Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

A leucemia mielóide crônica (LMC) é caracterizada pela translocação (9;22) que dá origem ao gene quimérico BCR-ABL. Este gene codifica uma proteína com atividade tirosina quinase, p210, constitutivamente ativa. O três mecanismos envolvidos na patogênese da LMC são o aumento da proliferação celular, alteração da adesão celular ao estroma e matriz medular e inibição da apoptose. A introdução do mesilato de imatinibe (MI), um inibidor de tirosina quinase, revolucionou o tratamento da LMC levando pacientes em fase crônica a remissões duráveis, porém uma parcela destes não responde ou perde a resposta ao longo do tratamento. Os mecanismos de resistência ao MI podem ser classificados como independentes de BCR-ABL (a1- glicoproteína ácida e genes de resistência a múltiplas drogas) ou dependentes de BCR-ABL (superexpressão de BCR-ABL e mutações do domínio quinase do gene ABL). Objetivo: avaliar a presença de mutações no domínio quinase do gene ABL e a expressão dos genes de resistência a múltiplas drogas MDR1 e BCRP em amostras pré-tratamento com MI, acompanhar estes pacientes mensalmente através da quantificação de transcritos BCR-ABL e quando ocorrer resistência reavaliar a presença de mutações do domínio quinase do ABL e a expressão dos genes de resistência a múltiplas drogas. Material e Métodos: Foram avaliados 61 pacientes com LMC em fase crônica. A pesquisa de mutações do domínio quinase foi realizada pela técnica de seqüenciamento direto e a expressão relativa dos genes de resistência a múltiplas drogas foi avaliada por PCR em tempo real. A quantificação absoluta do número de transcritos BCR-ABL foi realizada pela técnica de PCR em tempo real utilizando-se o sistema Taqman de sondas de hibridização. Resultados: Nas amostras pré-tratamento dos 61 pacientes estudados não foram detectadas mutações. Quando relacionamos o aumento da expressão dos genes MDR1 e BCRP à resposta citogenética completa aos 12 meses de tratamento não houve diferença estatística significativa (p>0,05). Quanto ao número de transcritos BCR-ABL, observamos que os pacientes que apresentaram menos de 1% pela escala internacional aos 3 meses de tratamento atingiram a RMM em período menor (7 meses) do que os que apresentaram mais de 1% (12 meses) com diferença estatística significativa (p = 0,03). Conclusões: As mutações do domínio quinase do gene BCR-ABL nas amostras pré-tratamento não foram detectadas ou pela sensibilidade da técnica de seqüenciamento direto (10%) ou porque tais mutações são mais freqüentes nas fases acelerada e blástica. A expressão dos genes de resistência a múltiplas drogas (MDR1) e BCRP) em pacientes com LMC-FC ao diagnóstico não apresentou correlação com o aparecimento de resistência secundária ao MI. Além disso a quantificação mensal dos transcritos BCR-ABL aos 3 meses pode ser considerada um marcador com valor prognóstico. / Chronic myeloid leukemia is characterized by t(9;22) translocation. The chimeric gene BCR-ABL encodes a p210BCRABL protein with constitutive tyrosine kinase activity which is directly related to CML pathogenesis. The imatinib mesylate, a tyrosine kinase inhibitor, is the first-choice treatment for patients in chronic phase but some patients show primary resistance or relapse after initial response. The mechanisms of resistance to the imatinib mesylate treatment are BCR-ABL dependent (amplification of BCR-ABL and mutation of kinase domain of BCR-ABL) or independent of BCR-ABL (1-acid glycoprotein and expression of multidrug resistance genes). Objective: The objective of this work was to evaluate the mechanisms of resistance (kinase domain mutation and MDR1 and BCRP genes expression) to imatinib mesylate in pretreatment samples, quantify of BCR-ABL transcript on a monthly follow up plan, and to re-evaluate the mechanisms of resistance in the absence or loss of treatment response. Patients and Methods: We have evaluated 61 pretreatment samples derived from chronic phase CML patients. The number of BCR-ABL transcripts was quantified by RTQ-PCR with taqman probes and MDR1 and BCRP expression were evaluated by RTQ-PCR with Syber Green. Mutations within the BCR-ABL kinase domain were screened by direct sequencing and we also have screened the T315I mutation in pretreatment samples by allele-specific PCR. Results:We detected no mutations in the 61 pretreatment samples. The correlation analysis between the expression of MDR1/BCRP genes and the cytogenetic response at 12 months of treatment revealed no significant statistical difference (p = > 0.05). The results of BCR-ABL quantification in the follow up of our cohort indicated that patients who had transcripts <1% by the international scale at 3 months of therapy are more likely to achieve rapid MMR (median of 7 months) than those who had >1% (median of 12 months) (p = 0,03). Conclusions: As expected, the kinase domain mutations of BCR-ABL in pretreatment samples of CML chronic phase patients are not detectable by direct sequencing because of the sensitivity of the assay (10%) and also because these mutations are more common in accelerated phase and blast crisis. About the expression of multidrug resistance genes MDR1 and BCRP, they showed no correlation with secondary resistance to imatinib mesylate. And finally the number of BCR-ABL transcripts at 3 months of treatment can be considered a marker with prognostic value.

