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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
411

Desenvolvimento e avaliação da atividade e farmacocinética de nanopartículas lipídicas sólidas contendo a associação de quinina e doxiciclina / Development and evaluation of the activity and pharmacokinetics of solid lipid nanoparticles loaded with the association of Quinine and Doxycycline

Brum Júnior, Liberato January 2011 (has links)
A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.
412

Desenvolvimento e validação de método de screening por LC-QTOF-MS/MS para análise de antibióticos da classe aminoglicosídeos em alimentos de origem animal

Arsand, Juliana Bazzan January 2015 (has links)
A presença de compostos de uso veterinário em produtos de origem animal é uma grande preocupação em termos de saúde pública. A utilização descontrolada de antibióticos como medicamentos veterinários pode levar ao desenvolvimento de resistência bacteriana, podendo prejudicar a eficácia destas drogas no uso em humanos. Os aminoglicosídeos são antibióticos extensivamente utilizados na criação de animais para o tratamento de infecções bacterianas ou para a promoção do crescimento. A União Europeia estabeleceu níveis máximos de resíduos permitidos (LMRs) para os aminoglicosídeos em vários alimentos de origem animal. O governo brasileiro implementou um plano de controle que avalia os resíduos de medicamentos veterinários em produtos de origem animal. Neste trabalho, foram desenvolvidos método quantitativo por cromatografia a líquido acoplada a espectrometria de massas triplo quadrupolo em modo tandem (LC-MS/MS) e método de screening utilizando cromatografia a líquido acoplada a espectrometria de massas com analisador por tempo-de-voo (LC-qTOF-MS) para a determinação simultânea dos aminoglicosídeos espectinomicina, tobramicina, gentamicina, kanamicina, higromicina, apramicina, estreptomicina, diidroestreptomicina, amicacina e neomicina em leite e em músculo bovino, de aves e suíno. Como método de extração utilizou-se procedimento com ácido tricloroacético e clean up por precipitação a baixa temperatura e C18 a granel. Os métodos por LC-MS/MS e LC-qTOF-MS foram validados de acordo com a Diretiva da Comissão Europeia 2002/657/CE. Os resultados obtidos no método quantitativo para recuperação, precisão, linearidade, especificidade, limites de decisão (CCα) e capacidades de detecção (CCβ) nestas matrizes foram considerados satisfatórios. Os dados de limite de detecção (LOD) e limite de quantificação (LOQ) foram estabelecidos a partir dos LMRs, variando de 5 a 100 ng g-1 para LOD e 12,5 a 250 ng g-1 para LOQ. As recuperações variaram de 36,8 a 98%, e os coeficientes de variação ficaram entre 0,9 e 20,2%, observando-se que todas as curvas foram feitas com nas próprias matrizes a fim de minimizar os efeitos de matriz que são intrínsecos para os casos estudados neste trabalho. Os valores de CCβ obtidos no método qualitativo foram entre 25 e 250 ng g-1, considerados satisfatórios para os analitos nessas matrizes. O método proposto mostrou-se simples, fácil e adequado para a análise de um grande número de amostras por dia a um baixo custo. / The presence of several substances in animal products has been a major public concern. The use of antibiotics drugs as veterinary medicines might lead to the development of bacterial resistance, which might undermine the efficacy of these drugs in human use. The aminoglycosides are antibiotics that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict maximum residue levels (MRLs) for aminoglycosides in several animal origin products. The Brazilian government implemented a control plan that assesses the residues of veterinary medicines in animal products. A quantitative method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) and a screening method by liquid chromatography mass spectrometry with time-of-flight system (LC-qTOF-MS) has been developed for simultaneous determination of the aminoglycosides spectonomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin in milk and in bovine, poultry and swine muscles. A simple extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. The LC–MS/MS and LC-qTOF-MS methods were validated according to the European Union Commission Directive 2002/657/EC. Adequate performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCβ) in all matrices tested. The detection limit (LOD) and limits of quantification (LOQ) was established from MRL ranging from 5 to 100 ng g-1 for LOD and 12.5 to 250 ng g-1 to LOQ. Recoveries ranged from 36.8 to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects which are intrinsic to the cases studied in this work. The CCβ values obtained in qualitative method were between 25 and 250 ng g-1, considered satisfactory for the analytes in those matrices. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.
413

Desenvolvimento e validação de método de screening por LC-QTOF-MS/MS para análise de antibióticos da classe aminoglicosídeos em alimentos de origem animal

