Philosophiae Doctor - PhD / With easy access to technology to generate expressed sequence tags (ESTs), several groups have sequenced from thousands to several thousands of ESTs. These ESTs benefit from consolidation and organization to deliver significant biological value. A number of EST projects are underway to extract maximum value from fragmented EST resources by constructing gene indices, where all transcripts are partitioned into index classes such that transcripts are put into the same index class if they represent the same gene. Therefore a gene index should ideally represent a non-redundant set of transcripts. Indeed, most gene indices aim to reconstruct the gene complement of a genome and their technological developments are directed at achieving this goal. The South African National Bioinformatics Institute (SANBI), on the other hand, embarked on the development of the sequence alignment and consensus knowledgebase (STACK) database that focused on the detection and visualisation of transcript variation in the context of developmental and pathological states, using all publicly available ESTs. Preliminary work on the STACK project employed an approach of partitioning the EST data into arbitrarily chosen tissue categories as a means of reducing the EST sequences to manageable sizes for subsequent processing. The tissue partitioning provided the template material for developing error-checking tools to analyse the information embedded in the error-laden EST sequences. However, tissue partitioning increases redundancy in the sequence data because one gene can be expressed in multiple tissues, with the result that multiple tissue partitioned transcripts will correspond to the same gene.Therefore, the sequence data represented by each tissue category had to be merged in order to obtain a comprehensive view of expressed transcript variation across all available tissues. The need to consolidate all EST information provided the impetus for developing a STACK human gene index, also referred to as a whole-body index. In this dissertation, I report on the development of a STACK human gene index represented by consensus transcripts where all constituent ESTs sample single or multiple tissues in order to provide the correct development and pathological context for investigating sequence variation. Furthermore, the availability of a human gene index is assessed as a diseasecandidate gene discovery resource. A feasible approach to construction of a whole-body index required the ability to process error-prone EST data in excess of one million sequences (1,198,607 ESTs as of December 1998). In the absence of new clustering algorithms, at that time, we successfully ported D2_CLUSTER, an EST clustering algorithm, to the high performance shared multiprocessor machine, Origin2000. Improvements to the parallelised version of D2_CLUSTER included: (i) ability to cluster sequences on as many as 126 processors. For example, 462000 ESTs were clustered in 31 hours on 126 R10000 MHz processors, Origin2000. (ii) enhanced memory management that allowed for clustering of mRNA sequences as long as 83000 base pairs. (iii) ability to have the input sequence data accessible to all processors, allowing rapid access to the sequences. (iv) a restart module that allowed a job to be restarted if it was interrupted. The successful enhancements to the parallelised version of D2_CLUSTER, as listed above, allowed for the processing of EST datasets in excess of 1 million sequences. An hierarchical approach was adopted where 1,198,607 million ESTs from GenBank release 110 (October 1998) were partitioned into "tissue bins" and each tissue bin was processed through a pipeline that included masking for contaminants, clustering, assembly, assembly analysis and consensus generation. A total of 478,707 consensus transcripts were generated for all the tissue categories and these sequences served as the input data for the generation of the wholebody index sequences. The clustering of all tissue-derived consensus transcripts was followed by the collapse of each consensus sequence to its individual ESTs prior to assembly and whole-body index consensus sequence generation. The hierarchical approach demonstrated a consolidation of the input EST data from 1,198607 ESTs to 69,158 multi-sequence clusters and 162,439 singletons (or individual ESTs). Chromosomal locations were added to 25,793 whole-body index sequences through assignment of genetic markers such as radiation hybrid markers and généthon markers. The whole-body index sequences were made available to the research community through a sequence-based search engine (http://ziggy.sanbi.ac.za/~alan/researchINDEX.html). / South Africa