Compte tenu des changements climatiques annoncés, les végétaux risquent d'être confrontés, dans un proche avenir, à des épisodes de sécheresse sévères de plus en plus fréquents. Afin de prédire l'impact de telles contraintes sur la productivité et le rendement des plantes cultivées, il convient d'étudier l'effet du manque d'eau sur l'activité photosynthétique, le transport et l'accumulation des sucres. Dans un premier temps, la croissance, le développement, la biomasse et la photosynthèse de huit écotypes d'Arabidopsis thaliana ont été caractérisés. Trois de ces écotypes (Col-0, Mt-0 et Shahdara) ont ensuite été soumis à une période de déficit hydrique. Une chute du contenu relatif en eau des rosettes (RWC), de la conductance stomatique (gs) et de l'assimilation nette (AN) a été observée précocement chez Mt-0, écotype initialement caractérisé par une très faible efficience de l'utilisation de l'eau (AN/E). En réponse au stress, l'analyse des courbes AN/Ci de chaque écotype montrait que la limitation stomatique s'accompagne rapidement de limitations métaboliques (baisse de la Vcmax). En fin de stress hydrique, une diminution des réserves en amidon a conduit dans tous les cas à une accumulation de saccharose dans les feuilles. Les expressions des gènes codant les principaux transporteurs foliaires de sucres (polyols, hexoses et saccharose) étaient quant à eux différemment affectés par le manque d'eau. Shahdara, qui est parvenu à conserver un RWC relativement élevé, est l'écotype qui a le mieux toléré la contrainte hydrique. À l'opposé, Mt-0 présentait, en fin de stress, une inhibition de l'AN couplée à des altérations majeures et irréversibles des photosystèmes II (chutes de qP et de Fv/Fm). / Considering the predicted climate changes, plants are likely to face, in the future, severe and frequent droughts. In order to evaluate the impact of such stress on productivity and crop yield, the effect of water shortage on photosynthetic activity, sugar transport and accumulation was investigated. Firstly, growth, development, biomass and photosynthesis of eight Arabidopsis thaliana ecotypes have been characterised. Secondly, three of them (Col-0, Mt-0 and Shahdara) were subjected to drought stress. The relative water content (RWC), the stomatal conductance (gs) and the net assimilation (AN) decreased early in rosette leaves of Mt-0 which was initially characterized by a very low water use efficiency (AN/E). In response to drought, AN/Ci curves analysis for each ecotype showed that stomatal limitation was quickly related with metabolic limitations (lower Vcmax). At the end of the stress, reduced starch content always led to a sucrose accumulation in leaves. The expression of genes encoding the main leaf sugar carriers (polyols, hexoses and sucrose) was differently impaired by the water shortage. Shahdara, that managed to maintain a relatively high RWC, was the most tolerant ecotype to water stress. In contrast, at the end of the stress, Mt-0 exhibited an AN inhibition together with significant and irreversible photosystem II alterations (drop of both qP and Fv/Fm).

La prédiction des paramètres pharmacocinétiques/toxicocinétiques (PK/TK), tels que la clairance intrinsèque (CLint) et la clairance hépatique (CLh) des médicaments, demeure un défi majeur en modélisation quantitative. Selon « l’hypothèse du médicament libre », seul le médicament libre peut traverser la membrane plasmique et la CLh de ce médicament est calculée en fonction de sa fraction libre (fup). Néanmoins, la captation hépatique facilitée par l’albumine (ALB) représente clairement une violation de « l’hypothèse du médicament libre ». Cette captation hépatique se base sur la possibilité que le complexe ALB-médicament puisse assurer un apport supplémentaire en médicament aux hépatocytes. Ainsi, cela pourrait expliquer en grande partie les sous-prédictions observées de CLh. Par ailleurs, certains médicaments peuvent se lier fortement à plusieurs protéines plasmatiques telles que l’ALB et l’alpha-1-glycoprotéine acide (AGP). Ainsi, la forte liaison d’un même médicament à l’ALB, à l’AGP, ou aux deux, pourrait avoir des répercussions bien distinctes sur la prédiction de ces paramètres PK/TK. Cependant, aucune étude n’a été faite pour simuler la différence entre leurs effets. L’objectif principal de cette thèse est donc d’évaluer (avec plus d’exactitude et de précision), pour une série de médicaments, en condition in vivo (ou in situ), ces répercussions en présence des deux protéines plasmatiques, conjointement ou séparément. En outre, il est indispensable de vérifier si une approche générique en modélisation peut être appliquée. Cette thèse est répartie en trois objectifs spécifiques. Le premier est de proposer un arbre décisionnel pour faciliter la sélection des approches prédictives appropriées de CLhin vivo pour des médicaments ayant des caractéristiques différentes. Le second est d’évaluer les répercussions de fortes liaisons aux deux protéines plasmatiques ALB et AGP sur la CLh de deux xénobiotiques choisis (perampanel (PER) et fluoxétine (FLU)) ; ces médicaments ont de fortes affinités pour les deux protéines et un métabolisme exclusif (ou prédominant) dans le foie. Et, le dernier est de développer et valider un nouveau modèle prédictif de CLh pour les xénobiotiques ayant le potentiel de se lier fortement dans le plasma, à l’ALB ainsi qu’à l’AGP. Dans un premier temps, des données in vitro rapportées chez l’humain ont été colligées pour 19 médicaments (substrats des transporteurs OAT2 et OATP1B1), et ont été ensuite utilisées dans six modèles d’extrapolation in vitro-in vivo (IVIVE) pour prédire lesdits paramètres. Après une comparaison statistique, les résultats ont montré que l’approche 2 (c’est-à-dire « fup-adjusted model ») qui se base sur la captation hépatique facilitée par l’ALB, avait la meilleure performance prédictive. Cependant, l’approche 5 (c’est-à-dire « Extended Clearance Model ») qui se base sur le transport facilité, en était une très pertinente à appliquer pour les substrats de transporteurs membranaires. Lesdits substrats seraient potentiellement moins affectés par l’ALB. Ainsi, un arbre décisionnel a été proposé pour choisir rapidement et judicieusement la meilleure approche IVIVE servant à prédire la CLhin vivo pour chaque xénobiotique en présence de l’ALB. Dans un deuxième temps, les médicaments PER et FLU ont été sélectionnés à partir d’une collecte de données (N= 1907 médicaments) en fonction de certains critères (avoir un métabolisme exclusif ou prédominant dans le foie, pas de transport facilité par les transporteurs membranaires, une haute affinité pour les deux protéines ALB et AGP, et un ratio de liaison à l’AGP sur celle à l’ALB proche de l’unité). Cette sélection a été réalisée pour faire des expériences sur des foies isolés et perfusés de rats (IPRL), en présence et en absence des protéines ALB et AGP (c’est-à-dire quatre scénarios IPRL). Les résultats IPRL ont démontré que PER est faiblement à moyennement métabolisé (extraction hépatique= 0,2-0,7), tandis que FLU est fortement métabolisé (extraction hépatique= 0,8-0,99). Le modèle Michaelis-Menten a été ajusté aux cinétiques métaboliques, et différents paramètres Vmax, Km et Km, u ont été obtenus de ce modèle. À de faibles concentrations libres pour les deux médicaments (c’est-à-dire à des concentrations thérapeutiques) et en présence des protéines plasmatiques, les valeurs de CLint non liée ont augmenté pour PER (avec l’ALB et le mélange des deux protéines (MIX)) et FLU (avec l’ALB, l’AGP et le MIX) par rapport à celles obtenues du scénario sans protéine (sauf pour PER avec AGP, lesdites valeurs ont diminué). Par ailleurs, les calculs des ratios CLint (SANS versus AVEC protéine) ont permis d’indiquer l’occurrence d’une