Arsand, Juliana Bazzan January 2015 (has links)
A presença de compostos de uso veterinário em produtos de origem animal é uma grande preocupação em termos de saúde pública. A utilização descontrolada de antibióticos como medicamentos veterinários pode levar ao desenvolvimento de resistência bacteriana, podendo prejudicar a eficácia destas drogas no uso em humanos. Os aminoglicosídeos são antibióticos extensivamente utilizados na criação de animais para o tratamento de infecções bacterianas ou para a promoção do crescimento. A União Europeia estabeleceu níveis máximos de resíduos permitidos (LMRs) para os aminoglicosídeos em vários alimentos de origem animal. O governo brasileiro implementou um plano de controle que avalia os resíduos de medicamentos veterinários em produtos de origem animal. Neste trabalho, foram desenvolvidos método quantitativo por cromatografia a líquido acoplada a espectrometria de massas triplo quadrupolo em modo tandem (LC-MS/MS) e método de screening utilizando cromatografia a líquido acoplada a espectrometria de massas com analisador por tempo-de-voo (LC-qTOF-MS) para a determinação simultânea dos aminoglicosídeos espectinomicina, tobramicina, gentamicina, kanamicina, higromicina, apramicina, estreptomicina, diidroestreptomicina, amicacina e neomicina em leite e em músculo bovino, de aves e suíno. Como método de extração utilizou-se procedimento com ácido tricloroacético e clean up por precipitação a baixa temperatura e C18 a granel. Os métodos por LC-MS/MS e LC-qTOF-MS foram validados de acordo com a Diretiva da Comissão Europeia 2002/657/CE. Os resultados obtidos no método quantitativo para recuperação, precisão, linearidade, especificidade, limites de decisão (CCα) e capacidades de detecção (CCβ) nestas matrizes foram considerados satisfatórios. Os dados de limite de detecção (LOD) e limite de quantificação (LOQ) foram estabelecidos a partir dos LMRs, variando de 5 a 100 ng g-1 para LOD e 12,5 a 250 ng g-1 para LOQ. As recuperações variaram de 36,8 a 98%, e os coeficientes de variação ficaram entre 0,9 e 20,2%, observando-se que todas as curvas foram feitas com nas próprias matrizes a fim de minimizar os efeitos de matriz que são intrínsecos para os casos estudados neste trabalho. Os valores de CCβ obtidos no método qualitativo foram entre 25 e 250 ng g-1, considerados satisfatórios para os analitos nessas matrizes. O método proposto mostrou-se simples, fácil e adequado para a análise de um grande número de amostras por dia a um baixo custo. / The presence of several substances in animal products has been a major public concern. The use of antibiotics drugs as veterinary medicines might lead to the development of bacterial resistance, which might undermine the efficacy of these drugs in human use. The aminoglycosides are antibiotics that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict maximum residue levels (MRLs) for aminoglycosides in several animal origin products. The Brazilian government implemented a control plan that assesses the residues of veterinary medicines in animal products. A quantitative method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) and a screening method by liquid chromatography mass spectrometry with time-of-flight system (LC-qTOF-MS) has been developed for simultaneous determination of the aminoglycosides spectonomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin in milk and in bovine, poultry and swine muscles. A simple extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. The LC–MS/MS and LC-qTOF-MS methods were validated according to the European Union Commission Directive 2002/657/EC. Adequate performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCβ) in all matrices tested. The detection limit (LOD) and limits of quantification (LOQ) was established from MRL ranging from 5 to 100 ng g-1 for LOD and 12.5 to 250 ng g-1 to LOQ. Recoveries ranged from 36.8 to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects which are intrinsic to the cases studied in this work. The CCβ values obtained in qualitative method were between 25 and 250 ng g-1, considered satisfactory for the analytes in those matrices. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.
414

Desenvolvimento e avaliação da atividade e farmacocinética de nanopartículas lipídicas sólidas contendo a associação de quinina e doxiciclina / Development and evaluation of the activity and pharmacokinetics of solid lipid nanoparticles loaded with the association of Quinine and Doxycycline

