Introdução: a ação antiestrogênica do tamoxifeno (TAM) é dependente da bioativação à endoxifeno (EDF) e 4-hidroxitamoxifeno (HTF). É bastante provável que sua eficácia terapêutica esteja relacionada ao alcance de um limiar nos níveis de EDF (>5,9 ng mL-1). Entretanto, as concentrações plasmáticas de EDF são altamente variáveis, em parte devido a polimorfismos no gene da CYP2D6 e ao uso de inibidores da enzima. A CYP3A4 também contribui para a formação do EDF e pode ser influenciada por interações medicamentosas e exposição solar. Recentemente, o polimorfismo CYP3A4*22 foi associado à redução da atividade da enzima. Entretanto, pouco se sabe sobre seu impacto na formação do EDF. Objetivo: Em virtude da alta variabilidade na resposta terapêutica e os múltiplos fatores associados ao metabolismo do TAM, o presente estudo objetivou avaliar o efeito dos polimorfismos da CYP2D6 e CYP3A4, interações medicamentosas e exposição à vitamina D na bioativação do TAM. Adicionalmente, dois métodos analíticos para a otimização do tratamento através da medida das razões metabólicas da CYP2D6 e quantificação do TAM e metabólitos em manchas de sangue seco (DBS) foram desenvolvidos. Patientes & métodos: Cento e dezesseis pacientes em tratamento adjuvante com o TAM forneceram amostras de plasma para dosagens do TAM, metabótilos e 25OHD3 no inverno e verão. As concentrações de TAM e metabólitos em plasma e DBS foram medidas por LCMS/ MS. Foram avaliados os genótipos da CYP2D6 e CYP3A4, bem como fenótipos obtidos pelas razões metabólicas determinadas após administração dos fármacos sonda dextrometorfano e omeprazol. As concentrações de vitamina D3 em plasma foram quantificadas por HPLC-UV. Foram obtidas informações sobre uso de inibidores ou indutores das enzimas e suplementação de vitamina D. Resultados: Cerca de 20% das pacientes apresentaram atividade metabólica reduzida para a CYP2D6 e 7% para a CYP3A4. Aproximadamente 30% das metabolizadoras lentas (ML), 56% das metabolizadoras intermediárias (MI) e 11.3% das metabolizadoras rápidas (MR) usavam fármaco inibidor da CYP2D6. As concentrações de EDF diminuiram proporcionalmente à redução da atividade metabólica da CYP2D6 (ML 2,79 ng mL-1, MI 5.36 ng mL-1 e MR 10,65 ng mL-1, P<0.01). A mediana das concentrações plasmáticas de TAM e HTF em pacientes CYP2D6 MI com metabolismo reduzido da CYP3A4 (161,50 ng mL-1 e 1,32 ng mL-1, respectivamente) foram superiores as encontradas nos pacientes CYP2D6 MI com metabolismo funcional da CYP3A4 (122,07 ng mL-1 e 0.61 ng mL-1, respectivamente, P<0.05). Adicionalmente, as concentrações de HTF e TAM foram aproximadamente 50% superiores em pacientes com genótipo CYP3A4*22 em comparação aos pacientes *1/*1. A sazonalidade também contribuiu para a variabilidade das concentrações dos metabólitos ativos, os níveis de EDF foram 24% e HTF 42% superiores no verão. Nas análises de DBS, foi possível identificar 96% dos pacientes com concentrações de EDF abaixo do limiar clínico, indicando seu potencial uso no monitoramento terapêutico do TAM. Conclusão: a CYP3A4 contribui para a bioativação do TAM através da formação de HTF, tornando-se mais importante em condições de atividade diminuída ou ausente da CYP2D6. Os níveis plasmáticos de EDF e HTF demonstraram ser influenciados pela sazonalidade, com aumento significativo no verão. Entretanto o mecanismo relacionado a associação da vitamina D, exposição solar e bioativação do TAM permanecem por ser elucidados. / Background: The therapeutic antiestrogenic effect of tamoxifen (TAM) requires metabolic activation to endoxifen (EDF) and 4-hydroxytamoxifen (HTF). Adequate therapeutic outcome seems to be dependent on the achievement of a threshold of EDF concentration (>5.9 ng mL-1). EDF plasma levels are highly variable among patients, which could be partly explained by polymorphisms in the CYP2D6 gene and the use of enzymes inhibitor drugs. In a lesser extent, CYP3A4 also contributes to EDF formation and can be influenced by drug interactions and sun exposure. From a genetic point of view, a recently described CYP3A4*22 