facilitation de la captation hépatique de médicaments par l’ALB ou l’AGP. Ces ratios ont aussi permis de vérifier si la cinétique métabolique pour PER et FLU suivait soit « l’hypothèse du médicament libre » soit celle de « la captation hépatique facilitée par les protéines plasmatiques ». Dans un dernier temps, une nouvelle approche prédictive de CLh (approche WO-to-MIX) est développée en se basant sur une nouvelle notion de liaison fractionnelle et en intégrant dans le « fup-adjusted model » de nouveaux paramètres tels que la fraction liée à l’ALB (fB-ALB) et celle liée à l’AGP (fB-AGP) à partir du scénario MIX. Ce modèle est basé sur la captation facilitée par l’ALB. Contrairement à l’approche WO-to-MIX, le « well-stirred model » (ou modèle conventionnel) est basé sur l’hypothèse du médicament libre. Ensuite, les paramètres Vmax et Km obtenus in situ pour PER et FLU lors des expériences IPRL sans protéines, ont été utilisés en combinaison avec le paramètre intrant de la fraction libre ajustée (fup-adjusted) pour le « fup-adjusted model » ou avec la fraction libre (fup) pour le « well-stirred model ». Une comparaison des performances prédictives globales des deux modèles a été faite. Les performances prédictives du nouveau modèle étaient prometteuses, en particulier pour FLU qui montrait le plus haut degré de captation hépatique médiée par l’ALB, par rapport au modèle conventionnel. L’approche WO-to-MIX est une première validation d’un nouveau modèle d’extrapolation proposé pour les médicaments comme FLU qui se lient à l’ALB et à l’AGP. Néanmoins, le modèle conventionnel reste utile à utiliser pour les médicaments comme PER. L’exactitude de prédiction était inférieure pour ce dernier médicament probablement parce que la captation hépatique par l’ALB ne semble pas être maximale, et, par conséquent, l’utilisation de fup-adjusted a surestimé la CLhin vivo. Par conséquent, plus de travail est nécessaire en particulier pour PER. Cette thèse démontre qu’une seule approche générique pour prédire la CLh n’existe pas. Néanmoins, le choix d’une approche IVIVE ayant une performance prédictive satisfaisante est maintenant possible. Les résultats de cette thèse contribuent à : 1) mieux comprendre les répercussions sur les paramètres PK/TK de la forte liaison des médicaments à l’ALB et à l’AGP ; 2) choisir la meilleure approche prédictive de CLh sur la base de l’affinité du xénobiotique (médicament ou contaminant) pour chacune des protéines plasmatiques et des mécanismes impliqués dans le foie ; et 3) prédire la CLh avec précision et exactitude des xénobiotiques qui se lient aux deux protéines plasmatiques. Ces approches IVIVE pour la CLh pourront assurément être intégrées dans des modèles PK/TK à base physiologique pour les xénobiotiques afin d’améliorer la prédiction de leur pharmacocinétique et d’accélérer le processus de développement de médicaments. / The prediction of pharmacokinetic/toxicokinetic (PK/TK) parameters such as intrinsic clearance (CLint) and hepatic clearance (CLh) for highly bound drugs is a major challenge in quantitative modeling. According to the ‘free drug hypothesis’, only the free drug can pass through the plasma membrane and the CLh of this drug is calculated according to its free fraction (fup). Nevertheless, the hepatic uptake facilitated by albumin (ALB) is a violation of the ‘free drug hypothesis’. This facilitated hepatic uptake is based on the possibility that the ALB-drug complex may provide additional drug intake to the hepatocytes. Thus, this could largely explain the underpredictions of CLh. In addition, some drugs can bind extensively in plasma, and to several plasma proteins such as ALB and alpha-1-glycoprotein acid (AGP). Thus, the high binding of the same drug to either ALB or AGP, or to both, could have distinct impacts on the prediction of these PK/TK parameters. However, no study has yet explored how to simulate the difference between these impacts. The main objective of this thesis is therefore to evaluate (with accuracy and precision) for a series of drugs, in the in vivo (or in situ) condition, these impacts in the presence of the two plasma proteins, jointly or separately. Also, it is important to verify if a generic model can be applied. This thesis is divided into three specific objectives. The first is to propose a decision tree to facilitate the selection of appropriate predictive approaches of CLhin vivo for drugs with different characteristics. The second is to assess the impacts of extensive binding to the two plasma proteins ALB and AGP on the CLh of two selected xenobiotics (perampanel (PER) and fluoxetine (FLU)); these drugs have strong affinities to both proteins and an