Chez les mammifères, l’alternance des états de vigilance nécessite la mise en jeu de mécanismes spéci ques qui facilitent les transitions entre l’éveil, le sommeil lent (SL) et le sommeil paradoxal (SP). L’objectif de cette thèse s’inscrit dans l’optique de disséquer chez la souris les processus neuronaux contrôlant l’alternance physiologique entre ces trois états de vigilance. Au cours de cette thèse, nous avons tout d’abord démontré par des approches complémentaires ex vivo et in vivo que le glucose peut favoriser l’endormissement par son action excitatrice directe sur les neurones promoteurs du SL localisés dans l’aire préoptique ventrolatérale (VLPO). Nous avons ensuite, par deux approches méthodologiques di érentes et complémentaires, contribué à préciser le rôle physiologique des neurones exprimant l’hormone de mélano-concentration (MCH) dans la régulation du cycle veille-sommeil, démontrant ainsi qu’en plus de faciliter le déclenchement et le maintien du SP lorsqu’ils sont activés, ils contrôlent certains aspects du SL en favorisant, au cours SL, un SL plus profond ainsi que la terminaison des épisodes de SL. Forts de ces nouveaux résultats supportant une contribution des neurones MCH à la régulation du SL, nous avons déterminé une voie potentielle pouvant sous-tendre cette fonction physiologique à travers leurs projections efférentes sur le VLPO. Nos résultats préliminaires indiquent que la stimulation optogénétique des axones des neurones MCH dans le VLPO favorise le déclenchement d’un état de transition entre SL et SP sans pour autant conduire au SP / In mammals, alternating between vigilance states requires some speci c processes that facilitate transitions between wake, Slow-Wave Sleep (SWS), and Paradoxical Sleep (PS). The objective of this thesis was to decipher, in mice, the neuronal mechanisms that control the alternation between these three vigilance states. During thus thesis, we first demonstrated using complementary ex vivo and in vivo approaches that glucose can facilitate sleep induction by directly exciting sleep- promoting neurons located within the ventrolateral preoptic nucleus (VLPO). Then, by developing two different and complementary approaches, we contributed to clarify the physiological role of melanin-concentrating hormone (MCH)-expressing neurons in sleep-wake regulation. Indeed, in addition to their PS-promoting effect when activated, we found that MCH neurons also contribute to the regulation of some aspects of SWS regulation by favouring the appearance of a deeper SWS and facilitating SWS episodes termination. These new results supporting a role of MCH neurons to SWS regulation led us to investigate a putative pathway underlying such an effect through efferent projections from MCH neurons to the VLPO. Preliminary results suggest that the optogenetic stimulation of axons from MCH neurons within the VLPO could facilitate the appearance of a transition state between SWS and PS without triggering PS onset

Tacrolimus (TAC) é um fármaco imunossupressor muito utilizado na prevenção de rejeição aguda após o transplante de órgãos. Essa droga apresenta um índice terapêutico muito baixo e grande variabilidade intra e interindividual, sendo necessário programas de monitorização terapêutica para se otimizar a eficácia e limitar a toxicidade. O TAC é um fármaco substrato do CYP3A5 e transportado pela proteína de efluxo PGP e acredita-se que o polimorfismos genéticos (SNPs) destas proteínas estejam relacionados a alta variabilidade farmacocinética desta droga. Neste estudo, investigamos a influência dos polimorfismos destas proteínas sobre alguns parâmetros farmacocinéticos do TAC e também, na incidência de lesões renais e rejeição em receptores de transplante renal. Pacientes recebendo TAC a no mínimo 12 meses (n=108) foram genotipados (PCR real time) para os polimorfismos do CYP3A5*3 (rs776746) e do gene ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) e 3435C>T (rs1045642). Dados da concentração