polymorphism has been associated with reduced enzyme activity. However, there is little knowledge about the impact of CYP3A4 polymorphisms on EDF formation. Objective: In view of the large variability on therapeutic response and the multiple factors associated to TAM metabolic activation, the present study aimed to evaluate the effect of CYP2D6 and CYP3A4 polymorphisms, drug interactions and vitamin D exposure on TAM metabolic activation. Additionally, two analytical methods for optimization of TAM treatment by measurement of CYP2D6 metabolic ratios and quantification of TAM and metabolites in dried blood sopts (DBS) were developed. Patients & methods: One hundred and sixteen patients under TAM therapy provided blood samples for measurement of TAM, NDT, EDF, HTF and 25OHD3 at Winter and Summer. TAM and metabolites were measured in plasma and DBS by LC-MS/MS. CYP2D6 and CYP3A4 genotypes and phenotypes, given according to [DMT]/[DTP] and [OME]/[OMS] metabolic ratios after administration of probe drugs, were also evaluated. Vitamine D3 was measured in plasma by HPLC-UV. Data on use of CYP2D6 and CYP3A4 inhibitor or inducer drugs and vitamin D supplementation were recorded. Results: About 20% of patients had reduced CYP2D6 metabolic activity and 7% CYP3A4 impaired metabolism. Approximately 30% of CYP2D6 poor metabolizers (PM), 56% of intermediate metabolizers (IM) and 11.3% of extensive metabolizers (EM) were using CYP2D6 inhibitor drugs. EDF levels diminished proportionally to the reduction of CYP2D6 metabolic activity (PM 2.79 ng mL-1, IM 5.36 ng mL-1 and EM 10.65 ng mL-1, P<0.01). Median plasma levels of TAM (161.50 ng mL-1) and HTF (1.32 ng mL-1) in CYP2D6 IM patients with reduced CYP3A4 metabolism were higher (P<0.05) than those from CYP2D6 IM patients with functional CYP3A4 metabolism (122.07 ng mL-1 and 0.61 ng mL-1, respectively). Indeed, HTF and TAM plasma levels were approximately 50% higher in patients with CYP3A4*22 genotype compared to patients with alleles *1/*1. Seasonality also contributed to EDF and HTF variability, summer concentrations were 24% and 42% higher compared to winter. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold and can be used in therapeutic monitoring of TAM. Conclusion: Our findings suggest that CYP3A4 contributes to the bioactivation of TAM through formation of HTF and becomes increasingly important in conditions of diminished or absent CYP2D6 activity. A significant variability on EDF and HTF exposure related to seasonality was identified, with considerable higher plasma concentrations during summer. The mechanism relating vitamin D status, seasonality and biotransformation of TAM still remains to be elucidated.

Introdução: a ação antiestrogênica do tamoxifeno (TAM) é dependente da bioativação à endoxifeno (EDF) e 4-hidroxitamoxifeno (HTF). É bastante provável que sua eficácia terapêutica esteja relacionada ao alcance de um limiar nos níveis de EDF (>5,9 ng mL-1). Entretanto, as concentrações plasmáticas de EDF são altamente variáveis, em parte devido a polimorfismos no gene da CYP2D6 e ao uso de inibidores da enzima. A CYP3A4 também contribui para a formação do EDF e pode ser influenciada por interações medicamentosas e exposição solar. Recentemente, o polimorfismo CYP3A4*22 foi associado à redução da atividade da enzima. Entretanto, pouco se sabe sobre seu impacto na formação do EDF. Objetivo: Em virtude da alta variabilidade na resposta terapêutica e os múltiplos fatores associados ao metabolismo do TAM, o presente estudo objetivou avaliar o efeito dos polimorfismos da CYP2D6 e CYP3A4, interações medicamentosas e exposição à vitamina D na bioativação do TAM. Adicionalmente, dois métodos analíticos para a otimização do tratamento através da medida das razões metabólicas da CYP2D6 e quantificação do TAM e metabólitos em manchas de sangue seco (DBS) foram desenvolvidos. Patientes & métodos: Cento e dezesseis pacientes em tratamento adjuvante com o TAM forneceram amostras de plasma para dosagens do TAM, metabótilos e 25OHD3 no inverno e verão. As concentrações de TAM