exclusive (or predominant) metabolism in the liver. And the last objective is to develop and validate a new predictive model of CLh for xenobiotics with the potential to bind extensively to ALB as well as to AGP. Firstly, in vitro data obtained in humans were collected for 19 drugs (i.e. substrates of OAT2 and OATP1B1 transporters) and were then used in six in vitro-to-in vivo (IVIVE) extrapolation models to predict these PK/TK parameters. After a statistical comparison, the results showed that the approach 2 (i.e. ‘fup-adjusted model’) that is based on the ALB-facilitated hepatic uptake, had the best predictive performance. However, the approach 5 (i.e. ‘Extended Clearance Model’) that is based on the membrane transporter-mediated uptake, was very relevant to apply for the substrates of membrane transporters. These substrates would potentially be less affected by ALB. Thus, a decision tree has been proposed to quickly and judiciously select the best IVIVE approach to predict CLhin vivo for each xenobiotic in the presence of ALB. Secondly, the PER and FLU drugs were selected from a data collection of 1907 drugs depending on certain criteria (exclusive or predominant metabolism in the liver, no transport facilitated by membrane transporters, high affinity for the two proteins ALB and AGP, and having a binding ratio between AGP and ALB close to the unity). This selection was made to conduct experiments using the isolated and perfused rat liver (IPRL) apparatus, in the presence, and in the absence of the ALB and AGP proteins (i.e. four IPRL scenarios). The IPRL results showed that PER is low to moderately metabolized (hepatic extraction= 0.2-0.7), while FLU is highly metabolized (hepatic extraction= 0.8-0.99). The Michaelis-Menten model was fitted to the obtained metabolic kinetics, and different parameters Vmax, Km and Km, u were obtained from the model. At low free concentrations for both drugs (i.e. therapeutic concentrations) and in the presence of plasma proteins, the values of unbound CLint increased for PER (with ALB and the mixture of the two proteins (MIX)) and FLU (with ALB, AGP and MIX); when compared to those obtained from the protein-free scenario (except for PER with AGP, the unbound CLint values decreased). In addition, the calculations of CLint ratios (WITHOUT versus WITH protein) indicated the occurrence of a hepatic uptake facilitated by ALB or AGP. These ratios also helped in verifying whether the metabolic kinetics for PER and FLU followed either ‘the free drug hypothesis’ or that of ‘plasma protein-facilitated hepatic uptake’. Finally, a new predictive approach of CLh (WO-to-MIX approach) was developed based on a new notion of fractional binding and incorporating new parameters such as the ALB bound fraction (fB-ALB) and the AGP bound fraction (fB-AGP) from the MIX scenario into the ‘fup-adjusted model’. This model is based on the ‘ALB-facilitated hepatic uptake’. Unlike the WO-to-MIX approach, the ‘well-stirred model’ is based on ‘the free drug hypothesis’. Then, the Vmax and Km parameters that were obtained in situ for PER and FLU from the protein-free IPRL experiments, were used in combination with the fup-adjusted input parameter for the ‘fup-adjusted model’ or with the free fraction (fup) for the ‘well-stirred model’. A comparison of the two models’ overall predictive performances was made. The predictive performances of the new model were promising for FLU, which showed the highest degree of ‘ALB-mediated hepatic uptake’, compared to the conventional model. This WO-to-MIX approach is a first validation of a novel extrapolation model suggested for drugs such as FLU that bind to both ALB and AGP. The well-stirred model remains however a useful tool to predict the clearance for drugs such as PER. The prediction accuracy was lower for the latter drug probably because the ALB-mediated hepatic uptake does not seem to be maximal, and, hence, the use of fup-adjusted has overestimated its CLhin vivo. Therefore, more work is needed particularly for PER. This thesis shows that a generic approach to predict the CLh in vivo does not exist. Nevertheless, the choice of an IVIVE approach with satisfactory predictive performances is now possible. The results of this thesis contribute to: 1) better understand the impacts on the PK/TK parameters of extensive drug binding to ALB and AGP; 2) choose the best predictive approach to CLh based on the affinity of xenobiotic (drug or contaminant) to each of the plasma proteins and the mechanisms involved in the liver; and 3) predict accurately and with precision the output CLh of xenobiotics that bind to the two plasma proteins. These IVIVE approaches for CLh can certainly be integrated into physiologically based PK/TK models for xenobiotics to improve the prediction of their pharmacokinetics and to accelerate the drug development process.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