plasmática de vale (Co; ng/mL), dose diária normalizada (mg/dia por Kg do paciente) e a concentração plasmática do fármaco normalizada pela dose ingerida (Co/dose, ng/mL por mg/dia por kg do paciente) de TAC foram obtidos dos prontuários médicos ao longo de três anos após o transplante renal. O desfecho clínico foi analisado avaliando-se a curva de sobrevida o enxerto, a função renal obtida pelo clearance de creatinina (Equação de Cockroft-Gault), e desenvolvimento de lesões renais e rejeição aguda e crônica com diagnósticos estabelecidos mediante suspeita clínica e confirmados pela avaliação das biópsias quanto a presença de necrose tubular aguda (NTA) e nefropatia crônica do enxerto (NFC) de acordo com a classificação de BANFF 07. Os haplótipos do gene ABCB1 foram inferidos estatisticamente utilizando o software PHASE (version 2.1). Diferenças foram consideradas significativas quando p<0,05. Não observamos desvios em relação ao esperado pelo equilíbrio de Hardy-Weinberg em nossa população para nenhum dos genes estudados. Frequências alélicas destes polimorfismos (6986G 74%; 1236C 60%; 3435C 59% and 2677G 64%) e haplótipos (49% 2677G-3435C-1236C e 31% 2677T-3435T-1236T) foram consistentes com outros estudos realizados na população brasileira. Indivíduos portadores de ao menos um alelo *1 do gene CYP3A5 necessitavam de maiores doses de TAC para obter níveis plasmáticos semelhantes aos dos indivíduos homozigotos para o alelo *3 (0,09 ± 0,03 vs. 0,06 ± 0,03; mg/dia/kg; p<0,001). Ao final do primeiro ano de transplante pacientes CYP*3/*3 apresentavam praticamente o dobro da razão Co/dose quando comparados com os pacientes CYP*1/*1 (144,60 ± 67,29 vs. 70,44 ± 56,05 ng*mL-1 /mg*kg- 1 /dia; p<0,001). Observou-se semelhante em relação quando indivíduos portadores dos alelos e haplótipo variante da PGP foram avaliados. Não encontramos associações significativas entre os genótipos e a sobrevida do enxerto ou clearance de creatinina. No entanto, observamos que os pacientes portadores do haplótipo GCC apresentaram maior incidência de desenvolvimento de NFC. Este estudo confirma o efeito dos polimorfismos do CYP3A5 e, em menor grau da PGP na farmacocinética do TAC. No entanto, não encontramos associações entre esses polimorfismos e desfechos clínicos significantes, sugerindo que a genotipagem para o CYP3A5 ou ABCB1 ainda não deva ser incorporada na prática clínica como uma ferramenta para o manejo de transplante renal. / Tacrolimus (TAC) is widely used to prevent acute rejection following solid-organ transplantation. This drug is characterized by a narrow therapeutic index and drug monitoring programs are required both to optimize efficacy and to limit toxicity. TAC is known to be substrate of cytochrome P450 (CYP) 3A5 and P-glycoprotein (PGP/ABCB) and its been suggested that genetic polymorphisms (SNPs) of these proteins are highly associated with variations in TAC pharmacokinetics. We investigated the influence of polymorphisms of CYP3A5 and ABCB1 gene on the pharmacokinetic parameters (PK) of TAC and on the incidence of kidney injuries and allograft rejection (AR) in renal transplant recipients. Patients receiving TAC for at least 12 months (n=108) were genotyped (real-time PCR) for CYP3A5*3 (rs776746) and for ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) and 3435C>T (rs1045642) polymorphisms. TAC predose concentration (Co; ng/mL), TAC daily dose (mg/day per kg body weight) and dose-normalized predose concentrations (Co/dose; ng/mL per mg/day per kg body weight) were retrieved from medical records up to 03 years after transplantation. Clinical outcomes were analyzed evaluating renal function in terms of creatinine clearance ( Cockroft-Gault equation) and allograft survival. Kidney injuries and AR diagnostics were established by clinical suspicion and in presence of histological findings in renal biopsies according to the 2007 Banff classification. ABCB1 gene haplotypes were statistically inferred using PHASE software (version 