Brum Júnior, Liberato January 2011 (has links)
A malária, causada por protozoários intracelulares do gênero Plasmodium, é uma das doenças tropicais mais devastadoras existentes. Mais de 3 bilhões de pessoas vivem em regiões endêmicas para a malária. Cinco espécies de Plasmodium (falciparum, vivax, ovale, malariae e knowlesi) causam doenças em humanos e a infecção com P. falciparum, o mais letal desses parasitas, resulta em mais de 1 milhão de mortes anualmente. O desenvolvimento de resistência aos fármacos antimaláricos tradicionais, leva ao uso de combinações de fármacos como a quinina (QN) e a doxiciclina (DOX). Nesse contexto, os objetivos deste trabalho foram desenvolver e caracterizar formulação de nanopartículas lipídicas sólidas (NLS) contendo a associação de QN/DOX, avaliar sua eficácia em um modelo in vivo de malária berghei, determinar a sua farmacocinética e o coeficiente de partição nos eritrócitos dos fármacos livres e nanoencapsulados. A formulação de NLS contendo QN/DOX (2,0/0,2 mg/mL) foi preparada pela técnica de homogeneização a alta pressão, utilizando polissorbato 80 e Lipoid® como emulsionantes e palmitato de cetila como matriz lipídica. No estudo preliminar de estabilidade, a formulação de NLS contendo QN/DOX apresentou tamanho de partícula adequado (152,8 ± 5,26 nm), índice de polidispersão (0,173 ± 0,006), potencial zeta (-38,6 ± 1,82 mV), alto conteúdo dos fármacos (95,9% ± 0,70/ 94,1% ± 2,41) e adequada eficiência de encapsulação (94,2% ± 1,14/83,0% ± 2,52), após 21 dias de armazenamento em temperatura ambiente. Para a análise do teor um método rápido e específico de cromatografia líquida-acoplada a espectrometria de massa (LC-MS/MS) foi desenvolvido e validado para a determinação simultânea de QN e DOX nas formulações. O método por LC-MS/MS utilizou coluna Waters Sun Fire C18 (50 mm x 3,0 mm de diâmetro) e a fase móvel foi composta de acetonitrila:ácido fórmico 0,1% (75:25, v/v), no fluxo de 0,45 mL/min (split 1:3). O volume de injeção foi de 10 μL. Ratos Wistar infectados por P. berghei foram utilizados para avaliar a eficácia da formulação de NLS contendo QN/DOX utilizando diferentes regimes de dose. As doses efetivas da formulação, i.v. (75/7,5 mg/kg/dia) e oral (105/10,5 mg/kg/dia), representam uma redução de quase 30% em comparação com os fármacos livres utilizados em associação. A farmacocinética foi avaliada após a administração dos fármacos livres ou nanoencapsulados pela vias i.v. (10/1 mg/ kg) e oral (25/2,5 mg/kg) em ratos Wistar infectados. Para a quantificação das amostras de plasma dos ratos, método por LC-MS/MS foi desenvolvido e validado. A QN, a DOX e a cimetidina (padrão interno, PI) foram extraídos do plasma através de precipitação de proteínas e a fase móvel consistiu de metanol/ácido fórmico 0,1% (70:30, v/v), no fluxo de 0,5 mL / min (split 1:3). A detecção foi realizada através da ionização por electrospray positivo, no modo de monitoramento de reações múltiplas, onde foram monitoradas as transições 325,0>307,0, 445,0>428,1 e 252,8>159,0, para QN, DOX e PI, respectivamente. A análise foi realizada em 2,0 min e o método foi linear na faixa de concentração plasmática entre 5-5000 ng/mL. Nenhuma alteração significativa dos parâmetros farmacocinéticos foi observada para ambos os fármacos e vias de administração, após a nanoencapsulação. O coeficiente de partição da QN nos eritrócitos infectados por P. berghei aumentou (5,53 ± 0,28) quando a formulação de NLS contendo QN/DOX foi usada em comparação com os fármacos livres em associação (3,81± 0,23). Nenhuma alteração significativa na penetração intraeritrocitária da DOX foi observada com a nanoencapsulação. Os resultados demonstram que a nanoencapsulação da QN/DOX em NLS diminui a dose efetiva para o tratamento da malária, sendo uma alternativa interessante a ser investigada para o tratamento da malária falciparum resistente. / Malaria is one of the most devastating tropical diseases caused by intracellular protozoan parasites of the genus Plasmodium. More than 3 billion people live in malarial endemic regions. Five species of Plasmodium (falciparum, vivax, ovale, malariae and knowlesi) cause disease in humans and infection with P. falciparum, the most deadly of these parasites, results in more than 1 million deaths annually. The development of resistance to traditional antimalarial drugs leads to the use of drug combinations such as quinine (QN)/doxycycline (DOX). In this context, the aims of this work were to develop and characterize solid lipid nanoparticles (SLN) loaded with QN/ DOX, to evaluate their efficacy in an in vivo model of berghei malaria, and to determine their pharmacokinetics and erythrocyte partition coefficient compared to the non-encapsulated (free) drug association. The SLN were prepared by high pressure homogenization technique using polysorbate 80 and Lipoid® as emulsifiers and cetyl palmitate as lipid matrix. In the preliminary stability study, QN/DOX-loaded SLN (2.0/0.2 mg/mL) presented adequate particle size (152.8 ± 5.26 nm), polydispersion index (0.173 ± 0.006), zeta potential (-38.6 ± 1.82 mV), high drug content (95.9% ± 0.70/94.1% ± 2.41) and appropriate encapsulation efficiency (94.2% ± 1.14/83.0% ± 2.52) after 21 days of storage at room temperature. For the assay analysis, a fast and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of QN and DOX. The LC-MS/MS method was carried out on a Sun Fire Waters C18 column (50 mm x 3.0 mm I.D.) and the mobile phase consisted of acetonitrile:0.1% formic acid (75:25, v/v), run at a flow rate of 0.45 mL/min (split 1:3). The injection volume was 10 μL. Plasmodium berghei infected Wistar rats were used to evaluate the efficacy of QN/DOX-loaded SLN using different dosing regimens. The effective QN/DOX-loaded SLN i.v. (75/7.5 mg/kg/day) and oral (105/10.5 mg/kg/day) doses represent an almost 30% reduction compared to the free drugs in association. Plasma pharmacokinetics was evaluated after administration of free or nanoencapsulated QN/DOX by i.v. (10/1 mg/kg) and oral (25/2.5 mg/kg) routes to infected Wistar rats. For the quantification of the rat plasma samples, a fast, sensitive and specific LC-MS-MS method was developed and validated for the determination of QN and DOX. QN, DOX and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation and the mobile phase consisted of methanol/formic acid 0.1% (70:30, v/v), run at a flow rate of 0.5 mL/min (split 1:3). Detection was carried out by positive Electrospray Ionization in multiple reaction monitoring mode, monitoring the transitions 325.0>307.0, 445.0>428.1 and 252.8>159.0, for QN, DOX and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range of 5-5000 ng/mL. No significant alteration of pharmacokinetic parameters was observed for both drugs and routes of dosing after nanoencapsulation. QN partition coefficient into P. berghei infected erythrocyte was increased (5.53 ± 0.28) when the QN/DOX-loaded SLN was used in comparison with the free drugs in association (3.81 ± 0.23). No significant alteration on DOX erythrocyte partition coefficient was observed. In summary, the results showed that QN/DOX nanoencapsulation into SLN allows the reduction of the effective antimalarial dose being an interesting alternative to be investigated for the treatment of falciparum resistant malaria.
415

