The increased risk of atherothrombosis in chronic kidney disease (CKD) has been under extensive examination for decades now. However, a treatment tailored for CKD patients is yet to be found. Current management plans can only tackle comorbidities and mostly fail. This thesis study examines the current literature related to CKD and thrombosis. The aim is to find a target suitable for therapeutic exploration. Normalizing the risk of thrombosis in CKD patients could curb a huge margin of their morbidity and mortality. In recent years, molecular biology studies attributed the extreme thrombogenicity in CKD to the retained uremic toxins. Indolic compounds are uremic toxins with a well described point of thrombotic activation. Of them, indoxyl sulfate is to be highlighted since it was shown to that it is one of the strongest pro-thrombotic uremic toxin. It is possible to therapeutically target this CKD specific cause of hyperthrombogenicity. Further research is very much needed in this area.

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Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-17T20:42:43Z No. of bitstreams: 1 Gabriela Gomes Cardoso Rodrigues.pdf: 623885 bytes, checksum: 3da3d1230fd77beffc9c12f00be889bb (MD5) / Made available in DSpace on 2016-05-17T20:42:43Z (GMT). No. of bitstreams: 1 Gabriela Gomes Cardoso Rodrigues.pdf: 623885 bytes, checksum: 3da3d1230fd77beffc9c12f00be889bb (MD5) Previous issue date: 2015-02-04 / Chronic kidney disease (CKD) is characterized by progressive and irreversible loss of renal function and often progresses with a muscular weakness, whose set of signs and symptoms is generally referred to as uremic myopathy. Possible risk factors for the uremic myopathy are the uremic toxins. Among uremic toxins, indoxyl sulfate (IS) is a derivative of tryptophan metabolism by intestinal bacteria. Because skeletal muscle tissue undergo constant remodeling due differentiation of myoblasts in myotubes, it is possible that uremic toxins have a deleterious effect to influence this process, exacerbating the uremic myopathy. Low level laser therapy (LLLT) is regarded as a growth promoter feature widely used in the treatment of chronic diseases and has shown positive effects on the modulation of skeletal muscle repair process and also in the process of inflammation. However, in the context of CKD, the LLLT has not yet been explored. The aim of this study was to evaluate the effects of the IS on cell viability and on oxidative stress on cellular differentiation in cultured C2C12 myoblasts. In addition, to verify the action of the LLLT as a protective alternative to the cells. The C2C12 myoblasts were cultured in DMEM culture medium containing 10% fetal bovine serum and were induced to differentiation process by adding 2% horse serum. Three different IS concentrations were used to mimic the plasma concentrations of normal individual, CKD patients with moderate uremia and CKD patients with advanced uremia (0.6 mg/l and 53 mg/l and 236 mg/l, respectively), at different times of incubation (24 h, 48 h and 72 h). Subsequently, the cells were subjected to treatment with LLLT GaAlAs 780 nm (output power 10 mW, 20 seconds application time and energy density of 0.5 J / cm2). In terms of analysis, we used MTT method to assess the viability of the cells, flow cytometry to assess the viability/cell death and oxidative stress, nitrite dosing to evaluate nitric oxide production and real-time PCR to analyze IL-6, myogenin and MyoD expression (inflammation and cell differentiation markers). The results demonstrate that the IS at the maximum concentration was toxic to C2C12 cells, because it significantly decreased cell viability by MTT and by flow cytometry and by increasing the percentage of necrosis. This effect was present throughout the three incubation periods. With respect to oxidative stress, was not any conclusion, probably by the time the samples, but do not rule out the possibility of IS induce this type of stress. Although the IS has induced death to C2C12 cells, the remaining had no change in cell differentiation markers. Treatment with BPL the IS sensitized cells, reducing cell viability. We conclude that the IS acts directly on C2C12 myoblasts with toxic effect and may be the one factor responsible for uremic myopathy. Treatment with LLLT was not effective in protecting the cells. / A doença renal crônica (DRC) é caracterizada pela perda progressiva e irreversível da função renal e que frequentemente cursa