Les dinoflagellés sont des eucaryotes unicellulaires retrouvés dans la plupart des écosystèmes aquatiques du globe. Ces organismes amènent une contribution substantielle à la production primaire des océans, soit en tant que membre du phytoplancton, soit en tant que symbiontes des anthozoaires formant les récifs coralliens. Malheureusement, ce rôle écologique majeur est souvent négligé face à la capacité de certaines espèces de dinoflagellés à former des fleurs d'eau, parfois d'étendue et de durée spectaculaires. Ces floraisons d'algues, communément appelées "marées rouges", peuvent avoir de graves conséquences sur les écosystèmes côtiers, sur les industries de la pêche et du tourisme, ainsi que sur la santé humaine. Un des facteurs souvent corrélé avec la formation des fleurs d'eau est une augmentation dans la concentration de nutriments, notamment l’azote et le phosphore. Le nitrate est un des composants principaux retrouvés dans les eaux de ruissellement agricoles, mais également la forme d'azote bioaccessible la plus abondante dans les écosystèmes marins. Ainsi, l'agriculture humaine a contribué à magnifier significativement les problèmes associés aux marées rouges au niveau mondial. Cependant, la pollution ne peut pas expliquer à elle seule la formation et la persistance des fleurs d'eau, qui impliquent plusieurs facteurs biotiques et abiotiques. Il est particulièrement difficile d'évaluer l'importance relative qu'ont les ajouts de nitrate par rapport à ces autres facteurs, parce que le métabolisme du nitrate chez les dinoflagellés est largement méconnu. Le but principal de cette thèse vise à remédier à cette lacune. J'ai choisi Lingulodinium polyedrum comme modèle pour l'étude du métabolisme du nitrate, parce que ce dinoflagellé est facilement cultivable en laboratoire et qu'une étude transcriptomique a récemment fourni une liste de gènes pratiquement complète pour cette espèce. Il est également intéressant que certaines composantes moléculaires de la voie du nitrate chez cet organisme soient sous contrôle circadien. Ainsi, dans ce projet, j'ai utilisé des analyses physiologiques, biochimiques, transcriptomiques et bioinformatiques pour enrichir nos connaissances sur le métabolisme du nitrate des dinoflagellés et nous permettre de mieux apprécier le rôle de l'horloge circadienne dans la régulation de cette importante voie métabolique primaire. Je me suis tout d'abord penché sur les cas particuliers où des floraisons de dinoflagellés sont observées dans des conditions de carence en azote. Cette idée peut sembler contreintuitive, parce que l'ajout de nitrate plutôt que son épuisement dans le milieu est généralement associé aux floraisons d'algues. Cependant, j’ai découvert que lorsque du nitrate était ajouté à des cultures initialement carencées ou enrichies en azote, celles qui s'étaient acclimatées au stress d'azote arrivaient à survivre près de deux mois à haute densité cellulaire, alors que les cellules qui n'étaient pas acclimatées mourraient après deux semaines. En condition de carence d'azote sévère, les cellules arrivaient à survivre un peu plus de deux semaines et ce, en arrêtant leur cycle cellulaire et en diminuant leur activité photosynthétique. L’incapacité pour ces cellules carencées à synthétiser de nouveaux acides aminés dans un contexte où la photosynthèse était toujours active a mené à l’accumulation de carbone réduit sous forme de granules d’amidon et corps lipidiques. Curieusement, ces deux réserves de carbone se trouvaient à des pôles opposés de la cellule, suggérant un rôle fonctionnel à cette polarisation. La deuxième contribution de ma thèse fut d’identifier et de caractériser les premiers transporteurs de nitrate chez les dinoflagellés. J'ai découvert que Lingulodinium ne possédait que très peu de transporteurs comparativement à ce qui est observé chez les plantes et j'ai suggéré que seuls les membres de la famille des transporteurs de nitrate de haute affinité 2 (NRT2) étaient réellement impliqués dans le transport du nitrate. Le principal transporteur chez Lingulodinium était exprimé constitutivement, suggérant que l’acquisition du nitrate chez ce dinoflagellé se fondait majoritairement sur un système constitutif plutôt qu’inductible. Enfin, j'ai démontré que l'acquisition du nitrate chez Lingulodinium était régulée par la lumière et non par l'horloge circadienne, tel qu'il avait été proposé dans une étude antérieure. Finalement, j’ai utilisé une approche RNA-seq