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Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (p. 91-99).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [104]-114).

The objective of this project was to develop a DNA hybridization sensor that can detect the presence of E. coli and reveal its resistance to the drug gentamicin. This probe will enable rapid and user-friendly diagnostics of E. coli infections and analysis of bacterial gentamicin-susceptibility profile by interrogation of a fragment of E. coli 16S rRNA bearing a substitution in the gentamicin-resistant cells. The sensor is promising for the point-of-care use to provide a timely UTI diagnostic solution. A quick diagnosis of E. coli infection and antibiotic resistance is crucial for treatment. To design a hybridization probe, we proposed a split approach for target interrogation and catalytic activity of a peroxidase-like deoxyribozyme (PDz) as a signal reporter. PDz contains a series of guanine residues in a strand and has been shown to form a parallel guanine-quadruplex (G4). This G4, with the addition of a hemin cofactor, catalyzes the reaction similar to that of horseradish peroxidase. If a colorless organic indicator is added to the G4-PDz-hemin containing solution and mixed H2O2, a colored oxidation product is formed (e.g., a dark blue/green). The color change reports the presence of the catalytically active G4, which occurs only when the nucleotide sequence of the target is a perfect match. When the target is not a perfect match, for example, in the case of the drug-causing nucleotide substitution, the G4 does not form, and there is no color change. The probes tested in this paper show promising results of such a sensor by being able to catalyze the described colorimetric reaction to generate a strong signal in the presence of a "gentamicin-susceptible" target and show selectivity against the "gentamicin-resistant" target.

O objetivo deste estudo foi avaliar a plataforma OpenArray para testes genéticos em doadores de sangue e determinar as frequências genotípicas e alélicas de alterações pontuais (SNPs) associadas à trombose venosa (G1691A e G20210A), à hiperhomocisteinemia (C677T, A1298C), e à hemocromatose hereditária (C282Y, H63D e S65C) na população de doadores de sangue de São Paulo, Brasil. Foram analisadas 400 amostras de sangue total coletadas de outubro a novembro de 2011. A detecção dos SNPs foi realizada utilizando a tecnologia de microarray em superfície sólida OpenArray. As amostras também foram analisadas utilizando a técnica de PCR em Tempo Real sistema FRET para comparação dos resultados e determinação da acurácia do sistema OpenArray. Observamos que houve 100% de concordância de resultados entre ambas as técnicas para todas as amostras, em todas as variantes pesquisadas, com exceção da mutação C282Y no gene HFE, a qual apresentou 99,75% de concordância. O resultado da amostra em questão foi posteriormente confirmado por sequenciamento direto (Sanger), que confirmou o resultado fornecido pelo método OpenArray. As frequências calculadas para cada SNP foram: FV G1691A 98,8% (G/G), 1,2% (G/A); FII G2021A 99,5% (G/G), 0,5% (G/A); MTHFR C677T 45,5% (C/C), 44,8% (C/T), 9,8% (T/T); MTHFR A1298C 60,3% (A/A), 33,6% (A/C), 6,1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78,1%(C/C), 20,3% (C/G), 1,6% (G/G); e HFE S65C 98,1% (A/A), 1,9% (A/T). Esses resultados descrevem as frequências de SNPs associados a doenças e são importantes para aprimorar o conhecimento atual do perfil genético da população de doadores de sangue brasileiros, embora um estudo maior seja necessário para determinar com a maior acurácia a frequência das mutações pesquisadas. Além disso, observamos que a plataforma OpenArray, demonstrou alta taxa de concordância com o método de PCR em Tempo Real sistema FRET. / The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs) associated with venous thrombosis (G1691A and G20210A), hyperhomocysteinemia (C677T, A1298C), and hereditary hemochromatosis (C282Y, H63D and S65C) in blood donors from Sao Paulo, Brazil. We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR-FRET system to compare the results and determine the accuracy of the OpenArray method. We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G), 1.2% (G/A); FII G2021A 99.5% (G/G), 0.5% (G/A); MTHFR C677T 45.5% (C/C), 44.8% (C/T), 9.8% (T/T); MTHFR A1298C 60.3% (A/A), 33.6% (A/C), 6.1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78.1%(C/C), 20.3% (C/G), 1.6% (G/G); and HFE S65C 98.1% (A/A), 1.9% (A/T).Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR

Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r��educe R��IP and r��ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 10, 2012 - Dec. 10, 2014

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 92-99).

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

O objetivo deste estudo foi avaliar a plataforma OpenArray para testes genéticos em doadores de sangue e determinar as frequências genotípicas e alélicas de alterações pontuais (SNPs) associadas à trombose venosa (G1691A e G20210A), à hiperhomocisteinemia (C677T, A1298C), e à hemocromatose hereditária (C282Y, H63D e S65C) na população de doadores de sangue de São Paulo, Brasil. Foram analisadas 400 amostras de sangue total coletadas de outubro a novembro de 2011. A detecção dos SNPs foi realizada utilizando a tecnologia de microarray em superfície sólida OpenArray. As amostras também foram analisadas utilizando a técnica de PCR em Tempo Real sistema FRET para comparação dos resultados e determinação da acurácia do sistema OpenArray. Observamos que houve 100% de concordância de resultados entre ambas as técnicas para todas as amostras, em todas as variantes pesquisadas, com exceção da mutação C282Y no gene HFE, a qual apresentou 99,75% de concordância. O resultado da amostra em questão foi posteriormente confirmado por sequenciamento direto (Sanger), que confirmou o resultado fornecido pelo método OpenArray. As frequências calculadas para cada SNP foram: FV G1691A 98,8% (G/G), 1,2% (G/A); FII G2021A 99,5% (G/G), 0,5% (G/A); MTHFR C677T 45,5% (C/C), 44,8% (C/T), 9,8% (T/T); MTHFR A1298C 60,3% (A/A), 33,6% (A/C), 6,1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78,1%(C/C), 20,3% (C/G), 1,6% (G/G); e HFE S65C 98,1% (A/A), 1,9% (A/T). Esses resultados descrevem as frequências de SNPs associados a doenças e são importantes para aprimorar o conhecimento atual do perfil genético da população de doadores de sangue brasileiros, embora um estudo maior seja necessário para determinar com a maior acurácia a frequência das mutações pesquisadas. Além disso, observamos que a plataforma OpenArray, demonstrou alta taxa de concordância com o método de PCR em Tempo Real sistema FRET. / The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs) associated with venous thrombosis (G1691A and G20210A), hyperhomocysteinemia (C677T, A1298C), and hereditary hemochromatosis (C282Y, H63D and S65C) in blood donors from Sao Paulo, Brazil. We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR-FRET system to compare the results and determine the accuracy of the OpenArray method. We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G), 1.2% (G/A); FII G2021A 99.5% (G/G), 0.5% (G/A); MTHFR C677T 45.5% (C/C), 44.8% (C/T), 9.8% (T/T); MTHFR A1298C 60.3% (A/A), 33.6% (A/C), 6.1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78.1%(C/C), 20.3% (C/G), 1.6% (G/G); and HFE S65C 98.1% (A/A), 1.9% (A/T).Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR