Dissertations / Theses on the topic 'Cytochrome P-450 Genetic aspects'

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: With knowledge of the human genome increasing constantly we are continually faced with new and potentially groundbreaking methods for managing, treating and/or identifying diseases and predisposition to diseases and conditions at a genetic level. The human cytochrome P450 (CYP) super-family of genes code for enzymes that can participate in metabolism of drugs and foreign chemicals and in steroid synthesis and metabolism. Mutations in these genes may contribute to clinically relevant diseases. In this study, the effects of mutations within four CYP genes were evaluated in two South African disease groups - variegate porphyria and breast cancer. Variegate porphyria (VP) has an unusually high incidence in South Africa due to the R59W founder mutation in the protoporphyrinogen oxidase (PPOX) gene that causes a disruption in the haem biosynthetic pathway. VP presents with variable clinical symptoms and has a relatively low penetrance. It is expected that environmental factors and modifier genes play a role in the clinical expression of VP. CYP genes are implicated as candidate modifier genes for the expression of VP due to the function they have in metabolising many drugs contraindicated in porphyria patients, and the necessity of haem binding to the apoprotein to produce a functional CYP enzyme. This is the first study to investigate CYPs as possible modifier genes for VP clinical expression. Six CYP polymorphisms (CYPIAlml, CYPIAlm2, CYPIA2 - 734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4), associated with four CYP loci, were genotyped in a VP population and a suitable control population. The results observed are suggestive of CYPIAlml and CYPIBI playing a role as modifiers for the clinical expression of VP as they were significantly associated (PA and CYPIBI 8372 A>C). This represents the first investigation of the potential role of CYPs as breast cancer risk modifiers in the two South African populations. Significant differences were observed (PC polymorphism in the population of mixed ancestry. Vast differences in allele frequencies were also observed between the two groups of breast cancer populations. These results emphasize the importance of population-based risk assessment when genetic testing and counselling for complex disease susceptibility is offered. The results of this study provide the first evidence suggesting a role for CYPs in modifying the clinical expression of VP and in acting as risk factors for developing breast cancer in a South African population.
AFRIKAANSE OPSOMMING: Met die konstante toename van kennis oor die mensgenoom kom ons voortdurend te staan voor nuwe metodes vir die beheer, behandeling en/of identifikasie van siektes en vatbaarheid vir siektes op 'n genetiese vlak. Die mens sitochroom P450 geensuperfamilie kodeer vir ensieme betrokke in die metabolisme van medisyne en ander chemiese stowwe en steroïed-sintese en -metabolisme. Mutasies in hierdie gene kan 'n bydrae lewer tot kliniese relevante siektes. In hierdie studie is die effek van mutasies in vier sitochroom gene bestudeer in twee Suid-Afrikaanse siekte groepe, variegate porfirie en borskanker. Variegate porfirie (VP) het 'n besonderse hoë frekwensie in Suid-Afrika as gevolg van die R59W stigter-mutasie in die protoporfirinogeen oksidase (PPOX) geen. Hierdie mutasie lei tot 'n versteuring in die heem biosintese padweg. VP presenteer met variërende kliniese simptome en het 'n betreklike lae penetrasie. Daar word vermoed dat omgewingsfaktore en kandidaat modifiserende gene 'n rol speel in die kliniese beeld van VP. Sitochroom P450 gene is geïdentifiseer as kandidaat modifiserende gene as gevolg van hulle rol in die metabolisme van verbode medikasie vir porfirie pasiënte, asook die binding van heem aan die apoproteïen wat noodsaaklik is vir die produksie van funksionele sitochroom P450 ensiem. Hierdie is die eerste studie wat sitochroom P450 gene as moontlike modifiserende gene vir die kliniese uitdrukking van VP ondersoek. Ses sitochroom P450 polimorfismes (CYPIAlml, CYPIAlm2, CYPIA2 -734 C>A, CYPIBI 8372 A>C, CYP2D6*3, CYP2D6*4) is ondersoek in beide 'n VP populasie en 'n geskikte kontrole populasie. Die resultate suggereer 'n rol vir CYPIAlml en CYPIBI in die modifisering van die kliniese uitdrukking van VP aangesien hulle betekenisvolle assosiasie (PA, CYPIBI 8372 A>C). Hierdie studie verteenwordig die eerste ondersoek na die potensiële rol van sitochroom P450s as risiko-modifiserende faktore vir borskanker in die twee populasies. Betekenisvolle verskille (PC polimorfisme in die gemengde herkoms populasie. Beduidende verskille in alleel frekwensies is ook waargeneem tussen die twee borskanker populasies. Hierdie resultate beklemtoon die belangrikheid van populasie gebaseerde risiko-beraming wanneer genetiese toetse en voorligting vir komplekse siekte-vatbaarheid aangebied word. Die resultate van hierdie studie bied die eerste getuienis dat sitochroom P450s 'n rol kan speel in die modifisering van die kliniese beeld van VP en ook kan optree as as risiko faktore vir die ontwikkeling van borskanker in 'n Suid-Afrikaanse populasie.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Epidemiology, School of Public Health." Discipline: Epidemiology; Department/School: Public Health.

Thesis (Ph. D.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains xiv, 150 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 104-120).

The cytochrome P450 (CYP) superfamily of enzymes catalyses the oxidative metabolism of lipophilic xenobiotics such as drugs and environmental chemicals, and also plays an essential role in the biosynthesis and metabolism of endogenous compounds such as cholesterol and bile acids, vitamins, steroids, arachidonic acid and eicosanoids. Cytochrome P450 2J2 (CYP2J2) is a recently identified member of the human CYP protein family that is highly expressed in the heart, vasculature, liver and other tissues. CYP2J2 metabolises arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs), which have a number of potent biological activities including cytoprotective, vasodilatory and anti-inflammatory effects. Given its widespread tissue distribution and the biological actions of EETs, CYP2J2 is likely to play an important role in cellular physiology, and altered expression of CYP2J2 may have pathophysiological consequences. Indeed, recent literature studies have indicated that CYP2J2 protein levels are decreased in vascular endothelial cells exposed to hypoxia and reoxygenation, and that maintenance of CYP2J2 expression enhances cell survival. Thus, CYP2J2 expression may be impaired in diseases or conditions associated with decreased oxygen availability, such as ischaemic heart disease, stroke and atherosclerosis, and this may contribute to their pathogenic consequences. Despite its likely importance in human physiology and pathophysiology, very little is known about the regulation of CYP2J2 gene expression. The aim of this study was to investigate the molecular mechanisms that control expression of the CYP2J2 gene. In particular, this study was designed to identify factors that regulate the expression of the CYP2J2 gene in the liver-derived HepG2 cell line during normoxia and hypoxia. A 2.4 kb fragment of the 5???-flanking region of the CYP2J2 gene (corresponding to nucleotides -2341 to +98, relative to the translation start site) was isolated from a human genomic library. Automated searching of the upstream regulatory region of CYP2J2 identified several putative binding sites for the transcription factor activator protein-1 (AP-1). Because AP-1 activity is altered in hypoxia, the possibility that AP-1 may participate in the regulation of CYP2J2 expression in hypoxia was explored. Cell culture studies examined the relationship between the expression of CYP2J2, and the AP-1 genes c-fos and c-jun, in HepG2 cells cultured in normoxia and hypoxia. Down-regulation of CYP2J2 mRNA and protein in hypoxic HepG2 cells was associated with the pronounced up-regulation of c-Fos protein from an undetectable level in normoxic cells; c-Jun protein levels were readily detectable in normoxia, and were also increased in hypoxia. Transient transfection studies revealed distinct effects of Fos and Jun proteins on CYP2J2 promoter activity. While the CYP2J2 promoter was strongly activated by c-Jun, c-Fos was inactive, and also abolished gene transactivation elicited by c-Jun. These results suggest that the constitutively expressed c-Jun is important in the maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia stimulates the formation of c-Fos/c-Jun heterodimers, which do not support CYP2J2 transcription, leading to gene down-regulation. Experiments with CYP2J2 promoter deletion constructs revealed that the region between -152 to -50 bp relative to the translation start site was crucial for activation of CYP2J2 by c-Jun. Electrophoretic mobility shift assays (EMSAs) and transfection studies identified two distinct elements within this region that were involved in c-Jun-dependent transactivation: an AP-1-like element at -56 to -63 bp, and an atypical AP-1 element at -105 to -95 bp. c-Jun homodimers interacted specifically with both elements. Separate mutagenesis of either element significantly impaired c-Jun-dependent transactivation of CYP2J2, while mutagenesis of both elements eliminated c-Jun-responsiveness. EMSAs established that c-Jun, but not c-Fos, interacted with both elements in normoxic HepG2 cells. Furthermore, mutagenesis of either c-Jun-response element significantly decreased the basal transcriptional activity of the CYP2J2 promoter in HepG2 cells, while mutagenesis of both elements almost completely suppressed basal promoter activity. These findings indicate a pivotal role for c-Jun in the maintenance of CYP2J2 expression in normoxic cells. Transfection studies indicated that c-Fos suppresses c-Jun-dependent activation of CYP2J2 at both the -56/-63 bp and -105/-95 bp c-Jun-response elements. However, c-Fos-dependent inhibition appears to be mediated by distinct mechanisms at these two regulatory elements. While both elements interacted with c-Jun homodimers, only the -105/-95 bp element was able to interact with c-Fos/c-Jun heterodimers. Thus, the up-regulation of c-Fos in hypoxia, and the shift from c-Jun homodimers to c-Fos/c-Jun heterodimers, directly decreased c-Jun binding and transactivation at the -56/-63 bp element. In contrast, up-regulation of c-Fos in hypoxia altered the composition of proteins bound at the -105/-95 bp element from c-Jun to c-Fos/c-Jun. Inhibition of promoter activity occurs because c-Fos/c-Jun heterodimers can occupy, but not transactivate, the CYP2J2 promoter via the -105/-95 bp element. In summary, this thesis provides novel information on the molecular mechanisms that control the differential expression of the human CYP2J2 gene in normoxia and hypoxia. In particular, this study has established that the AP-1 proteins c-Jun and c-Fos play a crucial role in modulating the transcriptional activation of the CYP2J2 promoter in response to cellular stress. Binding of c-Jun to two distinct c-Jun-response elements within the CYP2J2 proximal promoter induces transcriptional activation of the CYP2J2 gene and is essential for maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia promotes the formation of c-Fos/c-Jun heterodimers, which inhibit transcriptional activation of the CYP2J2 promoter by c-Jun, thus contributing to decreased CYP2J2 expression in hypoxia. Impaired expression of CYP2J2 may contribute to cellular injury in diseases such as atherosclerosis and stroke, and a greater understanding of the mechanisms responsible for mediating altered CYP2J2 expression may eventually lead to therapeutic strategies that manipulate the expression of this important human gene.

Thesis (Ph. D.)--West Virginia University, 2003.
Title from document title page. Document formatted into pages; contains xiv, 235 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 201-235).

Thesis (MSc)--Stellenbosch University, 2005.
ENGLISH ABSTRACT: This study describes: I. The cloning of the CVP 19 gene and construction of the intracellular expression vector pPIC3.5K-CYP19. II. The transformation of the yeast, Pichia pastoris with the constructed vector. III. The expression ofP450arom in Pichia pastoris. IV. The determination of enzyme activity and isolation of the protein from the Pichia pastoris cells. V. The expression of P450c 17 in Pichia pastoris. VI. The determination of kinetic constants for the conversion of progesterone to 170H-progesterone and 160H-progesterone by P450c17.