2.1). No deviation from Hardy-Weinberg equilibrium was observed in our study population for the polymorphic loci examined in CYP3A5 and ABCB1. Allelic frequencies of these polymorphisms (6986G 74%; 1236C 60%; 3435C 60% and 2677G 65%) and haplotypes (49% 2677G-3435C-1236C and 30% 2677T- 3435T-1236T) were consistent with other studies in the Brazilian population. Individuals carrying at least one CYP3A5*1 allele required higher TAC dose to achieve similar TAC blood levels as the homozygous individuals for the *3 allele (0.09 ± 0.03 vs.0.06 ± 0.03, mg/day per kg body weight, p<0.001). The presence of the CYP3A5*1 allele was also associated with lower TAC Co/dose compared to CYP*3 homozygous (84.9 ± 43.2 vs. 144.6 ± 66.7 ng/mL per mg/day per kg body weight, p<0.001). Regarding ABCB1 polymorphisms, individuals homozygous for the variant allele of each individual SNP and for the haplotype (TTT) showed higher Co/dose ratio. No associations were found between SNPs or haplotypes and allograft survival or creatinine clearance. We did find, though, that patients carrying GCC haplotype had a higher incidence of chronic rejection. Our findings confirm the effect of CYP3A5 and, less pronounced of ABCB1 polymorphisms, on the TAC pharmacokinetic. On the other hand, we did not find any association between these polymorphisms and relevant clinical outcomes, suggesting that CYP3A5 and ABCB1 genotyping must not be incorporated as a useful clinical tool on the management of kidney transplantation.

Tacrolimus (TAC) é um fármaco imunossupressor muito utilizado na prevenção de rejeição aguda após o transplante de órgãos. Essa droga apresenta um índice terapêutico muito baixo e grande variabilidade intra e interindividual, sendo necessário programas de monitorização terapêutica para se otimizar a eficácia e limitar a toxicidade. O TAC é um fármaco substrato do CYP3A5 e transportado pela proteína de efluxo PGP e acredita-se que o polimorfismos genéticos (SNPs) destas proteínas estejam relacionados a alta variabilidade farmacocinética desta droga. Neste estudo, investigamos a influência dos polimorfismos destas proteínas sobre alguns parâmetros farmacocinéticos do TAC e também, na incidência de lesões renais e rejeição em receptores de transplante renal. Pacientes recebendo TAC a no mínimo 12 meses (n=108) foram genotipados (PCR real time) para os polimorfismos do CYP3A5*3 (rs776746) e do gene ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) e 3435C>T (rs1045642). Dados da concentração plasmática de vale (Co; ng/mL), dose diária normalizada (mg/dia por Kg do paciente) e a concentração plasmática do fármaco normalizada pela dose ingerida (Co/dose, ng/mL por mg/dia por kg do paciente) de TAC foram obtidos dos prontuários médicos ao longo de três anos após o transplante renal. O desfecho clínico foi analisado avaliando-se a curva de sobrevida o enxerto, a função renal obtida pelo clearance de creatinina (Equação de Cockroft-Gault), e desenvolvimento de lesões renais e rejeição aguda e crônica com diagnósticos estabelecidos mediante suspeita clínica e confirmados pela avaliação das biópsias quanto a presença de necrose tubular aguda (NTA) e nefropatia crônica do enxerto (NFC) de acordo com a classificação de BANFF 07. Os haplótipos do gene ABCB1 foram inferidos estatisticamente utilizando o software PHASE (version 2.1). Diferenças foram consideradas significativas quando p<0,05. Não observamos desvios em relação ao esperado pelo equilíbrio de Hardy-Weinberg em nossa população para nenhum dos genes estudados. Frequências alélicas destes polimorfismos (6986G 74%; 1236C 60%; 3435C 59% and 2677G 64%) e haplótipos (49% 2677G-3435C-1236C e 31% 2677T-3435T-1236T) foram consistentes com outros estudos realizados na população brasileira. Indivíduos portadores de ao menos um alelo *1 do gene CYP3A5 necessitavam de maiores doses de TAC para obter níveis plasmáticos semelhantes aos dos indivíduos homozigotos para o alelo *3 (0,09 ± 0,03 vs. 0,06 ± 0,03; mg/dia/kg; p<0,001). Ao