e metabólitos em plasma e DBS foram medidas por LCMS/ MS. Foram avaliados os genótipos da CYP2D6 e CYP3A4, bem como fenótipos obtidos pelas razões metabólicas determinadas após administração dos fármacos sonda dextrometorfano e omeprazol. As concentrações de vitamina D3 em plasma foram quantificadas por HPLC-UV. Foram obtidas informações sobre uso de inibidores ou indutores das enzimas e suplementação de vitamina D. Resultados: Cerca de 20% das pacientes apresentaram atividade metabólica reduzida para a CYP2D6 e 7% para a CYP3A4. Aproximadamente 30% das metabolizadoras lentas (ML), 56% das metabolizadoras intermediárias (MI) e 11.3% das metabolizadoras rápidas (MR) usavam fármaco inibidor da CYP2D6. As concentrações de EDF diminuiram proporcionalmente à redução da atividade metabólica da CYP2D6 (ML 2,79 ng mL-1, MI 5.36 ng mL-1 e MR 10,65 ng mL-1, P<0.01). A mediana das concentrações plasmáticas de TAM e HTF em pacientes CYP2D6 MI com metabolismo reduzido da CYP3A4 (161,50 ng mL-1 e 1,32 ng mL-1, respectivamente) foram superiores as encontradas nos pacientes CYP2D6 MI com metabolismo funcional da CYP3A4 (122,07 ng mL-1 e 0.61 ng mL-1, respectivamente, P<0.05). Adicionalmente, as concentrações de HTF e TAM foram aproximadamente 50% superiores em pacientes com genótipo CYP3A4*22 em comparação aos pacientes *1/*1. A sazonalidade também contribuiu para a variabilidade das concentrações dos metabólitos ativos, os níveis de EDF foram 24% e HTF 42% superiores no verão. Nas análises de DBS, foi possível identificar 96% dos pacientes com concentrações de EDF abaixo do limiar clínico, indicando seu potencial uso no monitoramento terapêutico do TAM. Conclusão: a CYP3A4 contribui para a bioativação do TAM através da formação de HTF, tornando-se mais importante em condições de atividade diminuída ou ausente da CYP2D6. Os níveis plasmáticos de EDF e HTF demonstraram ser influenciados pela sazonalidade, com aumento significativo no verão. Entretanto o mecanismo relacionado a associação da vitamina D, exposição solar e bioativação do TAM permanecem por ser elucidados. / Background: The therapeutic antiestrogenic effect of tamoxifen (TAM) requires metabolic activation to endoxifen (EDF) and 4-hydroxytamoxifen (HTF). Adequate therapeutic outcome seems to be dependent on the achievement of a threshold of EDF concentration (>5.9 ng mL-1). EDF plasma levels are highly variable among patients, which could be partly explained by polymorphisms in the CYP2D6 gene and the use of enzymes inhibitor drugs. In a lesser extent, CYP3A4 also contributes to EDF formation and can be influenced by drug interactions and sun exposure. From a genetic point of view, a recently described CYP3A4*22 polymorphism has been associated with reduced enzyme activity. However, there is little knowledge about the impact of CYP3A4 polymorphisms on EDF formation. Objective: In view of the large variability on therapeutic response and the multiple factors associated to TAM metabolic activation, the present study aimed to evaluate the effect of CYP2D6 and CYP3A4 polymorphisms, drug interactions and vitamin D exposure on TAM metabolic activation. Additionally, two analytical methods for optimization of TAM treatment by measurement of CYP2D6 metabolic ratios and quantification of TAM and metabolites in dried blood sopts (DBS) were developed. Patients & methods: One hundred and sixteen patients under TAM therapy provided blood samples for measurement of TAM, NDT, EDF, HTF and 25OHD3 at Winter and Summer. TAM and metabolites were measured in plasma and DBS by LC-MS/MS. CYP2D6 and CYP3A4 genotypes and phenotypes, given according to [DMT]/[DTP] and [OME]/[OMS] metabolic ratios after administration of probe drugs, were also evaluated. Vitamine D3 was measured in plasma by HPLC-UV. Data on use of CYP2D6 and CYP3A4 inhibitor or inducer drugs and vitamin D supplementation were recorded. Results: About 20% of patients had reduced CYP2D6 metabolic activity and 7% CYP3A4 impaired metabolism. Approximately 30% of CYP2D6 poor metabolizers (PM), 56% of intermediate metabolizers (IM) and 11.3% of extensive metabolizers (EM) were using CYP2D6 inhibitor drugs. EDF levels diminished proportionally to the reduction of CYP2D6 metabolic activity (PM 2.79 ng mL-1, IM 5.36 ng mL-1 and EM 10.65 ng mL-1, P<0.01). Median plasma levels of TAM (161.50 ng mL-1) and HTF (1.32 ng mL-1) in CYP2D6 IM patients with reduced CYP3A4 metabolism were higher (P<0.05) than those from CYP2D6 IM patients with functional CYP3A4 metabolism (122.07 ng mL-1 and 0.61 ng mL-1, respectively). Indeed, HTF and TAM plasma levels were approximately 50% higher in patients with CYP3A4*22 genotype compared to patients with alleles *1/*1. Seasonality also contributed to EDF and HTF variability, summer concentrations were 24% and 42% higher compared to winter. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold and can be used in therapeutic monitoring of TAM. Conclusion: Our findings suggest that CYP3A4 contributes to the bioactivation of TAM through formation of HTF and becomes increasingly important in conditions of diminished or absent CYP2D6 activity. A significant variability on EDF and HTF exposure related to seasonality was identified, with considerable higher plasma concentrations during summer. The mechanism relating vitamin D status, seasonality and biotransformation of TAM still remains to be elucidated.

Introdução: a ação antiestrogênica do tamoxifeno (TAM) é dependente da bioativação à endoxifeno (EDF) e 4-hidroxitamoxifeno (HTF). É bastante provável que sua eficácia terapêutica esteja relacionada ao alcance de um limiar nos níveis de EDF (>5,9 ng mL-1). Entretanto, as concentrações plasmáticas de EDF são altamente variáveis, em parte devido a polimorfismos no gene da CYP2D6 e ao uso de inibidores da enzima. A CYP3A4 também contribui para a formação do EDF e pode ser influenciada por interações medicamentosas e exposição solar. Recentemente, o polimorfismo CYP3A4*22 foi associado à redução da atividade da enzima. Entretanto, pouco se sabe sobre seu impacto na formação do EDF. Objetivo: Em virtude da alta variabilidade na resposta terapêutica e os múltiplos fatores associados ao metabolismo do TAM, o presente estudo objetivou avaliar o efeito dos polimorfismos da CYP2D6 e CYP3A4, interações medicamentosas e exposição à vitamina D na bioativação do TAM. Adicionalmente, dois métodos analíticos para a otimização do tratamento através da medida das razões metabólicas da CYP2D6 e quantificação do TAM e metabólitos em manchas de sangue seco (DBS) foram desenvolvidos. Patientes & métodos: Cento e dezesseis pacientes em tratamento adjuvante com o TAM forneceram amostras de plasma para dosagens do TAM, metabótilos e 25OHD3 no inverno e verão. As concentrações de TAM e metabólitos em plasma e DBS foram medidas por LCMS/ MS. Foram avaliados os genótipos da CYP2D6 e CYP3A4, bem como fenótipos obtidos pelas razões metabólicas determinadas após administração dos fármacos sonda dextrometorfano e omeprazol. As concentrações de vitamina D3 em plasma foram quantificadas por HPLC-UV. Foram obtidas informações sobre uso de inibidores ou indutores das enzimas e suplementação de vitamina D. Resultados: Cerca de 20% das pacientes apresentaram atividade metabólica reduzida para a CYP2D6 e 7% para a CYP3A4. Aproximadamente 30% das metabolizadoras lentas (ML), 56% das metabolizadoras intermediárias (MI) e 11.3% das metabolizadoras rápidas (MR) usavam fármaco inibidor da CYP2D6. As concentrações de EDF diminuiram proporcionalmente à redução da atividade metabólica da CYP2D6 (ML 2,79 ng mL-1, MI 5.36 ng mL-1 e MR 10,65 ng mL-1, P<0.01). A mediana das concentrações plasmáticas de TAM e HTF em pacientes CYP2D6 MI com metabolismo reduzido da CYP3A4 (161,50 ng mL-1 e 1,32 ng mL-1, respectivamente) foram superiores as encontradas nos pacientes CYP2D6 MI com metabolismo funcional da CYP3A4 (122,07 ng mL-1 e 0.61 ng mL-1, respectivamente, P<0.05). Adicionalmente, as concentrações de HTF e TAM foram aproximadamente 50% superiores em pacientes com genótipo CYP3A4*22 em comparação aos pacientes *1/*1. A sazonalidade também contribuiu para a variabilidade das concentrações dos metabólitos ativos, os níveis de EDF foram 24% e HTF 42% superiores no verão. Nas análises de DBS, foi possível identificar 96% dos pacientes com concentrações de EDF abaixo do limiar clínico, indicando seu potencial uso no monitoramento terapêutico do TAM. Conclusão: a CYP3A4 contribui para a bioativação do TAM através da formação de HTF, tornando-se mais importante em condições de atividade diminuída ou ausente da CYP2D6. Os níveis plasmáticos de EDF e HTF demonstraram ser influenciados pela sazonalidade, com aumento significativo no verão. Entretanto o mecanismo relacionado a associação da vitamina D, exposição solar e bioativação do TAM permanecem por ser elucidados. / Background: The therapeutic antiestrogenic effect of tamoxifen (TAM) requires metabolic activation to endoxifen (EDF) and 4-hydroxytamoxifen (HTF). Adequate therapeutic outcome seems to be dependent on the achievement of a threshold of EDF concentration (>5.9 ng mL-1). EDF plasma levels are highly variable among patients, which could be partly explained by polymorphisms in the CYP2D6 gene and the use of enzymes inhibitor drugs. In a lesser extent, CYP3A4 also contributes to EDF formation and can be influenced by drug interactions and sun exposure. From a genetic point of view, a recently described CYP3A4*22 polymorphism has been associated with reduced enzyme activity. However, there is little knowledge about the impact of CYP3A4 polymorphisms on EDF formation. Objective: In view of the large variability on therapeutic response and the multiple factors associated to TAM metabolic activation, the present study aimed to evaluate the effect of CYP2D6 and CYP3A4 polymorphisms, drug interactions and vitamin D exposure on TAM metabolic activation. Additionally, two analytical methods for optimization of TAM treatment by measurement of CYP2D6 metabolic ratios and quantification of TAM and metabolites in dried blood sopts (DBS) were developed. Patients & methods: One hundred and sixteen patients under TAM therapy provided blood samples for measurement of TAM, NDT, EDF, HTF and 25OHD3 at Winter and Summer. TAM and metabolites were measured in plasma and DBS by LC-MS/MS. CYP2D6 and CYP3A4 genotypes and phenotypes, given according to [DMT]/[DTP] and [OME]/[OMS] metabolic ratios after administration of probe drugs, were also evaluated. Vitamine D3 was measured in plasma by HPLC-UV. Data on use of CYP2D6 and CYP3A4 inhibitor or inducer drugs and vitamin D supplementation were recorded. Results: About 20% of patients had reduced CYP2D6 metabolic activity and 7% CYP3A4 impaired metabolism. Approximately 30% of CYP2D6 poor metabolizers (PM), 56% of intermediate metabolizers (IM) and 11.3% of extensive metabolizers (EM) were using CYP2D6 inhibitor drugs. EDF levels diminished proportionally to the reduction of CYP2D6 metabolic activity (PM 2.79 ng mL-1, IM 5.36 ng mL-1 and EM 10.65 ng mL-1, P<0.01). Median plasma levels of TAM (161.50 ng mL-1) and HTF (1.32 ng mL-1) in CYP2D6 IM patients with reduced CYP3A4 metabolism were higher (P<0.05) than those from CYP2D6 IM patients with functional CYP3A4 metabolism (122.07 ng mL-1 and 0.61 ng mL-1, respectively). Indeed, HTF and TAM plasma levels were approximately 50% higher in patients with CYP3A4*22 genotype compared to patients with alleles *1/*1. Seasonality also contributed to EDF and HTF variability, summer concentrations were 24% and 42% higher compared to winter. The DBS method was able to identify 96% of patients with plasma EDF concentrations below the clinical threshold and can be used in therapeutic monitoring of TAM. Conclusion: Our findings suggest that CYP3A4 contributes to the bioactivation of TAM through formation of HTF and becomes increasingly important in conditions of diminished or absent CYP2D6 activity. A significant variability on EDF and HTF exposure related to seasonality was identified, with considerable higher plasma concentrations during summer. The mechanism relating vitamin D status, seasonality and biotransformation of TAM still remains to be elucidated.

No description available.

This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

No description available.

Parkinson's Disease affects more than 1 million people in the United States with 60,000 new cases being diagnosed each year. Currently, there is no cure for Parkinson's Disease, but there are several treatment options available. Currently the most popular surgical option is Deep Brain Stimulation. Microelectrode recording helps identify nuclei as the microelectrode passes through them. While the firing frequencies of the target nuclei are well defined, other nuclei are not. This study will attempt to characterize the Zona Incerta, which is the structure directly above the Subthalamic Nucleus, a target nucleus. Characterization of the firing frequency of the Zona Incerta will help aid Deep Brain Stimulation procedures. Looking at the Interspike Intervals for 25 files showed that the average firing frequency is 11.6Hz. A file recorded in the STN was used for comparison and to validate the methods used. This yielded an average firing frequency of 37.5Hz.

La thérapie de l'épilepsie par résection partielle exige l'identification des structures cérébrales qui sont impliquées dans la genèse des crises d'épilepsie focales. Plusieurs modalités telles que l'IRM, le PET SCAN, la sémiologie de la crise et l'électrophysiologie sont exploitées par les experts pour contribuer à la localisation de la zone épileptogène. L'EEG du scalp est la modalité qui procure la résolution temporelle à l'échelle des processus électrophysiologiques étudiés. Cependant du fait du positionnement des capteurs sur le scalp, sa résolution spatiale et, plus précisément, de profondeur est très médiocre. Dans certain cas (épilepsies pharmaco-résistantes), et pour palier à cette déficience spatiale, il est possible d'avoir recours à la SEEG. La SEEG permet des mesures électrophysiologiques intracérébrales : la résolution spatiale et donc anatomique est excellente dans l'axe de la microélectrode. La définition de la zone épileptogène, comme celle proposée par Talairach et Bancaud, est une définition électro-clinique basée sur les résultats d'enregistrements de SEEG intracérébraux. Elle tient compte non seulement de la localisation anatomique de la décharge épileptique partielle, mais également de l'évolution dynamique de cette décharge, c'est à dire les réseaux neurologiques actifs durant la période intercritique-critique et des symptômes cliniques. Récemment, il a été proposé une technique de diagnostic complémentaire de localisation de la zone épileptogénique employant la stimulation électrique cérébrale de profondeur (Deep Brain Stimulation). Cette source exogène peut activer les réseaux épileptiques et produire une réaction électrophysiologique telle qu'une crise d'épilepsie. Elle permet également de mettre en exergue les zones fonctionnelles cognitives. Cette source exogène est parfaitement définie spatialement et temporellement. Ainsi, la stimulation, couplée aux mesures SEEG, contribue à la modélisation de la propagation électrique cérébrale et, par voie de conséquence, à la compréhension du processus épileptique. De plus, ce travail sur le modèle de propagation directe apporte une aide à la résolution du problème inverse et donc à la localisation de sources. Les différentes tâches accomplies au cours de cette thèse sont les suivantes : création d'une base de données réelles à partir de 3000 stimulations et mesures SEEG pour 42 patients explorés ; extraction par séparation des signaux de propagation de la stimulation électrique (DBS) des mesures multidimensionnelles SEEG : 5 méthodes ont été développées ou adaptées et ont été validées au cours d'une première phase en simulation puis sur des signaux réels SEEG dans une seconde phase ; localisation des électrodes de SEEG dans le repère anatomique de l'IRM et du CT Scanner en y ajoutant une étape de segmentation de la matière grise et blanche, du liquide céphalorachidien et de l'os ; discussion sur de nombreux modèles de propagation réalistes ou non réalistes proposés dans la littérature, à la fois sur le plan du raffinement du modèle mais également sur les implantations numériques possibles : modèles de milieu, sphériques et réalistes infinis basés sur MRI et CT du patient ; comparaison entre les résultats générés par les modèles de sources et de milieux et les données obtenues après séparation de la stimulation électrique in vivo chez l'homme ; validation des modèles de tête FEM en intégrant les conductivités des milieux (CSF), gris et blancs céphalo-rachidiens et perspectives envisagées / The study of epilepsy requires the identification of cerebral structures which are involved in generation of seizures and connexion processes. Several methods of clinical investigation contributed to these studies : imaging (PET, MRI), electrophysiology (EEG, SEEG, MEG). The EEG provides a temporal resolution enough to analyze these processes. However, the localization of deep sources and their dynamical properties are difficult to understand. SEEG is a modality of intracerebral electrophysiological and anatomical high temporal resolution reserved for some difficult cases of pre-surgical diagnosis : drug-resistant epilepsy. The definition of the epileptogenic zone, as proposed by Talairach and Bancaud is an electro-clinical definition based on the results of intracerebral SEEG recordings. It takes into account not only the anatomical localization of partial epileptic discharge, but also the dynamic evolution of this discharge (active neural networks at the time of seizure) and clinical symptoms. Recently, a novel diagnostic technique allows an accurate localization of the epileptogenic zone using Depth Brain Stimulation (DBS). This exogenous source can activate the epileptic networks and generate an electrophysiological reaction. Therefore, coupling DBS with SEEG measurements is very advantageous : firstly, to contribute to the modeling and understanding of the (epileptic) brain and to help the diagnosis, secondly, to access the estimation of head model as an electrical conductor (conductive properties of tissues). In addition, supplementary information about head model improves the solution to the inverse problem (source localization methods) used in many applications in EEG and SEEG. The inverse solution requires repeated computation of the forward problem, i.e. the simulation of EEG and SEEG fields for a given dipolar source in the brain using a volume-conduction model of the head. As for DBS, the location of source is well defined. Therefore, in this thesis, we search for the best head model for the forward problem from real synchronous measurements of EEG and SEEG with DBS in several patients. So, the work of the thesis breaks up into different parts for which we need to accomplish the following tasks : Creation of database 3000 DBS measurements for 42 patients ; Extraction of DBS signal from SEEG and EEG measurements using multidimensional analysis : 5 methods have been developed or adapted and validate first in a simulation study and, secondly, in a real SEEG application ; Localization of SEEG electrodes in MR and CT images, including segmentation of brain matter ; SEEG forward modeling using infinite medium, spherical and realistic models based on MRI and CT of the patient ; Comparison between different head models and validation with real in vivo DBS measurements ; Validation of realistic 5-compartment FEM head models by incorporating the conductivities of cerebrospinal fluid (CSF), gray and white matters

Deep brain stimulation (DBS) surgery, a type of microelectrode-guided surgery, is an effective treatment for the movement disorders patients that can no longer be treated by medications. New rigid and non-rigid image registration methods were developed for the movement disorders patients that underwent DBS surgery. These new methods help study and analyze the brain shift during the DBS surgery and perform atlas-based segmentation of the deep brain structures for the DBS surgery planning and navigation. A diploë based rigid registration method for the intra-operative brain shift analysis during the DBS surgery was developed. The proposed method for the brain shift analysis ensures rigid registration based on diploë only, which can be treated as a rigid structure as opposed to the brain tissues. The results show that the brain shift during the DBS surgery is comparable to the size of the DBS targets and should not be neglected. This brain shift may further lengthen and complicate the DBS surgery contrary to the common belief that brain shift during the DBS surgery is not considerable. We also developed an integrated electrophysiological and anatomical atlas with eleven deep brain structures segmented by an expert, and electrophysiological data of four implant locations obtained from post-op MRI data of twenty patients that underwent DBS surgery. This atlas MR image is then non-rigidly registered with the pre-operative patient MR image, which provides initial DBS target location along with the segmented deep brain structures that can be used for guidance during the microelectrode mapping of the stereotactic procedure. The atlas based approach predicts the target automatically as opposed to the manual selection currently used. The results showed that 85% of the times, this automatic selection of the target location was closer to the target when compared to currently used technique.