Submitted by Filipe dos Santos (fsantos@pucsp.br) on 2017-10-19T12:02:19Z No. of bitstreams: 1 Rafael Nogueira Furtado.pdf: 1429930 bytes, checksum: ae438c7adb694c4e3a04faedbf462358 (MD5) / Made available in DSpace on 2017-10-19T12:02:19Z (GMT). No. of bitstreams: 1 Rafael Nogueira Furtado.pdf: 1429930 bytes, checksum: ae438c7adb694c4e3a04faedbf462358 (MD5) Previous issue date: 2017-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This doctoral thesis consists of an analysis of controversies about human gene editing, trying to reveal different conceptions of human constructed today. Contemporary scientific development has enabled unprecedented interventions on biological phenomena, while raising new technical, ethical and social challenges. Reflecting on these challenges, philosopher Peter Sloterdijk problematizes the effects of science and technology on society, facing the crisis of traditional humanist thinking. According to the author, humanism consisted of the main instrument of human formation, elaborated by Western culture. However, such an instrument would know its exhaustion today, leading Sloterdijk to question the role of biotechnology in the production of people. Grounded on the discussion established by the philosopher, this thesis analyzes controversies about human gene editing, having documents of public domain as its empirical material . The documents were selected from databases from January, 2015 to December, 2016. They consist of scientific papers published by the journals Nature, Science, Protein & Cell and The American Journal of Bioethics, as well as statements issued by the institutions UNESCO and Hinxton Group. The theorical approach of this research leans on analysis of discursive practices and the production of meanings, elaborated by the Center for Studies and Research on Discursive Practices in Daily Life (NUPRAD) of PUC-SP. It is intended to evidence the argumentative strategies present in the opposing and favorable discourses to gene editing, elucidating the effects of this practice for the understanding of what it is to be human today / Esta tese de doutorado consiste em uma análise de controvérsias sobre edição genética humana, buscando explicitar diferentes concepções de humano construídas na atualidade. O desenvolvimento científico contemporâneo tem possibilitado intervenções sem precedentes sobre os fenômenos biológicos, suscitando, entretanto, novos desafios técnicos, éticos e sociais. Refletindo acerca destes desafios, o filósofo Peter Sloterdijk problematiza os efeitos da ciência e da tecnologia para a sociedade, em face da crise do pensamento humanista tradicional. De acordo com o autor, o humanismo consistiu no principal instrumento de formação humana, elaborado pela cultura ocidental. Contudo, tal instrumento conheceria seu esgotamento na atualidade, levando Sloterdijk a se questionar sobre o papel da biotecnologia para a produção de pessoas. Partindo da discussão estabelecida pelo filósofo, a tese analisa controvérsias sobre edição genética humana, tendo como material empírico documentos de domínio público. Os documentos foram selecionados em bases de dados, entre o período de janeiro de 2015 a dezembro de 2016. Eles consistem em artigos científicos publicados pelas revistas Nature, Science, Protein & Cell e The American Journal of Bioethics, bem como em declarações emitidas pelas instituições UNESCO e Hinxton Group. Utiliza-se como abordagem a análise de práticas discursivas e produção de sentidos, elaborada pelo Núcleo de Estudos e Pesquisas sobre Práticas Discursivas no Cotidiano (NUPRAD) da PUC-SP. Procura-se evidenciar as estratégias argumentativas presentes nos discursos contrários e favoráveis à edição genética, elucidando os efeitos desta prática para a compreensão do que é ser humano hoje

Originally presented as the author's Thesis (doctoral--Universität Hamburg, 1994). / Includes bibliographical references (p. 280-298).

Copies of author's previously published works inserted. / Bibliography: leaves 321-370. / xv, 370, [21] leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of the project presented in this thesis is to isolate microsatellite markers and to construct a high resolution genetic map of the human X chromosome using these and pre-existing microsatellite markers. AC dinucleotide repeat markers are isolated from a bacteriophage library for application to the genetic localisations of X-linked disease genes, particularly those responsible for non-specific mental retardation (MRX). The genetic map is used to refine the location of the disease gene segregating in five families affected with X-linked mental retardation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1997

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1994. / Errata slip inserted at back. Includes bibliographical references (leaves 159-192).

Thesis (Ph.D.)--University of Melbourne, Howard Florey Institute and Dept. of Biochemistry and Molecular Biology, 2006. / Typescript. Includes bibliographical references (leaves 133-150).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Two plaque-pure phage lambda clones designated as λhtX-l and λhtX-2 that hybridized to unfractionated bovine liver tRNA were isolated from a human X chromosome-specific library. The λDNAs were characterized by restriction mapping and Southern blot hybridization techniques. The human DNA segment in λhtX-l contains five or more presumptive tRNA genes and at least one Alu family member. The 19-kilobase human DNA insert in λhtX-2 contains two or more presumptive tRNA genes and at least three Alu family members. Another human genomic clone designated λhVKV7 hybridized to mammalian valine tRNA IAC. The clone was characterized by physical mapping and Southern blot hybridization techniques. The 18.5-kilobase human DNA fragment in λhVKV7 contains a cluster of three tRNA genes and at least nine Alu family members.