final do primeiro ano de transplante pacientes CYP*3/*3 apresentavam praticamente o dobro da razão Co/dose quando comparados com os pacientes CYP*1/*1 (144,60 ± 67,29 vs. 70,44 ± 56,05 ng*mL-1 /mg*kg- 1 /dia; p<0,001). Observou-se semelhante em relação quando indivíduos portadores dos alelos e haplótipo variante da PGP foram avaliados. Não encontramos associações significativas entre os genótipos e a sobrevida do enxerto ou clearance de creatinina. No entanto, observamos que os pacientes portadores do haplótipo GCC apresentaram maior incidência de desenvolvimento de NFC. Este estudo confirma o efeito dos polimorfismos do CYP3A5 e, em menor grau da PGP na farmacocinética do TAC. No entanto, não encontramos associações entre esses polimorfismos e desfechos clínicos significantes, sugerindo que a genotipagem para o CYP3A5 ou ABCB1 ainda não deva ser incorporada na prática clínica como uma ferramenta para o manejo de transplante renal. / Tacrolimus (TAC) is widely used to prevent acute rejection following solid-organ transplantation. This drug is characterized by a narrow therapeutic index and drug monitoring programs are required both to optimize efficacy and to limit toxicity. TAC is known to be substrate of cytochrome P450 (CYP) 3A5 and P-glycoprotein (PGP/ABCB) and its been suggested that genetic polymorphisms (SNPs) of these proteins are highly associated with variations in TAC pharmacokinetics. We investigated the influence of polymorphisms of CYP3A5 and ABCB1 gene on the pharmacokinetic parameters (PK) of TAC and on the incidence of kidney injuries and allograft rejection (AR) in renal transplant recipients. Patients receiving TAC for at least 12 months (n=108) were genotyped (real-time PCR) for CYP3A5*3 (rs776746) and for ABCB1 1236C>T (rs1128503), 2677G>T/A (rs2032582) and 3435C>T (rs1045642) polymorphisms. TAC predose concentration (Co; ng/mL), TAC daily dose (mg/day per kg body weight) and dose-normalized predose concentrations (Co/dose; ng/mL per mg/day per kg body weight) were retrieved from medical records up to 03 years after transplantation. Clinical outcomes were analyzed evaluating renal function in terms of creatinine clearance ( Cockroft-Gault equation) and allograft survival. Kidney injuries and AR diagnostics were established by clinical suspicion and in presence of histological findings in renal biopsies according to the 2007 Banff classification. ABCB1 gene haplotypes were statistically inferred using PHASE software (version 2.1). No deviation from Hardy-Weinberg equilibrium was observed in our study population for the polymorphic loci examined in CYP3A5 and ABCB1. Allelic frequencies of these polymorphisms (6986G 74%; 1236C 60%; 3435C 60% and 2677G 65%) and haplotypes (49% 2677G-3435C-1236C and 30% 2677T- 3435T-1236T) were consistent with other studies in the Brazilian population. Individuals carrying at least one CYP3A5*1 allele required higher TAC dose to achieve similar TAC blood levels as the homozygous individuals for the *3 allele (0.09 ± 0.03 vs.0.06 ± 0.03, mg/day per kg body weight, p<0.001). The presence of the CYP3A5*1 allele was also associated with lower TAC Co/dose compared to CYP*3 homozygous (84.9 ± 43.2 vs. 144.6 ± 66.7 ng/mL per mg/day per kg body weight, p<0.001). Regarding ABCB1 polymorphisms, individuals homozygous for the variant allele of each individual SNP and for the haplotype (TTT) showed higher Co/dose ratio. No associations were found between SNPs or haplotypes and allograft survival or creatinine clearance. We did find, though, that patients carrying GCC haplotype had a higher incidence of chronic rejection. Our findings confirm the effect of CYP3A5 and, less pronounced of ABCB1 polymorphisms, on the TAC pharmacokinetic. On the other hand, we did not find any association between these polymorphisms and relevant clinical outcomes, suggesting that CYP3A5 and ABCB1 genotyping must not be incorporated as a useful clinical tool on the management of kidney transplantation.