AFRIKAANSE OPSOMMING: Hierdie studie beskryf: I. Die klonering van die CVP 19 geen en die konstruksie van die intrasellulêre uitdrukkingsplasmied, pPIC3.5K-CYPI9. II. Die transformasie van die gis, Pichia pastoris, met die gekonstrueerde plasmied. III. Die uitdrukking van aromatase in Pichia pastoris. IV. Die bepaling van ensiemaktiwiteit en die isolering van die proteïen vanuit Pichia pastoris. V. Die uitdrukking van P450c17 in Pichia pastoris. VI. Die bepaling van kinetiese konstantes vir die omsetting van progesteroon na 170H-progesteroon en 160H-progesteroon deur P450c17.

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Fundação de Amparo a Pesquisa do Estado de São Paulo
Introduction: The xenobiotics are exogenous substances to the organism, as N-nitrosamines, heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs), can which result in DNA adducts formation. Polymorphisms in genes involved in the metabolism of xenobiotics could contribute to this process and modulate the development of cancer. Objectives: To investigate the CYP1A1*2A (rs4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs2031920), CYP1E1*6 (rs6413432), Tyr113His EPHX1 (rs1051740) and His139Arg EPHX1 (rs2234922) polymorphisms related to the metabolism of xenobiotics, the risk of sporadic colorectal (SCRC) cancer, the interaction of these polymorphisms with lifestyle (smoking and drinking) and clinical and histopathological parameters and to evaluate the association of SCRC with socio-demographic factors. Methods: A case-control study was conducted in 641 subjects in the Brazilian population (241 patients with colorectal cancer and 400 controls (individuals without a history of cancer). Real-Time PCR and PCR-RFLP was performed for genotyping. Statistical analysis was performed using the chi-square tet and multiple logistic regression binary. Results: The results showed statistically significant differences between the case and control groups for age greater than 50 years (OR=8.21, 95%CI=5.49-12.28, p<0.01) and male gender (OR=0.50, 95%CI=0.32-0.87, p<0.01) The analysis of polymorphisms revealed an association between the alleles polymorphic CYP2E1*5B (OR=2.84, 95%CI=1.78-4.52, p<0.01, additive model) and CYP2E1*6 (OR=2.78, 95%CI=1.91-4.06, p<0.01, additive model) and the SCRC. Tumor size, lymph node involvement and disease primary site were not associated with polymorphisms. Conclusion: The CYP2E1*5B and CYP2E1*6 polymorphisms are involved in the risk of SCRC and individuals with age ≥ 50 years are more susceptible to this tumor type, of males are less susceptible.
Introducão: Os xenobióticos são substâncias exógenas ao organismo, tais como as N-nitrosaminas, aminas heterocíclicas (HAs) e hidrocarbonetos policíclicos aromáticos (HPAs), que podem formar adutos de DNA. Polimorfismos em genes envolvidos no metabolismo dos xenobióticos podem contribuir com este processo e, consequentemente, modular o desenvolvimento de câncer. Objetivos: Investigar os polimorfismos CYP1A1*2A (rs 4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs 2031920), CYP1E1*6 (rs 6413432), EPHX1 Tyr113His (rs1051740) e EPHX1 His139Arg (rs2234922), relacionados com o metabolismo dos xenobióticos, no risco de câncer de colorretal esporádico (CCRE), a interação desses polimorfismos com os hábitos de vida (tabagismo e etilismo) e parâmetros clínico-histopatológicos e avaliar a associação do CCRE com os fatores sócio-demográficos. Os Métodos: Um estudo caso-controle foi realizado em 641 indivíduos da população brasileira (241 pacientes com câncer de coloretal e 400 controles (indivíduos sem histórico de câncer). As técnicas de PCR em Tempo Real e PCR-RFLP foram realizadas para a genotipagem dos polimorfismos. A análise estatística utilizou os testes de Qui-Quadrado e Regressão Logística Múltipla Binária. Resultados: Os resultados mostraram diferenças estatisticamente significantes entre os grupos caso e controle para idade superior a 50 anos (OR=8,21; IC95%=5,49-12,28, p<0,01) e gênero masculino (OR=0,50; IC95%=0,32-0,87, p<0,01). A análise dos polimorfismos revelou associação entre os alelos polimórficos CYP2E1*5B (OR=2,84; IC95%=1,78-4,52; p<0,01, modelo aditivo) e CYP2E1*6 (OR=2,78; IC95%=1,91-4,06, p<0,01, modelo aditivo) e o CCRE. O tamanho do tumor, envolvimento de linfonodos e sítio primário da doença não foram associados com os polimorfismos. Conclusão: Os polimorfismos CYP2E1*5B e CYP2E1*6 estão envolvidos no risco de CCRE e indivíduos com idade superior ou igual a 50 anos são mais suscetíveis a este tipo tumoral, enquanto aqueles do gênero masculino são menos suscetíveis.

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Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão
INTRODUCTION: In Brazil, it is reported that a great part of the patients who arrive for the primary care service have as main complaint: sadness (depression) and / or anxiety, also with more complex disorders such as problems related to abuse of alcohol and serious and persistent mental disorders, such as schizophrenia and affective psychoses (bipolar mood disorder). In the pharmacogenetic studies of antidepressants and antipsychotics, cytochrome P450 (CYPs) enzymes have been shown to be one of the most significant targets in the interindividual changes in drug response kinetic parameters. OBJECTIVE: To characterize the frequency of CYP2D6 polymorphisms (*4, *6 and *17) in users of psychotropic therapy, especially tricyclic antidepressants and antipsychotics. METHODS: Cross - sectional, descriptive study carried out in the city of São Luis – MA. RESULTS: From a total of 105 charts, a sample of 43 patients was collected. The mean age was 40.98 (± 11.04) years, of which 24 (55.81%) were male and 19 (44, 19%) of females. Regarding the pharmacotherapy, all participants (100%) were on daily use of the antipsychotic haloperidol, seventeen (39.53%) patients used risperidone and one (2.32%) patient used amitriptyline (tricyclic antidepressant), all three drugs are substrates of the enzyme CYP2D6. For the polymorphism of CYP2D6, we had a prevalence of 11 (25.48%) for allele 4, and all the patients presented with this polymorphism were heterozygous, that means they had one polymorphic allele and one normal allele. For allele 17, we had a prevalence of 4 (9.3%), also all patients being heterozygous for this allele. Allele 6 did not appear in any patient in our study. We also had two patients who presented the polymorphisms for alleles * 4 and * 17 at the same time. CONCLUSION: This study was able to characterize the frequency of CYP2D6 (* 4, * 6 and 17 *) polymorphisms in users of psychotropic therapy, including tricyclic antidepressants and antipsychotics, in addition to contributing to a higher dose / response do medication, ensuring greater safety and efficacy in the use of medicines, improving the quality of life of patients.
INTRODUÇÃO: No Brasil, tem-se o relato de que grande parte dos pacientes que chegam para o atendimento na atenção primária apresentam, como principal queixa, tristeza (depressão) e/ou ansiedade, aparecendo também transtornos mais complexos como os problemas relacionados ao abuso de álcool, assim como os transtornos mentais graves e persistentes, como a esquizofrenia e as psicoses afetivas (transtorno bipolar do humor). Nos estudos farmacogenéticos de antidepressivos e antipsicóticos, as enzimas do citrocromo P450 (CYPs) tem se mostrado um dos alvos mais significativos nas alterações interindividuais dos parâmetros cinéticos de resposta às drogas. OBJETIVO: caracterizar a frequência de polimorfismos do CYP2D6 (*4, *6 e 17*) em usuários da terapêutica com psicotrópicos, principalmente antidepressivos tricíclicos e antipsicóticos. MÉTODOS: Estudo transversal, descritivo realizado no Município de São Luís (MA). RESULTADO: De um total de 105 prontuários, coletou-se amostra de 43 pacientes, a média de idade foi de 40,98 (±11,04) anos, sendo 24 (55,81%) do sexo masculino e 19 (44,19%) do sexo feminino. Em relação à farmacoterapia, todos os participantes (100%) estavam em consumo diário do antipsicótico haloperidol, dezessete (39,53%) pacientes faziam uso de risperidona e um (2,32%) paciente utilizava amitriptilina (antidepressivo tricíclico), todos os três medicamentos são substratos da enzima CYP2D6. Para o polimorfismo da CYP2D6, tivemos para o alelo 4 uma prevalência de 11 (25,48%), sendo que todos os pacientes que apresentaram esse polimorfismo são heterozigotos, ou seja, apresentaram um alelo polimórfico e outro normal. Para o alelo 17, tivemos uma prevalência de 4 (9,3%), sendo também todos os pacientes heterozigotos para esse alelo. O alelo 6 não apareceu em nenhum paciente do nosso estudo. Tivemos também dois pacientes que apresentaram os polimorfismos para os alelos *4 e *17 ao mesmo tempo. CONCLUSÃO: Este estudo pôde caracterizar a frequência de polimorfismos do CYP2D6 (*4, *6 e 17*) em usuários da terapêutica com psicotrópicos, incluindo antidepressivos tricíclicos e antipsicóticos, além de contribuir para uma maior adequação da relação dose/resposta ao medicamento, garantindo maior segurança e eficácia quanto ao uso de medicamentos, melhorando a qualidade de vida dos pacientes.

Neben Zigarettenrauchen und chronischem Alkoholkonsum scheinen genetische Faktoren bei der Entstehung und im Verlauf der Kopf-Hals-Karzinom Erkrankung bedeutsam zu sein. Genvariationen in den Enzymen, die zigarettenrauch-assoziierte Karzinogene (Glutathion-S-Transferase, GST, Cytochrom P450, CYP) metabolisieren, sowie Genvariationen in immunregulierenden Zytokinen (Tumor-Nekrose-Faktor) könnten Grund dieser Beeinflussung sein. Ziel dieser Studie war es, genetische Wirtsfaktoren zu identifizieren, die das Erkrankungsrisiko und das Tumorverhalten bzw. den Krankheitsverlauf von Plattenepithelkarzinomen im oberen Aerodigestivtrakt beeinflussen. Wir beschreiben den Zusammenhang zwischen Genotypen an GSTM1, M3, T1, P1, CYP2D6, 1A1, 2E1 und TNF Mikrosatelliten-Genorten und der Krankheitsentstehung sowie Tumorcharakteristika wie der initialen Tumorgröße, der Halslymphknotenmetastasierung, dem histologischen Differenzierungsgrad des Tumorgewebes und dem rezidivfreien Überleben bei 465 Patienten. Die Genotypen wurden aus der Leukozyten-DNA mittels PCR in Kombination mit Restriktionsenzymverdauungen und Elektrophoresetechniken identifiziert. Sowohl bei den Genvariationen in den entgiftenden Enzymen als auch bei den TNF Mikrosatelliten-Polymorphismen konnten Risikogenotypen festgestellt werden. Die Beeinflussung des Krankheitsverlaufs war teilweise deutlicher ausgeprägt, als die Beeinflussung der Krankheitsentstehung. Patienten mit Mehrfachkarzinomen wiesen die stärkste Anhäufung von Risikogenotypen auf. In dieser Studie konnte erstmals gezeigt werden, daß individuelle Genotypkonstellationen die Entstehung und das Verhalten von Plattenepithelkarzinomen im oberen Aerodigestivtrakt beeinflussen.
Cigarette smoking and alcohol consumption are the main risk factors for the development of head and neck cancer. Additionally, genetic factors seem to be influential. Gene variations in detoxifying enzymes such as glutathione S-transferase (GST) and cytochrome P450 (CYP) and variations in immune regulating proteins like tumor necrosis factor (TNF) are candidate genes. Accordingly, we have examined, in 465 patients with squamous cell carcinoma (SCC) of the head and neck, associations between GSTM1, GSTT1, GSTM3, GSTP1, CYP2D6, CYP1A1, CYP2E1 genotypes and TNF microsatellite polymorphisms and cancer susceptibility, outcome parameters like tumor extension, histological grade, and presence of lymph nodes, and tumor recurrence. Genotypes were determined by PCR; logistic regression and a step-wise model were used to investigate the influence of the individual genes. Individual genotypes in the detoxifying enzymes and TNF were associated with altered cancer risk. The influence on tumor behavior was partially stronger. Patients suffering multiple head and neck cancer showed the highest cumulation of risk mediating genotypes. The data demonstrate site-dependent associations between GST, CYP and TNF genotypes and tumor susceptibility, tumor extension, differentiation, and lymph node involvement, and tumor recurrence in SCC of the head and neck.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009.
Sutherlandia frutescens (S. frutescens), sub-species microphylla, is a member of the Fabacea family and is used as a herbal remedy for the treatment of several ailments which include influenza, diabetes, cancer, tuberculosis, chronic fatigue syndrome, rheumatoid arthritis, anxiety, clinical depression, and more recently, those living with human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS) (1-4). Many of the symptoms of these ailments are associated with a perturbation of the stress response which may be associated with disorders of the endocrine system. Of all the traditional plants in South Africa, S. frutescens is regarded the most profound in that it is a multipurpose traditional remedy. The plant has enjoyed a long history of use and reports indicating its efficacy as a safe treatment for various health conditions have added to the popularity of this medicinal plant. The extracts of S. frutescens have been shown to exhibit anti-proliferative effects on cancer cells, antioxidant activity, and to possess anti-diabetic and anti-inflammatory potential (5, 6), providing scientific evidence for its therapeutic use in the treatment of cancer and diabetes. However, this study focuses on the potential use of this medicinal plant in the treatment of stress and stress related diseases. Chronic stress is characterized by elevated plasma levels of glucocorticoids. These steroid hormones are synthesized in the adrenal cortex in a series of reactions involving the steroidogenic enzymes. The major aim of this thesis was the determination of the influence of S. frutescens extracts on the adrenal cytochrome P450 enzymes. Aqueous, methanol and chloroform S. frutescens extracts were prepared and the interaction with the cytochrome P450 enzymes was investigated. The effect of these extracts towards progesterone (PROG), deoxycortisol and deoxycorticosterone (DOC) binding to the cytochrome P450 enzymes as well as their influence on the metabolism of these steroid substrates was investigated. A similar study (7) showed that compounds from the S. frutescens extracts could interact with these enzymes and possibly affect adrenal steroidogenesis. This study further investigates the bioactive properties of the plant material in terms of the influence of S. frutescens on the cytochrome P450 enzymes and the effect of the manufacturing process on the bioactivity of the plant.

Introdução: A grande variabilidade individual em resposta a fármacos antitabagismo sugere que tratamentos específicos podem ser mais efetivos em determinados subgrupos de fumantes. No contexto de medicina personalizada, o principal objetivo do presente estudo foi avaliar se polimorfismos nos genes CHRNA4, CHRNB2, CYP2B6 e ANKK1 estão associados com a resposta às terapias de cessação tabágica em pacientes provenientes de um programa de assistência ao fumante. Métodos: Estudo de coorte com 483 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona em monoterapia ou coadministrada com terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. Os polimorfismos CHRNA4 (rs1044396 e rs2236196), CHRNB2 (rs2072660 e rs2072661) e ANKK1 (rs1800497) foram genotipados pela análise da curva de melting e os polimorfismos CYP2B6 *9 (rs3745274), *4 (rs2279343), *5 (rs3211371) foram genotipados por restrição enzimática. Resultados: Os pacientes com o genótipo CC para o polimorfismo CHRNA4 (rs10443196) obtiveram menor taxa de sucesso no tratamento com vareniclina (29,5%) em comparação com os portadores dos genótipos CT ou TT (50,9%) (P=0,007; n=167). Os genótipos CT ou TT foram associados com maior odds ratio para o sucesso (OR=1,67; IC 95%=1,10-2,53; P=0,02), em um modelo multivariado. Os pacientes com o genótipo AA para o polimorfismo CYP2B6 (rs2279343) obtiveram maior taxa de sucesso no tratamento com bupropiona (48,0%) em comparação com portadores dos genótipos AG ou GG (35,5%) (P=0,05; n=237). O genótipo AA foi associado com maior odds ratio para o sucesso no tratamento (OR=1,92; IC 95%=1,08-3,42; P=0,03), em um modelo multivariado. Não foram observadas diferenças significativas nos escores FTND e Issa com relação aos polimorfismos estudados. Conclusão: Os polimorfismos CHRNA4 (rs1044396) e CYP2B6 (rs2279343) estão associados com a cessação tabágica em indivíduos tratados com vareniclina e bupropiona, respectivamente. Sugere-se que estes polimorfismos influenciam a resposta farmacológica e podem ser importantes para o desenho de uma farmacoterapia individualizada
Background: The large individual variability in response to drugs for smoking cessation suggests that specific treatments can be more effective in particular subgroups of smokers. In the context of personalized medicine, the main aim of the present study was to evaluate whether the CHRNA4, CHRNB2, CYP2B6 and ANKK1 polymorphisms are associated with response to smoking cessation therapies in patients from a smoker assistance program. Methods: This cohort study enrolled 483 smoking patients patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion in monoterapy or plus nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores were analyzed for nicotine dependence. The CHRNA4 (rs1044396 and rs2236196), CHRNB2 (rs2072660 and rs2072661) and ANKK1 rs1800497 polymorphisms were genotyped by high resolution melting analysis and the CYP2B6 *9 (rs3745274), *4 (rs2279343) and *5 (rs3211371) were genotyped by restriction fragment lenght polymorphisms. Results: Patients with CHRNA4 rs1044396 CC genotype had lower success rate in treatment with varenicline (29.5%) compared with carriers of CT or TT genotypes (50.9%) (P=0.007, n=167). The CT or TT genotypes were associated with higher odds ratio for success (OR=1.67, 95%CI=1.10-2.53, P=0.02), in a multivariate model. Patients with CYP2B6 rs2279343 AA genotype had higher success rate in treatment with bupropion (48.0%) compared with carriers of AG or GG genotypes (35.5%) (P=0.05, n=237). The AA genotype was associated with higher odds ratio for success (OR=1.92, 95%CI=1.08-3.42, P=0.03), in a multivariate model. We did not observe significant differences in the FTND and Issa scores according to the studied polymorphisms. Conclusion: The CHRNA4 rs1044396 and CYP2B6 rs2279343 are associated with smoking cessation in individuals on varenicline and bupropion terapies, respectively. We suggest that these polymorphisms influence the pharmacological response of these drugs and it might be important in the design of individualized pharmacotherapy