Determinação multiclasse de resíduos de agrotóxicos e medicamentos veterinários em carnes por LC-MS/MS / Multiclass determination of pesticides and veterinary drugs residues in meat by LC-MS/MS

Friggi, Caroline do Amaral 23 March 2012 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The agribusiness has been incorporating new technologies in the production of meat, including good management of farms, health, balanced diet, breeding and integrated production. Allied to this, there is a growing concern with issues related to residues and contaminants in animals intended for food production. In the study, were compared four extraction methods based on the method QuEChERS for the determination of 79 pesticides and 15 veterinary drugs analyzed by Liquid Chromatography coupled to tandem Mass Spectrometry in samples of meat and pork liver, chicken and fish. The parameters evaluated were: linear range (6 concentration levels and 6 injections each), limit of detection (LOD), limit of quantification (LOQ), decision limit (CCα), capability of detection (CCβ) and matrix effect, as well as precision and accuracy, in terms of recovery. For this, we performed the fortification of matrices previously homogenized with solutions containing compounds at 3 levels of fortification (20, 50 and 100 μg kg-1), 6 replicates for each level, were performade. The modified QuEChERS extraction method consists in the extraction of 10 g of the matrix with 10 mL of acetonitrile containing 1% (v/v) of acetic acid and shaking vigorously by hand for 1 min. After, 4 g of anhydrous magnesium sulfate and 1.7 g anhydrous sodium acetate were added and the agitation repeated. After the extract was centrifugated for 8 min (3500 rpm) and 2 mL of the extract was transferred to another tube containing 300 mg of anhydrous magnesium sulfate and 50 mg of sorbent PSA, repeating the agitation and centrifugation. The extract was diluted (1:1 v/v) in the mobile phase and then analyzed by LC-MS/MS. Analytical curves prepared in solvent and in extracts of matrices evaluated showed adequate linearity between 5 and 200 μg kg-1, with coefficients of determination greater than 0.99. The method showed satisfactory recovery values between 70 and 120% (RSD ≤ 20%) to about 95% of compounds in all matrices, except for liver showed satisfactory values to about 80% of the compound at levels of 20, 50 and 100 μg kg-1. The LOQ of method was 10 μg kg-1. CCα values were between 1.1 and 102.9 μg kg-1 and CCβ values were in the range 1.9 to 105.9 μg kg-1. The results of this study showed that the modified method QuEChERS is fast, simple and efficient and can be applied to the determination of pesticides and veterinary drugs in samples of meat and liver pork, chicken and fish fillet for routine analysis. Furthermore, the use of LC-MS/MS was efficent, allowing the determination and confirmation of a large number of compounds simultaneously at residues livels. / O agronegócio nacional vem incorporando novas tecnologias na produção de carnes, incluindo bom manejo das granjas, sanidade, alimentação balanceada, melhoramento genético e produção integrada. Aliado a este fato, há uma preocupação crescente com questões relacionadas à presença de resíduos e contaminantes em animais destinados a produção de alimentos. Neste estudo, comparou-se quatro métodos de extração baseados no método QuEChERS para a determinação de 79 agrotóxicos e 15 medicamentos veterinários por Cromatografia Líquida acoplada à Espectrometria de Massas em Série em amostras de carne e fígado suíno, carne de frango e filé de peixe. Os parâmetros avaliados foram: faixa de linear das curvas analíticas (6 níveis de concentração e 6 injeções cada), limite de detecção (LOD), limite de quantificação (LOQ), limite de decisão (CCα), capacidade de detecção (CCβ) e efeito matriz, bem como precisão e exatidão, em termos de recuperação. Para isso, efetuou-se a fortificação das matrizes previamente homogeneizadas, com soluções contendo os compostos em 3 níveis de fortificação (20, 50 e 100 μg kg-1), 6 réplicas para cada nível, e aplicou-se o método proposto. A extração pelo método QuEChERS modificado consistiu na extração de 10,0 g da matriz com 10 mL de acetonitrila contendo 1% (v/v) de ácido acético sob agitação manual e vigorosa por 1 min. Acrescentou-se 4,0 g de sulfato de magnésio anidro e 1,7 g de acetato de sódio anidro, repetindo-se a agitação. O extrato foi centrifugado por 8 min (3500 rpm) e após 2 mL de extrato foram transferidos para outro tubo contendo 300 mg de sulfato de magnésio anidro e 50 mg de sorvente PSA, repetindo-se a agitação e a centrifugação. O extrato foi diluído (1:1) na fase móvel e em seguida analisado por LC-MS/MS. As curvas analíticas, preparadas no solvente e nos extratos das matrizes avaliadas, apresentaram linearidade adequada entre 5 e 200 μg L-1, com valores de coeficiente de determinação maiores que 0,99. O método apresentou valores de recuperação satisfatórios entre 70 e 120% e (RSD ≤ 20%) para cerca de 95% dos compostos em todas as matrizes, exceto para fígado que apresentou valores satisfatórios para cerca de 80% dos compostos nos níveis de 20, 50 e 100 μg kg-1. O valor de LOQ do método foi de 10 μg kg-1. Os valores de CCα ficaram entre 1,1 e 102,9 μg kg-1 e os valores de CCβ ficaram na faixa de 1,9 a 105,9 μg kg-1. Os resultados desse estudo mostraram que o método QuEChERS modificado é rápido, simples e eficiente, podendo ser aplicado para a determinação de agrotóxicos e medicamentos veterinários em amostras de carne e fígado suíno, carne de frango e filé de peixe em análise de rotina. Além disso, o emprego da LC-MS/MS mostrou-se eficiente, permitindo a determinação e confirmação de um grande número de compostos simultaneamente em níveis de resíduos.
416