As enzimas CYP2D6, CYP2C19 e CYP2C9 são responsáveis pelo metabolismo de aproximadamente metade dos 200 medicamentos mais prescritos nos EUA. Padronizamos ensaios de genotipagem baseados na discriminação alélica com o sistema TaqMan® em 198 indivíduos. Para o gene CYP2D6, os alelos *1 e *2 foram os mais freqüentes, seguidos pelos alelos *4, *41, *35, *17, *5, *10, *6, *29 e *9. Desenvolvemos também uma nova metodologia para a determinação do número de cópias do gene CYP2D6. Para o gene CYP2C19, o alelo *1 foi o mais frequente, seguido pelos alelos *17, *2 e *3. Nosso estudo foi o primeiro a determinar a freqüência alélica do gene CYP2C19 no Brasil. Para o gene CYP2C9, o alelo *1 foi o mais frequente, seguido pelos alelos *2 e *3. Desenvolvemos uma metodologia reprodutível e acessível para a genotipagem dos polimorfismos principais dos genes CYP2D6, CYP2C19 e CYP2C9. A identificação precoce de indivíduos suscetíveis a efeitos adversos, bem como de metabolizadores rápidos pode trazer grandes benefícios aos pacientes possibilitando assim uma medicina personalizada. / The enzymes CYP2D6, CYP2C19 and CYP2C9 metabolize approximately half of the 200 most prescribed drugs in the USA. We standardized genotyping tests based on allelic discrimination, using TaqMan® genotyping system in 198 samples. For the CYP2D6 gene, allele *1 and *2 were the most frequent, followed by alleles *4, *4, *35, *17, *5, *10, *6, *29 and *9. We have also developed a new methodology for determining the copy number variations of the CYP2D6 gene. For the CYP2C19 gene, the allele *1 was the most common, followed by the alleles *17, *2 and *3. In our concern, our study was the first to determine the allele frequency of the CYP2C19 gene in Brazil. For CYP2C9 gene, the allele *1 was the most common followed by the alleles *2 and *3. We developed a methodology reproducible and accessible for genotyping the most important polymorphisms of the genes CYP2D6, CYP2C19 and CYP2C9. The previous identification of individuals at risk to develop adverse drug reactions as well as ultrarapid-metabolizers may bring benefits to the patients, leading to a personalized therapy.

<p>The studies in this thesis describe the development of a microarray based minisequencing system and its application to highly parallel genotyping of single nucleotide polymorphisms. The technical developments included identification of a three-dimensional microarray surface coating with high binding capacity for oligonucleotides modified with amino groups as the most optimal one for the system. The system was also established for multiplexed, reproducible quantitative analysis of SNP alleles both on the level of DNA and RNA. The sensitivity of the system to distinguish SNP alleles present as a minority in a mixed sample was found to be 1-6%. </p><p>The microarray based minisequencing system was applied in a pharmacogenetic study on antihypertensive drug response. A panel of 74 SNPs located in candidate genes related to blood pressure regulation were genotyped in DNA samples from hypertensive patients that had been treated with the antihypertensive drugs irbesartan or atenolol. Multiple regression analysis of the genotype data against the reduction in blood pressure identified genotype combinations of four to five SNPs that explain 44-56% of the reduction in blood pressure in the two treatment groups. The genotypes of two individual SNPs in the angiotensinogen (AGT) gene and a SNP in the low density lipoprotein receptor (LDLR) gene appeared to be associated to reduced blood pressure after treatment with atenolol, while a SNP in the apolipoprotein B (APOB) gene was associated to blood pressure reduction after irbesartan treatment. The genotype of one SNP in the adrenergic alpha-2A-receptor gene (ADRA2A) was related to the reduction in left ventricular mass following atenolol treatment while the genotypes of two SNPs, one in the APOB gene and one in the AGT gene were related to the reduction in left ventricular mass in the patients treated with irbesartan.</p>

