Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 153 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 136-149).

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Tesis para optar al Grado Académico de Magíster en Bioquímica Área de Especialización: Toxicología y Diagnóstico Molecular y Memoria para optar al Título Profesional de Bioquímica / Los médicos prescriben fármacos sobre la base de sus características farmacológicas y sobre la posibilidad de obtener resultados clínicamente reproducibles. Sin embargo, muchos de los fármacos son eficaces en sólo 25% a 60% de los pacientes, por lo tanto, muchos pacientes no responden a los tratamientos e incluso experimentan reacciones adversas e intoxicaciones. Existen variaciones individuales en las respuestas a fármacos que pueden deberse a diversos efectos, pero en la actualidad se sabe que muchas de estas variaciones se encuentran predeterminadas genéticamente; éste es el blanco de estudio de la farmacogenética. La debrisoquina-4-hidroxilasa (CYP2D6) es una de las enzimas más importante del metabolismo de fármacos que actúan tanto a nivel del sistema nervioso central (SNC) como del sistema circulatorio. Los polimorfismos de CYP2D6 presentan grandes diferencias en las frecuencias alélicas según la población y etnia. En este estudio se analizaron a través de PCR-AS semianidada, PCR RFLP y PCRL las variables CYP2D6*2, CYP2D6*3, CYP2D6*4 y la duplicación del gen CYP2D6 en 321 voluntarios sanos. Las frecuencias alélicas encontradas son muy similares a las registradas para la población española: para *2 un 40,5%, para *3 un 1,09%, para *4 un 11,8%. No se encontraron duplicaciones del gen CYP2D6 en un subgrupo de 20 voluntarios analizados. Para determinar con exactitud la influencia de los polimorfismos del gen CYP2D6 en su actividad debrisoquina hidroxilasa, se seleccionaron 23 voluntarios del grupo en estudio que presentaban genotipos relevantes, los cuales debieron ingerir el marcador metabólico debrisoquina. Se recolectaron las muestras de orina de 0-8 horas y se analizaron a través de HPLC. Presentaron el fenotipo esperado 18 voluntarios, de los cuales el más relevante es el genotipo *4/*4 que presentó una actividad catalítica muy disminuida en comparación al grupo wt/wt. Se obtuvo una correlación genotipo-fenotipo para algunos voluntarios logrando el objetivo fundamental que busca un estudio farmacogenético, es decir, la determinación de la respuesta individual

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Includes bibliography.

Thesis (Ph. D.)--Washington State University, December 2009. / Title from PDF title page (viewed on Dec. 11, 2009). "School of Molecular Biosciences." Includes bibliographical references.

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Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well studied eutherian mammals. This poses a problem in applying data from metabolic studies with eutherians to marsupials. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. As a result, there is a clear need for improved understanding of the metabolic capabilities of Australian marsupials, particularly at the molecular level. The current PhD candidature therefore focused on characterising the important xenobiotic-metabolising enzyme superfamily, cytochrome P450, with particular emphasis on the CYP3A subfamily, in Australian marsupials, namely koala (Phascolarctos cinereus), tammar wallaby (Macropus eugenii), Eastern grey kangaroo (Macropus giganteus) and the Southern hairy-nosed wombat (Lasiorhinus latifrons). / Expression of CYP3A-like protein using hepatic microsomes was detected by western blot analysis in all four marsupial species studied. Female koalas were observed to express higher levels of CYP3A-like protein than male koalas. CYP3A activity for each marsupial species was determined in hepatic microsomes using erythromycin, a known human CYP3A4 substrate. Erythromycin N-demethylation activity was detected in all marsupial hepatic microsomes, with highest activity observed in koala. Koalas displayed gender differences in activity with female koalas showing a significant 2-fold increase. Inhibition studies with troleandomycin showed decreased erythromycin activity in both female and male koalas. Erythromycin activity in wallaby and kangaroo microsomes was notably lower than observed in koala. No gender differentiation was noted in wallaby or kangaroo. This observed difference in CYP3A activity between species may be indicative of the koala's eucalyptus diet. / Full-length CYP3A cDNAs were isolated from both koala and Eastern grey kangaroo. These clones are the first CYP3A sequences to be cloned from any marsupial species. Given the significant role that CYP3A enzymes play in the metabolism of both endogenous and exogenous compounds, these clones provide an important step in elucidating the metabolic capacity of marsupials. / The CYP2C subfamily was also investigated in koala using two previously cloned CYP2C members, CYP2C47 and CYP2C48. Site-directed mutagenesis was used to engineer the CYP2C48 cDNA into a suitable form for expression. Stable cell lines were generated for both CYP2C and CYP3A full-length cDNAs using a mammalian expression system. These cell lines were used to determine catalytic activity of the marsupial CYPs. / Multiple protein alignments were used to identify substrate recognition sites and critical residues involved in the metabolism of a variety of substrates. Sequence analysis of the deduced amino acid sequence of the CYP3A clones has highlighted important species-specific features, for example a Thr residue at position 119 which has only been found in a limited number of species, including koala, and has been shown to influence steroid metabolism. / Modelling of all marsupial CYP2C and CYP3A full-length cDNAs and phylogenetic analysis of all known marsupial cDNA sequences was performed. These studies highlight the need for inclusion of marsupial information when assessing mammalian evolution. / Collectively, the work presented here provides valuable insights into the marsupial CYP2C and CYP3A subfamilies and highlights the significance of species differences in xenobiotic metabolism. / Thesis (PhDPharmacy)--University of South Australia, 2005.