Spectroelectrochemical determination of the antioxidant properties of carpobrotus mellei and carpobrotus quadrifidus natural products

Maoela, Manki Sarah January 2009 (has links)
Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins,various flavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine flavonoids in C. mellei and C. quadrifidus. 8 flavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect flavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants.UV-visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 μmol L-1 and 1.44 μA(μmol L-1)-1, respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively. May 2009
417

Diagnosis of acute and chronic enteric fever using metabolomics / Diagnos av akut och kronisk enterisk feber med hjälp av metabolomik

Näsström, Elin January 2017 (has links)
Enteric (or typhoid) fever is a systemic infection mainly caused by Salmonella Typhi and Salmonella Paratyphi A. The disease is common in areas with poor water quality and insufficient sanitation. Humans are the only reservoir for transmission of the disease. The presence of asymptomatic chronic carriers is a complicating factor for the transmission. There are major limitations regarding the current diagnostic methods both for acute infection and chronic carriage. Metabolomics is a methodology studying metabolites in biological systems under influence of environmental or physiological perturbations. It has been applied to study several infectious diseases, with the goal of detecting diagnostic biomarkers. In this thesis, a mass spectrometry-based metabolomics approach, including chemometric bioinformatics techniques for data analysis, has been used to evaluate the potential of metabolite biomarker patterns for diagnosis of enteric fever at different stages of the disease. In Paper I, metabolite patterns related to acute enteric fever were investigated. Human plasma samples from patients in Nepal with culture-confirmed S. Typhi or S. Paratyphi A infection were compared to afebrile controls. A metabolite pattern discriminating between acute enteric fever and afebrile controls, as well as between the two causative agents of enteric fever was detected. The strength of using a panel of metabolites instead of single metabolites as biomarkers was also highlighted. In Paper II, metabolite patterns for acute enteric fever, this time focusing only on S. Typhi infections, were investigated. Human plasma from patients in Bangladesh with culture-positive or -negative but clinically suspected S. Typhi infection were compared to febrile controls. Differences were found in metabolite patterns between the culture-positive S. Typhi group and the febrile controls with a heterogeneity among the suspected S. Typhi samples. Consistencies in metabolite patterns were found to the results from Paper I. In addition, a validation cohort with culture-positive S. Typhi samples and a control group including patients with malaria and infections caused by other pathogens was analysed. Differences in metabolite patterns were detected between S. Typhi samples and all controls as well as between S. Typhi and malaria. Consistencies in metabolite patterns were found to the primary Bangladeshi cohort and the Nepali cohort from Paper I. Paper III focused on chronic Salmonella carriers. Human plasma samples from patients in Nepal undergoing cholecystectomy with confirmed S. Typhi or S. Paratyphi A gallbladder carriage were compared to non-carriage controls. The Salmonella carriage samples were distinguished from the non-carriage controls and differential signatures were also found between the S. Typhi and S. Paratyphi A carriage samples. Comparing metabolites found during chronic carriage and acute enteric fever (in Paper I) resulted in a panel of metabolites significant only during chronic carriage. This work has contributed to highlight the potential of using metabolomics as a tool to find diagnostic biomarker patterns associated with different stages of enteric fever.
418