The main forms of inflammatory bowel diseases (IBD) are Crohns disease and ulcerative colitis. These are chronic diseases, with periods of progression and remission. They are mostly characterized by digestive symptoms such as diarrhea, abdominal pain and weight loss. They affect young individuals and their frequencies have increased for the last decades. The etiology of these pathologies is not well understood, however genetic and environmental factors are involved. The treatment of IBD aims to control the inflammation and to extend periods of clinical remission. Infliximab is an anti-TNF-α antibody, leading to a clear improvement of the symptomatology. However, about 30 % of the patients do not response to this treatment. Genetic factors are certainly involved in these inter-individual differences. The purpose of our work was to find: 1- genetic factors implicated in the response to Infliximab in Crohns disease and 2- genetic factors predisposing to IBD. First we could show that both genes LTA and TNF, which are closely related, are not associated with the answer to Infliximab in Crohns disease. However, different polymorphisms of the ADAM17 gene were associated with a response to the treatment in our Belgian cohort. Second, we could demonstrate an association between an insertion/deletion in the IRF5 gene and IBD. The insertion allele, predisposing to IBD, is expected to create a new binding site for the SP1 transcription factor.

Ovarian cancer is one of the most common female cancer diseases in the world today and in Sweden more than 800 new cases are diagnosed every year. The standard treatment consists of chemotherapy with paclitaxel in combination with carboplatin after initial cytoreductive surgery. The response to treatment and the severity of adverse drug reactions after chemotherapy varies greatly among individuals, and one of the most important factors responsible for these differences is now recognized to be the genetic variability. One of the major obstacles to successful treatment is drug resistance. Several potential mechanisms have been suggested for the resistance to paclitaxel, such as mutations in the target protein β-tubulin, single nucleotide polymorphisms (SNPs) in the gene ABCB1, which encodes the transport protein P-glycoprotein. P-glycoprotein can mediate efflux of various drugs from cancer cells as well as from the circulation into the intestinal lumen, and overexpression and/or high activity leads to drug resistance and/or increased elimination. Another reason might be the high interindividual variability of paclitaxel plasma concentrations, which has been suggested to be influenced by variability in metabolic enzymes, such as CYP2C8 and CYP3A4, and transport proteins e.g. P-glycoprotein. In the studies constituting this thesis we have investigated the possibilities of predicting the pharmacokinetics of paclitaxel as well as the tumor response and adverse drug reactions after chemotherapy in the preparation of personalized chemotherapy. We studied the correlation between the response and the presence of mutations in the dominant β-tubulin gene and SNPs in ABCB1. DNA from 40 ovarian tumors was screened for sequence variations in the β-tubulin gene without finding any, showing that β-tubulin mutations are rare and unlikely to be a clinically relevant resistance mechanism for paclitaxel. The SNPs G2677T/A and C3435T in the ABCB1 gene were determined in 53 ovarian cancer tumors from patients with poor (progressive disease or relapse within one year) or good (disease-free survival of more than one year) response to paclitaxel-carboplatin chemotherapy. Patients homozygously mutated for G2677T/A had a higher probability of responding to chemotherapy. There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment. No correlation was found for the C3435T variant. By using a newly developed quantitative LC/MS method for the simultaneous determination of paclitaxel and its hydroxymetabolites in human plasma we assessed the individual elimination of paclitaxel in 33 ovarian cancer patients. The patients were genotyped for SNPs in the ABCB1, CYP2C8 and CYP3A4 genes and their in vivo CYP3A4 enzyme activity, tumor response and toxicity, especially the neurotoxicity, were determined. Patients heterozygous for G/A in position 2677 in ABCB1 had a significantly higher clearance of paclitaxel than patients with the wild type or homozygously mutated, but not compared to patients carrying the G/T alleles. A lower clearance of paclitaxel was also found for patients heterozygous for CYP2C8*3 when stratified according to the ABCB1 G2677T/A genotype. The CYP3A4 enzyme activity in vivo affected the relative influence of CYP2C8 and CYP3A4 on the metabolism, but not the total clearance of paclitaxel. The exposure to paclitaxel was correlated to the neurotoxicity, but not to the treatment response. In conclusion, our findings suggest that the SNP G2677T/A in the ABCB1 gene, but not β-tubulin mutations, might be a predictor for paclitaxel response and that the interindividual variability in paclitaxel pharmacokinetics might be predicted by ABCB1 and CYP2C8 genotypes and provide useful information for individualized chemotherapy. / Ovarialcancer (äggstockscancer) är en av de vanligaste cancerformerna hos kvinnor i Sverige idag. Behandlingen består vanligen av tumörreducerande kirurgi följd av kemoterapi med paklitaxel och karboplatin. Målsättningen med detta avhandlingsarbete har varit att förbättra cytostatikabehandlingen (cellgiftsbehandlingen) med framförallt paklitaxel vid ovarialcancer genom att lägga grunden för individualisering av doser och förutsäga tumörsvaret vid behandlingen. Ett problem med dagens cancerbehandling är att många cancerceller så småningom blir resistenta mot olika cytostatika. För att angripa den mest resistenta cellen innan den induceras att öka uttrycket av, eller utveckla, fler resistensmekanismer vore det en fördel om vi före behandlingen kunde prediktera vilken dos av cytostatika som är bäst lämpad för individen samt om tumören kommer att reagera på behandlingen eller ej. En av de viktigaste faktorerna för skillnader i behandlingseffekt tros vara genetiska variationer mellan olika individer. I våra studier har vi använt genetiska metoder för att studera om vi kan prediktera tumörsvaret vid behandlingen genom att bestämma mutationer i genen för paklitaxels målprotein, β-tubulin, samt bestämma genetiska variationer i ABCB1-genen, kodande för transportproteinet P-glykoprotein. Tanken är att ett förändrat målprotein eller en förändrad förmåga hos cancercellerna eller kroppen att transportera ut paklitaxel skulle leda till en skillnad i påverkan på tumören. DNA från 40 ovarialtumörer analyserades utan att en enda sekvensvariation hittades i genen för β-tubulin, vilket tyder på att genetiska förändringar i genen för β-tubulin sannolikt inte är en klinisk relevant resistensmekanism. De normalt förekommande genetiska variationerna G2677T/A och C3435T i ABCB1-genen bestämdes i DNA från 53 ovarialtumörer där behandlingen endera givit en bra (tumörfri minst ett år) eller dålig (progression av tumören eller tumörfri mindre än ett år) anti-tumöreffekt. Patienter som var dubbelmuterade i position 2677 dvs hade endera T/T eller T/A (A/A hittades inte i materialet) i denna position hade en högre sannolikhet att få ett bra anti-tumörsvar vid behandlingen. Även antalet muterade baser påverkade utfallet, ju fler muterade baser i position 2677, desto högre sannolikhet att få ett bra svar på behandlingen. Andelen T eller A var också högre i den grupp av patienter som fått en lyckad behandling. För att kunna prediktera patientens individuella förmåga att bryta ner paklitaxel studerade vi inverkan av sekvensvariationer i generna för de nedbrytande enzymerna, CYP2C8 och CYP3A4, och transportproteinet P-glykoprotein (genen ABCB1) på eliminationen av läkemedlet i kroppen. Vi utvecklade en metod för att mäta paklitaxelkoncentrationerna i blodet och använde den för att studera hur snabbt 33 ovarialcancer patienter eliminerade cytostatikat från blodbanan. Hos dessa patienter bestämde vi förekomsten av kända genetiska variationer i generna ABCB1, CYP2C8 och CYP3A4 samt deras CYP3A4 enzymaktivitet i kroppen. Biverkningarna och tumörsvaret vid behandlingen utvärderades också. Eliminationen av paklitaxel hos dessa patienter var beroende av vilken bas som fanns i position 2677 i ABCB1-genen och förekomsten av den genetiska varianten CYP2C8*3. Enzymaktiviteten hos CYP3A4 kunde inte påvisas påverka eliminationen av paklitaxel utan snarare vilket enzym, CYP2C8 eller CYP3A4, som var relativt dominant i respektive patient. Exponeringen av paklitaxel korrelerade till den neurologiska påverkan som patienten orsakades av cytostatikat, men kunde inte korreleras till tumörsvaret vid slutet av cytostatikabehandlingen. Sammanfattningsvis ger patientens genetiska variationer i ABCB1, men inte β-tubulin, information om behandlingsutfallet. Genetiska variationer i CYP2C8 och ABCB1 påverkar patientens förmåga att eliminera paklitaxel och kan förhoppningsvis användas för att individualisera doserna. Vår förhoppning är att resultaten i denna avhandling skall kunna användas för att individualisera och ytterligare förbättra cytostatikabehandlingen vid ovarialcancer.

No description available.

No description available.

Au cours de la dernière décennie, la pharmacogénomique est devenue le mantra révolutionnaire de nombreux chercheurs et de certains porte-paroles de l'industrie. L'intérêt que porteront les compagnies bio-pharmaceutiques du secteur privé à la recherche et au développement de nouveaux médicaments pharmacogénomiques sera déterminé par la facilité à obtenir du financement et les perspectives de retombées économiques. Dans cette perspective, le droit de la propriété intellectuelle (plus spécifiquement le droit des brevets) a toujours été l'instrument de prédilection pour motiver la recherche et le développement des produits pharmaceutiques. Cependant, l'extension de ce droit au domaine de la pharmacogénomique est controversé. Cette étude évalue l'applicabilité du système international des brevets au domaine de la pharmacogénomique. Suite à une analyse comparative du droit et des principaux textes normatifs, applicables aux brevets pharmaceutiques et biotechnologiques, ainsi qu'à une revue de la doctrine, l'étude soutient que le système de brevets reste une solution viable pour encourager la recherche et le développement dans le domaine de la pharmacogénomique. Cependant, certains ajustements sont nécessaires pour empêcher que des brevets trop larges, ayant des fondements juridiques douteux, ne soient octroyés sur des nouveaux tests de diagnostic pharmacogénomiques et sur des nouveaux outils de diagnostic pharmacogénomiques, ce qui serait néfaste à la recherche et limiterait l'accès aux soins de santé. Plusieurs stratégies sont proposées pour promouvoir un système de brevets applicable au domaine des biotechnologies qui, tout en donnant la motivation nécessaire aux inventeurs et à l'industrie, protégerait nos valeurs humaines fondamentales. / In the last decade, pharmacogenomics has become the "revolution" mantra for numerous researchers and industry representatives. The research interest of the industry for pharmacogenomics will be determined by financing possibilities and prospective economic benefits. In this perspective, the intellectual property system (more specifically patents), has always been the privileged tool to motivate research and development of pharmaceutical products. However, its application to pharmacogenomics is controversial. This study evaluates the applicability of the international patent system to the area of pharmacogenomics. A comparative review and analysis of international laws and guidelines applicable to biotechnology and pharmaceutical patents as well as a review of the literature was carried out. Our study found that the patent system remains a viable solution to promote research and development of pharmacogenomics. However, some adjustments are needed to ensure that overbroad patent having a weak legal basis are not granted on both new pharmacogenomic research tools and diagnostic tests since this could be detrimental to research and limit access to healthcare. Strategies are suggested to promote a patent system, applicable to the field of biotechnology, that will give the necessary incentive to inventors and industry while protecting our fundamental human values. / "Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maîtrise en droit (LL.M.) Option droit, Biotechnologies et société" / Texte du mémoire également publié dans Lex Electronica, vol. 10 n°2 (Été/Automne 2005)