Intrdodução: As terapias com anticonvulsivantes de anel aromático (ACA) são freqüentemente associadas a reações adversas. No entanto, reações de hipersensibilidade (RH) não-imediatas (tardias) a estes fármacos são raras, imprevisíveis e geralmente relacionadas à alta morbidade e mortalidade. Foi demonstrado que estas RH aos ACA estão fortemente associadas ao Antígenio de Leucócitos Humanos (HLA)-B*1502 em pacientes chineses e ao HLA-A*3101 em caucasianos. Polimorfismos de genes do metabolismo do Citocromo P450 (CYP)2C9 foram mais associados a estas reações em pacientes orientais. Objetivo: Nosso objetivo é analisar a associação das reações de hipersensibilidade a anticonvulsivantes de anel aromático com os polimorfismos descritos e de interesse, bem como realizar a tipificação de HLA em uma população de São Paulo, Brasil. Métodos: Estudo tipo caso-controle com genotipagem dos polimorfismos de interesse por reação em cadeia da polimerase (PCR) em tempo real e tificação de HLA A, B, C, DRB, DQA, DQB por PCR seguido de deteção utilizando método LuminexR. A avaliação fenotípica se baseou em sistemas de escores padronizados, utilizando um questionário adaptado da ENDA (Rede Européia de Alergia a Medicamentos), em registros médicos e no acompanhamento clínico. O teste de contato com o medicamento suspeito foi realizado de acordo com as recomendações da ENDA, nos pacientes que apresentaram reação. Resultados: Foram estudados 506 pacientes, 65% do gênero feminino e a idade média foi de 43,6 anos. Oitenta por cento era de etnia mista. Polimorfismos de HLA-A*3101, HLA-B*1502, CYP2C9, CYP2C19 e CYP3A5 foram analisados de 55 indivíduos com reações de hipersensibilidade (RH) a antiepilépticos, de 85 tolerantes e de 366 controles sadios. Dos 55 casos foram validados como RH, 32 apresentaram Reação a Drogas com eosinofilia e sintomas sistêmicos (DRESS), 12 Síndrome de Stevens-Johnson (SSJ) e 11 exantema maculo-papular. De todos os 46 testes de contato com medicamento, 29 (63%) foram positivos, tanto em SSJ como em DRESS. Houve associação significativa entre polimorfismo de HLA-A*1502 e casos. Nenhum de nossos grupos de estudo apresentou associação positiva com polimorfismos de HLAA* 3101. Verificamos uma forte associação entre a atividade normal do CYP3A5 e indivíduos tolerantes quando comparado com casos (p = 0,0002, OR = 4,8). A tipificação de HLA demonstrou associação significante de HLA-A*31, HLA-A*74, HLA-B*35 e HLA-B*53 com reações graves aos ACA e de HLA-B*44 e HLA-C*03 com indivíduos tolerantes. Conclusão: Estes resultados sugere fortemente a existência de fatores genéticos de risco e/ou de proteção a RH a ACA em indivíduos brasileiros, mas não devem ser considerados de forma isolada. Assim, a relevância deste estudo extrapola o objetivo de estudo caso-controle e sugere um modelo como forma de prevenção primária às RH aos ACA.
Background: Antiepileptics with aromatic ring (AAR) therapies are frequently associated with adverse reactions. Nevertheless non-immediate (late) hypersensitivity reactions (HR) to these drugs are rare, unpredictable and usually related with high morbidity and mortality. A strong pharmacogenetic association has been reported in Chinese patients with these HR and Human Leukocyte Antigen (HLA)-B*1502 and with HLA- A*3101 in caucasians. Polymorphism of genes of P450 Cytocrome (CYP)2C9 has been related to these reactions in patients of oriental origin. Objective: Our aim is to analyze the association between hypersensitivity reactions due to AAR and the described polymorphisms, as well as perform the typification of HLA in a population of São Paulo, Brazil. Methods: Case-control study genotyping the polymorphisms of interest by polymerase chain reaction (PCR) real time and typifying HLA A, B, C, DRB, DQA, DQB by PCR followed by LuminexR .The phenotype evaluation was based on standardized scoring systems using an adapted ENDA (European Network of Drug Allergy) questionnaire, medical records and on the clinical follow-up in our Allergy Clinic. The patch test with the culprit drug was performed in patients who experienced HR according to the ENDA recommendations. Results: We studied 506 subjects, 65% female and mean age was 43,6 years. Eighty percent had mixed ethnicity. Polymorphisms of HLA-B*1502, HLA- A*3101, CYP2C9, CYP2C19 e CYP3A5 were studied in 55 subjects with antiepileptics HR, 85 tolerants, and 366 control subjects. Of 55 cases were validated as AHR, 32 presented Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), 12 Stevens-Johnson Syndrome (SJS) and 11 maculopapular exanthema. Of all 46 drug patch tests, 29 (63%) were positive, in both SJS and DRESS. A significant association between polymorphism of HLA-A*1502 and cases was found. None of our study groups presented positive association with HLA-A*3101 polymorphisms. We found a strong association between the normal activity of CYP3A5 and tolerants subjects when compared to HR (p=0.0002, OR=4.8). The HLA typification showed a significant association between HLA-A*31, HLA-A*74, HLAB* 35 e HLA-B*53 and severe AAR reactions and HLA-B*44 and HLA-C*03 in tolerants subjects. Conclusion: These results strongly suggests the existence of genetic risk and/or protective factors to the development of HR to AAR AAR in Brazilian subjects, but it should not be considered in a isolated manner. So, the relevance of this study extrapolates the aim of a case-control study and suggests a system of primary prevention to HR due to AAR

"July 2001"
Includes bibliographical references (leaves 144-183)
xv, 193 leaves : ill., plates (col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

Lui, Ming Yin.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 117-129).