Liquid chromatography–mass spectrometry in drug metabolism studies

Rousu, T. (Timo) 29 May 2012 (has links)
Abstract Drug metabolite profiling and identification studies are nowadays regularly conducted with liquid chromatography (LC) coupled with mass spectrometry (MS) as an analytical tool. The speed, selectivity and sensitivity of modern LC–MS instruments have been significantly increased in recent years. Especially the use of ultra-high-performance LC (UHPLC) in combination with a modern high-resolution MS instrument offers high full scan detection sensitivity, mass accuracy and the detection of both expected and unexpected metabolites in a single LC–MS run. The present study showed that no single LC–MS conditions were suitable for the analysis of a large group of structurally diverse compounds. The testing of optimum conditions for each individual compound led to more high-quality data when chromatographic retention behavior and mass spectrometric ionization efficiency for in vitro metabolite profiling were considered. The developed LC–MS methods were applicable for measuring both the disappearance of the parent compound and the formation of metabolites. Tentative metabolite identification was based on the measured accurate mass time-of-flight (TOF) MS data. In the second part, a rapid and sensitive assay was designed and built for the trapping, screening and characterization of reactive metabolites in vitro. In total, 78 trapped reactive metabolite conjugates were detected and identified based on accurate mass data using 12 structurally different test compounds. The majority of the detected conjugates were reported for the first time. Amine-containing compounds, that formed methylated and cyanide-trapped products after CYP-mediated reaction steps in human liver microsomal (HLM) incubations, were studied further. The observed methylated cyano conjugates were shown to be experimental artifacts, i.e., metabonates. The study also describes the use of traditional high-performance LC (HPLC) and the more modern UHPLC coupled to time-of-flight, triple quadrupole and hybrid linear ion trap mass spectrometers in drug metabolism studies, and reviews on how to choose the most suitable LC–MS system for metabolite profiling purposes in drug discovery and early drug development. / Tiivistelmä Nestekromatografia (LC) yhdistettynä massaspektrometriaan (MS) on nykyaikana yleisesti käytetty analyysimenetelmä lääkeaineiden aineenvaihduntatuotteiden (metaboliittien) havaitsemisessa ja tunnistamisessa. Modernien LC–MS -laitteiden nopeus, selektiivisyys ja herkkyys ovat merkittävästi parantuneet viime vuosina. Käytettäessä ultrakorkean suorituskyvyn nestekromatografia (UHPLC) yhdessä nykyaikaisen korkean massaresoluution MS-laitteen kanssa on mahdollista havaita kaikki sekä odotetut että odottamattomat metaboliitit yhdellä kertaa. Tutkimalla suurta joukkoa rakenteellisesti erilaisia yhdisteitä voitiin todeta, että yksittäiselle yhdisteelle optimoidut mittausolosuhteet johtivat korkealaatuisempaan dataan kuin yleiset ei-optimoidut olosuhteet, kun arvioitiin sekä kromatografista piikin profiilia ja pidättymistä että ionisaatiotehokkuutta. Yksikään yksittäinen analyysiolosuhde ei myöskään soveltunut kaikille yhdisteille. Tutkimuksessa kehitetyillä LC–MS -analyysimenetelmillä tutkittiin sekä kanta-aineen häviämistä että metaboliatuotteiden muodostumista in vitro -menetelmillä. Alustava metaboliatuotteiden tunnistus perustui tarkan massan mittaukseen lentoaikamassaspektrometrillä (TOFMS). Tutkimustyön seuraavassa vaiheessa kehitettiin nopea ja herkkä analyysimenetelmä reaktiivisten metaboliittien pyydystämiseen, havaitsemiseen ja tunnistamiseen ihmisen maksamikrosomivalmisteista in vitro -menetelmin. 12 testiyhdisteelle havaittiin kaikkiaan 78 erilaista reaktiivisen metaboliitin konjugaatiotuotetta, jotka tunnistettiin tarkan massan perusteella. Suurin osa tunnistetuista konjugaatiotuotteista raportoitiin ensimmäistä kertaa. Amiineja sisältäville testiyhdisteille havaittiin muodostuvan sytokromi P450 (CYP) entsyymien katalysoimien reaktioiden välityksellä metyloituneita ja syanidianionilla konjugoituneita metaboliatuotteita. Tarkempien tutkimusten jälkeen näiden todettiin olevan koejärjestelyistä johtuvia artefaktoja, toisin sanoen metabonaatteja, eivätkä todellisia reaktiivisten metaboliittien konjugaatiotuotteita. Tässä tutkimuksessa arvioitiin myös perinteiseen korkean suorituskyvyn nestekromatografiin (HPLC) sekä uudempaan UHPLC-laitteistoon kytkettyjen lentoaika-, kolmoiskvadrupoli- ja hybridimallisten ioniloukkumassaspektrometrien soveltuvuutta aikaisen lääkekehitysvaiheen metaboliatutkimuksiin.
419

