Global ETD Search

11	Metagenomic discovery and characterisation of restriction endonuclease from Kogelberg Biosphere Reserve Mtimka, Sibongile 05 1900 (has links) Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research. / Chemistry / M. Sc. (Life Sciences) Bacteriophage Fosmid library Functional screening Kogelberg Nature Reserve Restriction endonuclease Restriction enzymes Soil metagenome 572.786 Restriction enzymes, DNA Bacteriophages -- Genetics Endonucleases
12	Triagem funcional de genes envolvidos no processo de manutenção da inativação do cromossomo X em humanos / Functional screening of genes involved in the maintenance of X chromosome inactivation in humans Naja Vergani 01 April 2014 (has links) A compensação da dosagem gênica entre fêmeas XX e machos XY em mamíferos é adquirida através de um complexo mecanismo epigenético que resulta na inativação de grande parte de um dos cromossomos X nas células femininas. O processo de inativação do cromossomo X (XCI) se inicia cedo durante a embriogênese, concomitantemente à diferenciação celular, e envolve a aquisição de modificações epigenéticas características do cromossomo X inativo (Xi). Uma estabelecido, o padrão de inativação é estavelmente mantido através de todas as mitoses celulares subsequentes e por toda a vida do organismo (exceto para células germinativas que sofrem reativação do Xi). Os mecanismos envolvidos na iniciação e estabelecimento da XCI foram extensivamente estudados, especialmente em camundongos. Embora algumas características epigenéticas associadas à manutenção da XCI tenham sido descritas, a identidade e modo específico de ação de fatores envolvidos durante essa fase da XCI são aspectos ainda não bem compreendidos. Além disso, o processo de XCI apresenta diferenças importantes entre humanos e camundongos e estudos direcionados para a identificação de novos componentes envolvidos na manutenção da XCI em humanos tornam-se de fundamental importância. Triagens funcionais genômicas por bibliotecas de shRNAs constituem uma ferramenta poderosa para a identificação de genes envolvidos em diferentes mecanismos celulares e vias bioquímicas. Sendo assim, utilizamos essa ferramenta para triar genes envolvidos na manutenção da XCI em humanos. Células somáticas femininas primárias HPRT+/-/HPRT- foram transduzidas com uma biblioteca lentiviral de shRNAs e posteriormente tratadas em meio de cultura contendo a droga HAT para seleção de células HPRT+ nas quais esperava-se que o cromossomo Xi presente tivesse sofrido reativação em decorrência do knockdown de genes envolvidos na manutenção da XCI. Essa estratégia nos permitiu identificar 20 novos genes candidatos a estarem envolvidos na manutenção da XCI. Esses candidatos deverão ser avaliados individualmente para confirmar seu papel no processo de controle epigenético do cromossomo X / Transcriptional dosage compensation between mammalian XX females and XY males is acquired through a complex epigenetic mechanism that leads to the inactivation of most part of one of the X chromosomes in the female cells. The X chromosome inactivation (XCI) process takes place early during embryogenesis and involves the acquisition of epigenetic modifications that are characteristic of the inactive X chromosome (Xi). Once silencing is established, the inactivation pattern is maintained in through all the subsequent mitosis and the same X chromosome remains stably silenced in all the descendant cells and throughout the life of the organism (except for the germ line cells that undergo X chromosome reactivation). The initiation of XCI has been studied extensively, especially in mice. Although some epigenetic features associated with the maintenance of XCI have already been described, the identity and specific mode of action of the factors involved in this phase of XCI are largely unknown. Moreover, the XCI process presents important differences between mice and humans, and studies directed to the identification of new players involved in the maintenance of human XCI are fundamentally important. Functional genome-wide screens using multiplex shRNA libraries are a powerful tool for the identification of genes involved in different cellular mechanisms and biochemical pathways. In order to screen for genes involved in the maintenance of XCI in humans, a population of HPRT+/-/HPRT- primary somatic female cells were transduced with a multiplex lentiviral shRNA library and subsequently treated in HAT medium to select for HPRT+ cells in which we expected that the Xi would undergo reactivation as a result of the knockdown of genes involved in the maintenance of XCI. As a result, we identified 20 new candidate genes that could potentially be involved in the maintenance of XCI. These candidates should be individually evaluated in order to confirm their role in the epigenetic control of the X chromosome Epigenética ICX Inativação do cromossomo X Sequenciamento de próxima geração Triagem genômica funcional Epigenetics Genomic functional screening Next generation sequencing NGS X chromosome inactivation XCI
13	Season Long Changes in Performance Outcome Measures Using the Functional Preparticipation Examination Sabol, Todd C. 16 June 2017 (has links) No description available. Sports Medicine Health Care Single-leg reach SLAR Single-leg hop SLHOP PPE, FPPE, functional screening
14	Identifikation von Genen und Mikroorganismen, die an der dissimilatorischen Fe(III)-Reduktion beteiligt sind / Isolation of Genes and Microorganisms Involved in Dissimilatory Fe(III)-Reduction Özyurt, Baris 21 January 2009 (has links) No description available. 500 Naturwissenschaften allgemein Mathematics and Computer Science Metagenomik dissimilatorische Fe(III)-Reduktion Dissimilatory iron reduction DIR dissimilatory oxido-reduction of iron dissimilatory iron(III) reduction metal reduction ferricironreduction microbial iron respiration bacterial respiration microorganisms for energy production dissimilatory iron reducing bacteria dissimilatory metal reducing bacteria DMRB energy production electricity energy transduction microbial electricity electron donor electron acceptor electron transfer carbon source carbohydrate mannit mannitol biofilm mineral mineral formation microbial degradation ribosomal proteins cytochromes hypothetical proteins enzymes oxidation reduction ferric iron iron oxide Fe(II) Fe(III) soluble iron insoluble iron ferric reductase activity iron mineral formation mineralisation <i>E. coli</i> Escherichia coli environmental microorganisms life in extreme environments uncultivable bacteria soil microbial ecology anaerobic growth anaerobic incubation environmental sequences environmental DNA DNA extraction functional screening metagenomic metagenome meta-genome microbial ecology environmental genomics soil metagenome BACs sequencing bioinformatics microbial communities 16S rRNA phylogenetics 30 42.30 42.30 58.30 42.49 58.30 42.13 RA W

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