No description available.

by Peter, Wei Gong Chang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 103-124). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / ABBREVIATIONS --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Human genome project --- p.5 / Chapter 1.3 --- Human adult heart cDNA library --- p.7 / Chapter 1.4 --- Human fetal heart cDNA library --- p.8 / Chapter 1.5 --- Sequencing of a human heart cDNA clone --- p.9 / Chapter 1.6 --- Knowledge of the role of selenium --- p.13 / Chapter 1.7 --- Mouse 56kDa selenium binding protein and acetaminophen-binding protein --- p.16 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Plating out the cDNA library --- p.20 / Chapter 2.1.1 --- "Mediums, buffers and solutions" --- p.20 / Chapter 2.1.2 --- Bacteriophage clones preparation --- p.21 / Chapter 2.2 --- cDNA clone amplification by PCR --- p.23 / Chapter 2.3 --- Cycle sequencing of PCR products --- p.25 / Chapter 2.3.1 --- "Media, buffers and solutions" --- p.25 / Chapter 2.3.2 --- Preparation of sequencing reaction --- p.25 / Chapter 2.4 --- Gel electrophoresis using an automated A.L.F sequencer --- p.27 / Chapter 2.5 --- DNA sequence analysis --- p.29 / Chapter 2.6 --- Preparation of competent E. coli for transformation --- p.30 / Chapter 2.7 --- Transformation of plasmid into competent E. coll --- p.31 / Chapter 2.8 --- Mini-preparation of plasmid DNA --- p.32 / Chapter 2.9 --- Large scale plasmid DNA preparation by QIAGEN --- p.34 / Chapter 2.10 --- Cloning the human 56 kDa selenium binding protein (hSP56) into the pAED4 vector --- p.36 / Chapter 2.10.1 --- Bacterial strains and vector --- p.36 / Chapter 2.10.2 --- "Media, buffers and solutions" --- p.38 / Chapter 2.10.3 --- Primers design and PCR --- p.42 / Chapter 2.10.4 --- Purification of PCR products by Geneclean --- p.43 / Chapter 2.10.5 --- Restriction digestion of purified PCR product and pAED4 --- p.44 / Chapter 2.10.6 --- Ligation and transformation of hSP56 --- p.45 / Chapter 2.10.7 --- Screening and purification ofpAED4hSP56. --- p.47 / Chapter 2.11 --- Expression of hsp56 --- p.50 / Chapter 2.11.1 --- Induction of hSP56 expression --- p.50 / Chapter 2.11.2 --- SDS-PAGE and protein detection --- p.51 / Chapter 2.12 --- Northern hybriddization of hSP56 --- p.53 / Chapter 2.12.1 --- Animals & human tissue --- p.53 / Chapter 2.12.2 --- "Mediums, buffers and solutions" --- p.53 / Chapter 2.12.3 --- Preparation of total RNA --- p.56 / Chapter 2.12.4 --- Formaldehyde agarose gel electrophoresis --- p.57 / Chapter 2.12.5 --- Preparation of radioactive probe --- p.58 / Chapter 2.12.6 --- RNA transfer and Northern hybridization --- p.59 / Chapter 2.13 --- Chromosomal mapping of the hSP56 gene --- p.62 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- The sequencing results of 553 cDNA clones --- p.63 / Chapter 3.2 --- Catalogues of genes expressed --- p.65 / Chapter 3.3 --- Sequence analysis of hSP56 --- p.71 / Chapter 3.4 --- Northern hybridization of hSP56 --- p.84 / Chapter 3.5 --- Cloning of hSP56 into pAED4 --- p.87 / Chapter 3.6 --- Expression of the hSP56 in E. coli --- p.89 / Chapter 3.7 --- Chromosomal mapping of the hSP56 gene --- p.92 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- General discussion --- p.94 / Chapter 4.2 --- The possible roles of hSP56 and mSP56 --- p.101 / Chapter 4.3 --- Future prospects --- p.102 / REFERENCES --- p.103 / APPENDIX 1 --- p.125 / APPENDIX.2 --- p.127