Evaluation of new laboratory methods for routine use

Lehto, T. (Tiina) 12 January 2016 (has links)
Abstract Laboratory medicine is under constant pressure from changes in the operating environment. Organisational changes and tendering processes have led to a trend towards shorter turn-around times and more cost-effective choices. Analysis tools that were previously only available at research laboratories, such as the mass spectrometer and polymerace chain reaction (PCR), have now made their way to university hospital laboratories and even mid-sized laboratories. Organisational changes have increased the need to monitor the pre-analytical steps. The specimen can be drawn from the patient in a satellite laboratory, which may be located several hours from the central laboratory. The increased transportation times may change the analytical properties of the specimens, which is why the stability of different analytes should be investigated thoroughly in different temperatures. It should be born in mind that doctors are treating the patients based on the results they receive from the laboratory. To avoid possible malpractice, the analytical properties should remain reliable. Traditionally, some analyses have been carried out manually, which is known to be time-consuming and carries the possibility of wide intra-observatory mistakes. For that reason, it would be reasonable to perform some manual analyses, such as body fluid analysis, in an automated manner. Automating the manual steps taken in the laboratory would release labour for other tasks and may increase the cost-effectiveness of the work. Organisational changes have redirected the needs of a clinical laboratory towards automated options instead of manual ones and finding more economically-based alternatives to replace or complement traditional methods. / Tiivistelmä Laboratoriolääketiede on jatkuvan muutospaineen alla. Organisaatiomuutokset ja kilpailutus ovat saaneet aikaan sen, että laboratorioiden analytiikkatarjonnan tulee olla kilpailukykyistä niin hinnan kuin tulosten vastausnopeuden suhteen. Aikaisemmin pelkästään tutkimuskäytössä olleet menetelmät, kuten PCR ja massaspektrometri, ovat jalkautuneet jo keskussairaalatasoiseen tutkimusvalikoimaan. Organisaatiomuutokset ovat saaneet aikaan myös sen, että näytteet voidaan ottaa potilaasta alueellisissa toimipisteissä ja kuljettaa päivän aikana keskuslaboratorioon analysoitavaksi. Kuljetusmatkat ja -ajat saattavat olla hyvinkin pitkiä. Tämän johdosta on erittäin tärkeää selvittää näytteiden säilyvyys niin, että tulokset pysyvät luotettavina eikä potilaan hoito kärsi. Perinteisesti osa tutkimuksista, kuten punktionesteen solut, on tehty käsin mikroskopoimalla, jonka tiedetään olevan aikaa vievää ja näin ollen myös kallista analysointia. Kyseisen tutkimuksen siirtäminen analysaattoreille tehtäväksi voi tuoda laboratoriolle taloudellisen säästön lisäksi työvoiman vapautumista manuaalisesti suoritettavalta mikroskopoinnilta. Muutospaineet laboratoriotoiminnoissa ovat saaneet aikaan tarpeen automatisaation lisääntymiselle ja taloudellisempien vaihtoehtojen löytämiselle perinteisten menetelmien rinnalle tai niiden sijaan.
420

Alterations of the epigenome in brain cancer: loss of 5-hydroxymethylcytosine / Altérations de l’épigénome dans le cancer du cerveau: perte de 5-hydroxyméthylcytosine