Abstracts in English and Chinese.
Acknowledgement --- p.I
Abstract --- p.III
Abstract in Chinese --- p.V
List of Abbreviations --- p.VII
List of Figures --- p.X
List of Tables --- p.XII
Table of Contents --- p.XIII
Chapter Chapter 1: --- Introduction
Chapter 1.1 --- Overview on type 2 diabetes --- p.1
Chapter 1.1.1 --- Definition of diabetes --- p.1
Chapter 1.1.2 --- Diagnostic criteria --- p.2
Chapter 1.1.3 --- Prevalence and societal impact --- p.2
Chapter 1.1.4 --- Risks factors for type 2 diabetes --- p.4
Chapter 1.1.4.1 --- Metabolic syndrome --- p.4
Chapter 1.1.4.2 --- Genetics of type 2 diabetes --- p.6
Chapter 1.1.4.3 --- "Environmental risk factors, lifestyle and energy imbalance" --- p.8
Chapter 1.1.5 --- Pathophysiology of type 2 diabetes --- p.9
Chapter 1.1.5.1 --- Insulin secretion and signaling --- p.9
Chapter 1.1.5.1.1 --- Insulin Secretion --- p.9
Chapter 1.1.5.1.2 --- Insulin signaling --- p.11
Chapter 1.1.5.2 --- Natural history of type 2 diabetes --- p.12
Chapter 1.1.5.3 --- Insulin resistance --- p.13
Chapter 1.1.5.4 --- Impairment in insulin secretion --- p.15
Chapter 1.1.5.5 --- Endocannabinoid system: A new target for energy balance and metabolism --- p.16
Chapter 1.1.5.6 --- Effects of diabetes mellitus and its complications --- p.16
Chapter 1.2 --- Biology of prostacyclin synthase (PTGIS) --- p.18
Chapter 1.2.1 --- Molecular information of PTGIS --- p.18
Chapter 1.2.2 --- Transcriptional control of PTGIS --- p.19
Chapter 1.2.3 --- Protein structure of PGIS --- p.21
Chapter 1.2.4 --- Sub-cellular localization and tissue distribution --- p.22
Chapter 1.2.5 --- Function of PGIS --- p.25
Chapter 1.2.5.1 --- Function of PGI2 in blood vessels --- p.26
Chapter 1.2.5.2 --- Role of PGh in embryo development --- p.26
Chapter 1.2.5.3 --- Role of PGI2 in apoptosis --- p.27
Chapter 1.2.5.4 --- Targeted knock-out mice phenotype --- p.27
Chapter 1.2.6 --- Relationship between PTGIS and diseases --- p.28
Chapter 1.2.6.1 --- Genetic association --- p.28
Chapter 1.2.6.2 --- Inactivation and tyrosine nitration of PGIS by peroxynitrite --- p.29
Chapter 1.3 --- Biology of peroxisome proliferator-activated receptor delta (PPARD) --- p.30
Chapter 1.3.1 --- Molecular information of PPARD --- p.30
Chapter 1.3.2 --- Transcriptional control of PPARD --- p.31
Chapter 1.3.3 --- Translational control and protein structure --- p.32
Chapter 1.3.4 --- Sub-cellular localization and tissue expression --- p.35
Chapter 1.3.5 --- Function of PPARδ --- p.37
Chapter 1.3.5.1 --- Mechanisms of action --- p.37
Chapter 1.3.5.2 --- Ligands for PPARδ --- p.38
Chapter 1.3.5.3 --- PPARδ in lipoprotein metabolism --- p.39
Chapter 1.3.5.4 --- PPARδ action in adipose tissue --- p.39
Chapter 1.3.5.5 --- PPARδ action in skeletal and cardiac muscle --- p.40
Chapter 1.3.5.6 --- PPARδ action in liver --- p.42
Chapter 1.3.5.7 --- PPARδ and endocannabinoid system --- p.42
Chapter 1.3.5.8 --- PPARδ action in inflammation --- p.43
Chapter 1.3.5.9 --- Targeted knock-out mice phenotype --- p.44
Chapter 1.3.5.10 --- Disease association --- p.44
Chapter 1.4 --- Functional relationship of PGIS and PPARδ: possible role in energy metabolism --- p.46
Chapter 1.5 --- Methods for studying genetics of type 2 diabetes and linkage analysis results --- p.47
Chapter 1.5.1 --- Genome-wide scan --- p.47
Chapter 1.5.2 --- Candidate gene approach --- p.48
Chapter 1.6 --- Hypothesis and objectives --- p.49
Chapter 1.7 --- Long-term significance --- p.49
Chapter Chapter 2: --- Association Study of Prostacyclin Synthase and Peroxisome Proliferator-Activated Receptor Delta Gene Polymorphisms with Type2 Diabetes and Related Metabolic Traits
Chapter 2.1 --- Introduction and research design --- p.50
Chapter 2.2 --- Study population --- p.52
Chapter 2.2.1 --- Ethics approval --- p.52
Chapter 2.2.2 --- Subjects --- p.52
Chapter 2.2.3 --- Clinical assessments --- p.52
Chapter 2.3 --- Materials and methods --- p.55
Chapter 2.3.1 --- DNA samples --- p.55
Chapter 2.3.2 --- Marker selection --- p.55
Chapter 2.3.3 --- Genotyping --- p.57
Chapter 2.3.4 --- Statistical analysis --- p.59
Chapter 2.4 --- Results and Discussion --- p.60
Chapter 2.4.1 --- Clinical characteristics of the study population --- p.60
Chapter 2.4.2 --- Genotyping and LD analysis --- p.60
Chapter 2.4.3 --- Association with type 2 diabetes and related metabolic traits --- p.61
Chapter 2.4.3.1 --- Single SNP association with type 2 diabetes --- p.61
Chapter 2.4.3.2 --- Single SNP association with metabolic traits --- p.64
Chapter 2.4.3.3 --- Gene-gene interaction on type 2 diabetes --- p.74
Chapter 2.4.3.4 --- Gene-gene interaction on metabolic traits --- p.74
Chapter 2.5 --- Limitation and improvement --- p.79
Chapter 2.6 --- Conclusions --- p.79
Chapter Chapter 3: --- Functional Studies of Prostacyclin Synthase rs508757-A/G Intronic Polymorphism
Chapter 3.1 --- Introduction and research design --- p.80
Chapter 3.2 --- Materials and methods --- p.81