pour vérifier si certains transcrits de composantes impliquées dans le métabolisme du nitrate de Lingulodinium étaient sous contrôle circadien. Non seulement ai-je découvert qu’il n’y avait aucune variation journalière dans les niveaux des transcrits impliqués dans le métabolisme du nitrate, j’ai aussi constaté qu’il n’y avait aucune variation journalière pour n’importe quel ARN du transcriptome de Lingulodinium. Cette découverte a démontré que l’horloge de ce dinoflagellé n'avait pas besoin de transcription rythmique pour générer des rythmes physiologiques comme observé chez les autres eukaryotes. / Dinoflagellates are unicellular eukaryotes found in most aquatic ecosystems of the world. They are major contributors to carbon fixation in the oceans, either as free-living phytoplankton or as symbionts to corals. Dinoflagellates are also infamous because some species can form spectacular blooms called red tides, which can cause serious damage to ecosystems, human health, fisheries and tourism. One of the factors often correlated with algal blooms are increases in nutrients, particularly nitrogen and phosphorus. Nitrate is one of the main components of agricultural runoffs, but also the most abundant bioavailable form of nitrogen in marine environments. Thus, agricultural activities have globally contributed to the magnification of the problems associated with red tides. However, bloom formation and persistence cannot be ascribed to human pollution alone, because other biotic and abiotic factors are at play. Particularly, it is difficult to assess the relative importance of nitrate addition over these other factors, because nitrate metabolism in dinoflagellate is mostly unknown. Filling part of this gap was the main goal of this thesis. I selected Lingulodinium polyedrum as a model for studying nitrate metabolism, because this dinoflagellate can easily be cultured in the lab and a recent transcriptomic survey has provided an almost complete gene catalogue for this species. It is also interesting that some molecular components of the nitrate pathway in this organism have been reported to be under circadian control. Thus, in this project, I used physiological, biochemical, transcriptomic and bioinformatic approaches to enrich our understanding of dinoflagellate nitrate metabolism and to increase our appreciation of the role of the circadian clock in regulating this important primary metabolic pathway. I first studied the particular case of dinoflagellate blooms that occur and persist in conditions of nitrogen depletion. This idea may seems counterintuitive, because nitrogen addition rather than depletion, is generally associated with algal blooms. However, I discovered that when nitrate was added to nitrogen-deficient or nitrogen-sufficient cultures, those that had been acclimated to nitrogen stress were able to survive for about two months at high cell densities, while non-acclimated cells died after two weeks. In conditions of severe nitrogen limitation, cells could survive a little bit more than two weeks by arresting cell division and reducing photosynthetic rates. The incapacity to synthesize new amino acids for these deprived cells in a context of on-going photosynthesis led to the accumulation of reduced carbon in the form of starch granules and lipid bodies. Interestingly, both of these carbon storage compounds were polarized in Lingulodinium cells, suggesting a functional role. The second contribution of my thesis was to identify and characterize the first nitrate transporters in dinoflagellates. I found that in contrast to plants, Lingulodinium had a reduced suite of nitrate transporters and only members of the high-affinity nitrate transporter 2 (NRT2) family were predicted to be functionally relevant in the transport of nitrate. The main transporter was constitutively expressed, which suggested that nitrate uptake in Lingulodinium was mostly a constitutive process rather than an inducible one. I also discovered that nitrate uptake in this organism was light-dependent and not a circadian-regulated process, as previously suggested. Finally, I used RNA-seq to verify if any transcripts involved in the nitrate metabolism of Lingulodinium were under circadian control. Not only did I discovered that there were no daily variations in the level of transcripts involved in nitrate metabolism, but also that there were no changes for any transcripts present in the whole transcriptome of Lingulodinium. This discovery showed that the circadian timer in this species did not require rhythmic transcription to generate biological rhythms, as observed in other eukaryotes.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.