Lowry, Carolyne Mary January 2017 (has links)
Abstract : 5-Methylcytosine is an epigenetic mark, which can be oxidized to 5-hydroxymethylcytosine (5hmC) in DNA by ten-eleven translocation (TET) oxygenases. It is an initial step in the demethylation of 5mC. Levels of 5hmC is relatively high in the brain compared to other organs, but these levels are known to be significantly reduced during the development of a brain tumor, especially in glioblastoma multiforme (GBM). However, no known mechanisms may fully explain this abnormality. The objectives of my project were to (1) understand the implications of the demethylation pathway mediated by TET, and (2) gain a deeper insight in the epigenetic make-up of brain tumors. (1) U87 cells were incubated with 5mC, 5hmC, 5-formylcytosine (5fC) or co-incubated of 5hmC with 3,4,5,6-tetrahydro-2’-deoxyuridine (dTHU) over a timeline of 0, 24, 48 and 96 hours. (2) 130 brain tumors (GBM= 79; grade II/III= 51) were obtained directly from surgery and immediately suspended in DNA extraction buffer. Both cell samples and tumor tissues underwent DNA extraction and DNA digestion protocols. The percent per cytosine (%/C) was obtained by quantification of 5mC, 5hmC, 5fC, 5-hydroxymethyluracil (5hmU) and 5formyluracil (5fU) using LC-MS/MS. (1) Cellular incubations showed that it is possible to increase levels of 5hmC in DNA, but also a slight increase in 5mC levels throughout the experiment. 5HmC levels dramatically increased by 1.9-fold after 96h. On the other hand, no increase was observed in 5fC levels. Both 5hmC and 5fC incubations were accompanied by high increases in 5hmU and 5fU levels respectively. The addition of dTHU to the 5hmC incubation decreased 5hmU incorporation by 65%. (2) The average levels of 5mC, 5hmC and 5fC, in brain tumors, were 4.0, 0.15 and 0.021 %/C respectively. 5HmU and 5fU levels were present at comparable levels of 5hmC and 5fC. Levels of 5hmC, 5hmU and 5fU were significantly lower in the DNA of GBM specimens. There was a strong correlation between 5mC with 5hmC and 5fC in GBM, but this was absent in low grade tumors. The presence of 5hmU and 5fU in brain tumor and the increase in their levels during cell incubations indicate a deamination activity in these cancerous cells, which may impinge on the cellular levels of 5hmC, in particular. Furthermore, upon the incubations with 5hmC, downstream levels of 5fC did not increase suggesting a TET malfunction. TET activity is maintained in GBMs, but impaired in low grade tumors due to isocitrate dehydrogenase-1 (IDH1) mutations. Therefore, in brain tumors, a strong deamination activity and TET impairment may lead to epigenetic reduction of 5hmC. / Résumé : 5Mtéthylcytosine (5mC) est une marque épigénétique qui peut être oxydée en 5-hydroxyméthylcytosine (5hmC) par ten-eleven translocation (TET) oxygénases. Ceci amène à l’étape initiale de la déméthylation de 5mC. Le niveau de 5hmC est plus élevé dans le cerveau par rapport aux autres organes. Par contre, ce niveau a une réduction marquante au cours du développement d’une tumeur cérébrale, notamment le glioblastome multiforme (GBM). Toutefois, il n’y a aucun mécanisme connu pour expliquer cette anomalie. Les objectifs de ce projet étaient de (1) discerner l’implication de la voie de déméthylation obtenu par médiation de TET et (2) d’avoir une compréhension plus profonde de la constitution épigénétique des tumeurs cérébraux. (1) Des cellules U87 ont été incubées avec 5mC, 5hmC, 5-fomylcytosine (5fC) et une co-incubation de 5hmC avec 3,4,5,6-tétrahydro-2’-déoxyuridine (dTHU). Les échantillons ont été récoltés à 0, 24, 48, 96 heures. (2) 130 tumeurs cérébraux (GBM = 79; grade II/III = 51) étaient obtenus directement de chirurgie et mise en suspension dans un tampon d’extraction d’ADN le jour même. Les échantillons d’U87 et de tissu tumoral ont subi les protocoles d’extraction et de digestion d’ADN. Le pourcentage par cytosine (%/C) était calculé par la quantification de 5mC, 5hmC, 5fC, 5-hydroxyméthyluracile (5hmU) et 5-formyluracile (5fU) en utilisant LC-MS/MS. (1) Les incubations d’U87 ont démontrées la possibilité augmenter les niveaux génomique de 5hmC, mais aussi une légère augmentation des niveaux de 5mC. Les niveaux de 5hmC ont accru de 1.9 fois après 96hrs. Par contre, aucune variation n’a été observée dans les niveaux de 5fC. Les incubations de 5hmC et 5fC ont été accompagnées par une élévation des niveaux de 5hmU et 5fU respectivement. L’addition de dTHU avec 5hmC avait diminué l’incorporation de 5hmU par 65%. (2) Dans les tumeurs cérébraux, les niveaux moyens de 5mC, 5hmC et 5fC étaient de 4.0, 0.15 et 0.021%/C respectivement. Les quantités de 5hmU et 5fU étaient grandement plus faible dans le GBMs que dans les tumeurs de bas grades. La présence de 5hmU et 5fU dans les tumeurs et leur augmentation durant l’incubation des U87 indiquent une activité de désamination, qui peut, en particulier, entraver les niveaux de 5hmC. En outre, malgré l’incubation avec 5mC, les niveaux de 5hmC et 5fC n’ont pas augmentés suggérant un dysfonctionnement de TET. L’activité de TET est maintenue dans GBM, mais altérée dans les tumeurs de bas grades à cause de mutation isocitrate dehydrogenase-1 (IDH1). Par conséquent, la forte activité de désamination et la déficience en TET peuvent conduire à une réduction épigénétique.

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