La Tomographie par Émission de Positons (TEP) est une méthode d'imagerie médicale nucléaire permettant de mesurer l'activité métabolique d'un organe par la dégradation d'un radio-traceur injecté. Cette méthode d'imagerie peut être utilisée pour l'observation de l'activité métabolique cérébrale à l'aide d'un radio-traceur adéquat, tel que le 18F-Fluorodésoxyglucose. Dans le cadre d'une étude clinique, des patients cérébro-lésés ayant des troubles de la conscience ont eu une chirurgie d'implantation d'électrodes de Stimulation Cérébrale Profonde (SCP). Afin d'effectuer un suivi des patients avant et après la procédure de SCP, et parce qu'elle est compatible avec la présence d'électrode, l'imagerie TEP est utilisée. Nous nous posons la question suivante, comment caractériser les variations entre deux imageries TEP afin de mesurer précisément l'éffet d'un traitement ? Par construction les valeurs obtenues en imagerie TEP dépendent de nombreux facteurs. Si le poids du patient ainsi que la quantité injectée de radio-traceur marqué sont classiquement normalisés en utilisant la méthode des 'Standard Uptake Value' (SUV), la glycémie, entre autre ne l'est pas. Pour cette raison, calculer les variations d'activités entre deux imageries TEP est un problème délicat. Nous proposons une fonction pour calculer les cartes de variation métabolique de deux acquisitions TEP basée sur une approche voxel du ratio des imageries TEP. Nous l'appliquons à l'étude des patients stimulés (SCP) avec troubles de la conscience. Plus spéciffiquement, nous nous intéressons à la comparaison des imageries TEP intra-patient (avant versus après SCP), mais aussi à la comparaison interpatient (patient versus référence). Dans le processus de création des cartes intra-patient, les imageries TEP sont recalées rigidement avec une acquisition pondérée T1 d'Imagerie par Résonance Magnétique (IRM) structurelle. Du fait de déformations majeures liées aux lésions cérébrales, un masque cérébral précis est créé manuellement par un expert clinique. Dans le processus de création des cartes inter-patient, les imageries TEP des patients sont recalées de manière élastique à une imagerie de référence, un atlas (groupe témoin), que nous construisons. Dans ce cas, un masque semi-automatique de l'intérieur de la boîte crânienne est réalisé. Les résultats peuvent être affinés par l'application supplémentaire d'un masque manuel déformé. Un des points clefs de la méthode est de calculer une normalisation spécifique à chaque imagerie, les rendant comparables, afin de calculer une caractérisation quantitative des variations métaboliques cérébrales. Les cartes de variation métabolique cérébrale obtenues sont ensuite comparées aux évaluations et effets cliniques observés afin de juger de leur pertinence. / Positron Emission Tomography is a nuclear medicine imaging method, allowing measure of an organe metabolic activity through degradation of an injected radio-tracer. This methode can be used, with the appropriate radio-tracer, such as 18F-Fluorodeoxyglucose, for observation of cerebral metabolic activity. Through a clinical study, brain damaged patients with counciousness disorders had an implantation surgery of Deep Brain Stimulation (DBS) electrodes. To be able to do the follow up of the patient before and after the DBS procedure, and because it's compatible with electrodes, PET imaging is used. We ask ourself the following question, how to characterize variations between two PET images, to precisely mesure the impact of a treatment ? By construction, PET imaging obtained values depend of numerous factors. If patient weight and injected radio-tracer are classicaly normalized, using the `Standard Uptake Value' (SUV) method, glycemia for exemple is not. For this reason, compute activity variations between two PET images is a delicate problem. We propose a specific function to allow computation of metabolic variation maps for two PET acquisitions, based on a voxel approach of the PET imaging ratio. We apply it to the study of stimulated patients (DBS) with counciousness disorders. More specifically, we are interested in intra-patient PET imaging comparison (before versus after DBS), but also in inter-patient comparison (patient versus reference). During the intra-patient maps creation process, PET patient images are rigidly registered with a T1 weighted structural Magnetic Resonance Imaging (MRI) acquisition. Due to major deformation caused by cerebral injuries, a precise brain mask is created by a clinical expert. During the inter-patient maps creation process, PET patient imaging are non-rigidly registered to a reference imaging, an Atlas we build. In this case, a semi automatic mask of the inside skull is computed. Results can be further improved by the supplementary application of a deformed manual mask. One of the method key elements, is to estimate a specific normalization for each imaging, making them comparable, in order to calculate quantitative charaterisation of cerebral metabolic variations. Cerebral metabolic variation maps obtained are then compared to observed clinical assesments and effects to judge their relevance.