapas funcionais, nos quais são posicionados genes transcritos ou regiões próximas aos genes. Este tipo de mapeamento é importante para estudos de associação gene-característica, e identificação de genes candidatos. Este trabalho objetivou a construção de bibliotecas de cDNA de diferentes tecidos (painel, látex e folha) e tratamentos (exposição ao frio e infecção controlada por Microcyclus ulei) de seringueira para desenvolver sequências EST e marcadores moleculares gene-direcionados a partir destas sequências, para aumentar a saturação de um mapa integrado baseado em microssatélites, no qual identificaram 18 quantitative trait loci (QTLs) para características de crescimento, construído previamente em nosso laboratório. Foram sequenciados 10.464 clones, gerando 8.551 ESTs de alta qualidade que agrupadas formaram 5.211 unigenes. Destes, 3.582 (68,7%) apresentam similaridade com uma proteína hipotética ou expressa. Foram desenvolvidos 173 marcadores EST-SSR e 43 marcadores SNP para H. brasiliensis. 150 EST-SSRs (87%) podem estar associados a genes funcionais, e 98,8% foram transferidos para outras espécies de Hevea, sugerindo que o gênero seja um complexo formado pelas diferentes espécies. Os SNPs foram identificados em 13 ESTs similares a proteínas de resposta a estresse, desenvolvimento e síntese de látex. Seis sequências foram abundantes nas bibliotecas de exposição ao frio e análises de expressão demonstraram que a expressão de cinco sequências aumentou durante o experimento, principalmente a expressão de duas sequências que foi aumentada mais de 70 vezes. Dos EST-SSRs desenvolvidos, 46 foram genotipados na população segregante F1 com 270 indivíduos, e estes marcadores foram adicionados ao mapa genético de seringueira, totalizando 330 marcadores. O programa OneMap foi usado para a construção do mapa que possui 3.068,9 cM de extensão e 22 grupos de ligação (LGs). Cinco locos foram mapeados em regiões QTL, e os transcritos de três são similares a proteínas de resposta a estresse e desenvolvimento. Estes locos podem ser genes candidatos para estudos relacionados a características de crescimento em seringueira. Até o momento, este é o primeiro trabalho em seringueira que combina análises de ESTs de diferentes tecidos e tratamentos, e análises sobre a exposição a baixas temperaturas, em vários genótipos de seringueira. Os novos marcadores adicionados ao mapa poderão auxiliar na identificação de genes de interesse e de QTLs para outras características de importância agronômica. Os vários marcadores gene-direcionados desenvolvidos serão utilizados para mapeamento e posicionamento de possíveis genes em outras populações de mapeamento que estão sendo avaliadas no Laboratório de Análise Genética e Molecular / Abstract: Rubber tree (Hevea brasiliensis), native species of the Amazon, is world¿s major source of natural rubber. Rubber tree breeding programs have been fundamental for the selection of desirable traits. However, the breeding cycle is time consuming (around 30 years), which makes the development of new techniques for early evaluation a necessity. cDNA libraries and Expressed Sequence Tags (ESTs) are very important tools in molecular biology: they enable the identification of genes preferentially expressed in tissues or cellular types and are also a valuable resource of polymorphic markers, powerful instruments for genotyping and molecular mapping. The use of EST-derived markers allows the construction of functional maps, wherein expressed genes or regions near genes are positioned. This type of mapping is important for gene-trait association studies and candidate genes identification. The present study aimed at the construction of cDNA libraries from different tissues (panel, latex and leave) and treatments (cold exposure and Microcyclus ulei controlled infection) of rubber tree for the development of EST sequences and gene-targeted molecular markers, to raise the saturation of a microsatellite-based integrated genetic map previously constructed in our laboratory, in which 18 quantitative trait loci (QTLs) related to growth traits were identified. Sequencing of 10.464 clones generated 8,551 high quality ESTs that were clustered into 5,211 unigenes. Among these, 3,582 (68.7%) showed similarity to a hypothetical or expressed protein. A total of 173 EST-SSR and 43 SNP markers were developed for H. brasiliensis. 150 SSRs (87%) could be associated with functional genes, and 98.8% were transferred to other Hevea species, suggesting that the genus is a complex formed by different species. The SNP markers were identified in 13 ESTs that showed similarity to stress response, development and latex biosynthesis proteins. Six sequences were highly abundant in the cold exposure libraries and expression analyses demonstrated that five sequences were up-regulated during the exposure, with emphasis to two sequences with more than 70-fold increase in expression. From the developed EST-SSRs, 46 were genotyped in the segregating F1 population comprised of 270 plants. These markers were added to the genetic map, which know contains a total of 330 markers. The OneMap software was used for the map construction that now has 3,068.9 cM and 22 linkage groups. Five loci were mapped into QTLs, and transcripts of three of them present similarity to proteins involved in stress response and developmental processes. These loci may be candidate genes for studies related to rubber tree growth traits. To our knowledge, this is the first work in rubber tree that combines analyses of ESTs from different tissues and treatments, and to analyze sequences under cold stress, in several H. brasiliensis genotypes. The new positioned markers may help in the identification of genes of interest and QTLs for other agronomic important traits. The several gene-targeted markers developed here will be used in the mapping and positioning of possible genes in other mapping populations that are now being evaluated at Genetics and Molecular Analysis Laboratory / Doutorado / Genetica Vegetal e Melhoramento / Doutora em Genética e Biologia Molecular

Transcriptome analysis based on deep expressed sequence tag (EST) sequencing allows quantitative comparisons of gene expression across multiple species. Using pyrosequencing, we generated over 7 million ESTs from four stages of developing seeds of Ricinus communis, Brassica napus, Euonymus alatus and Tropaeolum majus, which differ in their storage tissue for oil, their ability to photosynthesize and in the structure and content of their triacylglycerols (TAG). The larger number of ESTs in these 16 datasets provided reliable estimates of the expression of acyltransferases and other enzymes expressed at low levels. Analysis of EST levels from these oilseeds revealed both conserved and distinct species-specific expression patterns for genes involved in the synthesis of glycerolipids and their precursors. Independent of the species and tissue type, ESTs for core fatty acid synthesis enzymes maintained a conserved stoichiometry and a strong correlation in temporal profiles throughout seed development. However, ESTs associated with non-plastid enzymes of oil biosynthesis displayed dissimilar temporal patterns indicative of different regulation. The EST levels for several genes potentially involved in accumulation of unusual TAG structures were distinct. Comparison of expression of members from multi-gene families allowed the identification of specific isoforms with conserved function in oil biosynthesis. In all four oilseeds, ESTs for Rubisco were present, suggesting its possible role in carbon metabolism, irrespective of light availability. Together, these data provide a resource for use in comparative and functional genomics of diverse oilseeds. Expression data for more than 350 genes encoding enzymes and proteins involved in lipid metabolism are available at the 'ARALIP' website ().