Chapter 3.2.1 --- Bioinformatics --- p.81
Chapter 3.2.1.1 --- Cross-species alignment --- p.81
Chapter 3.2.1.2 --- BLAST search and open reading frame prediction --- p.81
Chapter 3.2.1.3 --- Transcription factor binding sites prediction --- p.82
Chapter 3.2.2 --- PCR amplification from cDNA --- p.82
Chapter 3.2.3 --- Culture of mammalian cell --- p.83
Chapter 3.2.3.1 --- Cell line --- p.83
Chapter 3.2.3.2 --- Medium and supplement --- p.83
Chapter 3.2.3.3 --- Cell culture wares --- p.83
Chapter 3.2.3.4 --- Cell culture conditions --- p.84
Chapter 3.2.4 --- Construction of reporter vectors with rs508757 flanking sequence --- p.84
Chapter 3.2.4.1 --- Cloning and vector preparation --- p.84
Chapter 3.2.4.2 --- Site-directed mutagenesis --- p.84
Chapter 3.2.5 --- Dual-luciferase reporter assay --- p.85
Chapter 3.2.5.1 --- Transfection of VSMC --- p.85
Chapter 3.2.5.2 --- Cell lysis and luminescence measurement --- p.86
Chapter 3.2.6 --- Circular Dichroism --- p.87
Chapter 3.2.6.1 --- Introduction to DNA quardruplex structure and circular dichroism --- p.87
Chapter 3.2.6.1.1 --- DNA quardruplex --- p.87
Chapter 3.2.6.1.2 --- Circular dichroism --- p.88
Chapter 3.2.6.2 --- Circular dichroism measurement --- p.89
Chapter 3.2.6.2.1 --- DNA samples --- p.89
Chapter 3.2.6.2.2 --- CD spectroscopy --- p.89
Chapter 3.2.7 --- Statistical analysis --- p.90
Chapter 3.3 --- Results and Discussion --- p.91
Chapter 3.3.1 --- Cross-species alignment --- p.91
Chapter 3.3.2 --- BLAST search and ORF prediction --- p.92
Chapter 3.3.3 --- PCR results on testing the presence of a new transcript --- p.93
Chapter 3.3.4 --- Effect of rs508757 flanking sequence on SV40 promoter activity --- p.94
Chapter 3.3.5 --- Circular dichroism experiment on rs508757 flanking sequence --- p.96
Chapter 3.3.6 --- DNA slipping model --- p.98
Chapter 3.3.7 --- Transcription factor binding site prediction --- p.99
Chapter 3.4 --- Limitation and improvement --- p.107
Chapter 3.5 --- Conclusions --- p.107
Chapter Chapter 4: --- "General Discussion, Conclusion and Future Perspectives"
Chapter 4.1 --- General discussion --- p.108
Chapter 4.2 --- Future perspectives --- p.115
Chapter 4.2.1 --- "Association on type 2 diabetes and molecular interaction between transcription factors, PTGIS and PPARD" --- p.115
Chapter 4.2.2 --- Association with diabetic nephropathy --- p.115
Chapter 4.2.3 --- Study tissue or cell type specific actions of PGIS and PPARδ --- p.116
Chapter 4.3 --- Conclusions to my project --- p.116
Chapter Chapter 5: --- Bibliography --- p.117
Appendix --- p.130

Indiana University-Purdue University Indianapolis (IUPUI)
Cytochrome P450 2D6 (CYP2D6) is an important drug metabolizing enzyme that is involved in the metabolism of 20-25% of commonly prescribed drugs. There is interindividual variability in CYP2D6 enzyme activity and this leads to compromised metabolism of many drugs. Genetic and environmental factors explain only a part of the interindividual variability; the other factors that contribute to this variability are largely unknown. Hence, it becomes important to study CYP2D6 to understand the endogenous and exogenous factors that control its activity. The specific objective of this research was to determine the contribution of genetic and epigenetic factors in the regulation of CYP2D6 expression and activity. The specific aims were: (1) to identify the common CYP2D6 polymorphisms in Vietnamese and Filipino women with breast cancer and evaluate its association with plasma concentrations of endoxifen (an active metabolite of the breast cancer therapeutic drug, tamoxifen); (2) to identify the CYP2D6 copy number variations (CNVs) in these women and evaluate their association with endoxifen concentration; and (3) to identify microRNAs (miRNAs) that regulate the expression of CYP2D6 directly or indirectly. The results of this study indicated that: (1) in Vietnamese and Filipino women, the reduced function allele CYP2D6*10 was frequent (~55%) and it was significantly associated with reduced endoxifen concentration; (2) in these women, only 39% carried two copies of the CYP2D6 gene, the rest had a genomic imbalance for CYP2D6, primarily involving the CYP2D6(*36)n-*10 allele. However, carrying multiple copies of CYP2D6*36 allele did not significantly affect CYP2D6 activity, suggesting that multiple copies of a gene does not always translate to additive effects; and (3) microRNAs were identified to target HNF4A, a transcriptional factor that regulates CYP2D6 expression. These miRNAs are likely to play an important role in the indirect regulation of CYP2D6. Taken together, these results emphasize on the role of polymorphisms, CNVs and possibly miRNAs in the regulation of CYP2D6. These clinically important biomarkers will help to improve the efficacy and reduce the side effects of many CYP2D6 substrate drugs and thus contribute to personalization of drug therapy.