com um quadro de fraqueza muscular, cujo conjunto de sinais e sintomas é globalmente designado como miopatia urêmica. Possíveis fatores predisponentes para a miopatia urêmica são as toxinas urêmicas. Dentre as toxinas urêmicas, o indoxil sulfato (IS) é uma derivada do metabolismo do triptofano presente em bactérias intestinais. Devido ao fato do tecido muscular esquelético sofrer constante remodelação graças à diferenciação de mioblastos em miotubos, é possível que toxinas urêmicas tenham um efeito deletério por influenciar este processo, agravando a miopatia urêmica. A terapia a laser de baixa potência (LBP) é considerada como um recurso bioestimulante amplamente utilizado no tratamento de doenças crônicas e tem demonstrado efeitos positivos sobre a modulação do processo de reparo muscular esquelético e também no processo da inflamação. Entretanto, no contexto de DRC, o LBP não foi ainda explorado. O objetivo do presente estudo foi avaliar dos efeitos do IS sobre a viabilidade celular, sobre o estresse oxidativo e sobre a diferenciação celular em cultura de mioblastos C2C12. Além disso, verificar a ação do LBP como forma de proteção às células. Os mioblastos C2C12 foram cultivados em meio de cultura de DMEM, contendo 10% de soro fetal bovino e foram induzidos ao processo de diferenciação por meio da adição de 2% soro de cavalo. Três diferentes concentrações de IS foram usadas para mimetizar as concentrações plasmáticas de indivíduo normal, paciente DRC com uremia moderada e paciente DRC com uremia avançada (0,6 mg/l; 53 mg/l e 236 mg/l, respectivamente), em diferentes períodos de incubação (24 h, 48 h e 72 h). Posteriormente, as células foram submetidas ao tratamento com laser de baixa potência AsGaAl 780 nm (potência de saída de 10 mW, tempo de aplicação de 20 segundos e densidade de energia de 0,5 J/cm2). Como análise, foi utilizado o método MTT para acessar a viabilidade das células, citometria de fluxo para avaliar a viabilidade/mortalidade das células, bem como o estresse oxidativo, dosagem de nitrito para avaliar a produção de óxido nítrico e PCR em tempo real para analisar a expressão de IL-6, miogenina e MyoD (marcadores de inflamação e diferenciação celular). Os resultados demonstram que o IS na concentração máxima foi tóxico para as células C2C12, pois diminuiu significativamente a viabilidade das células, tanto por MTT como por citometria de fluxo, aumentando a porcentagem de necrose. Este efeito foi presente nos três períodos de incubação. Com relação ao estresse oxidativo, não foi possível nenhuma conclusão, provavelmente pelo tempo das amostras , porém não descartamos a possibilidade do IS induzir este tipo de estresse. Embora o IS tenha induzido morte às células C2C12, as remanescentes não tiveram alteração dos marcadores de diferenciação celular. O tratamento com LBP sensibilizou as células ao IS, diminuindo a viabilidade das células. Concluímos que o IS age diretamente sobre mioblastos C2C12 com efeito tóxico, podendo ser um dos responsáveis pela miopatia urêmica. O tratamento com LBP não foi eficiente na indução de proteção às células.

La synthèse tout-en-un de 4-quinolones et d'indoxyles a été réalisée par couplage de Sonogashira carbonylant suivi d'une cyclisation. Une étude en catalyse homogène a révélé que les catalyseurs présents contrôlaient la sélectivité vers l'un ou l'autre des composés. Si la première étape est pallado-catalysée, l'étape de cyclisation est catalysée par des nucléophiles organiques. Ainsi, des 4-quinolones ont été préparées par multi-catalyse {[Pd]+amine} et des indoxyles par catalyse tandem {[Pd]/phosphine}. Les systèmes catalytiques ont été hétérogénéisés par fonctionnalisation de silices mésoporeuses de type SBA. Deux stratégies ont été utilisées pour contrôler la localisation de la fonctionnalité. Différents complexes de Pd ont été intégrés dans les pores du matériau par greffage post-synthétique ou dans les murs par synthèse directe. Plusieurs amines et une phosphine ont été immobilisées par greffage post-synthétique donnant ainsi des catalyseurs nucléophiles fonctionnalisés dans leurs pores. Les activités catalytiques de ces matériaux ont été évaluées dans la synthèse de la 2-phényl-4-quinolone et du 2-benzylidène-indoxyle. La première a été préparée dans de bons rendements et le système {[Pd]@SBA+amine@SBA} est recyclable sur 3 cycles. Tous les matériaux ont montré une lixiviation du Pd mais leur utilisation permet de diminuer la contamination en Pd du produit final comparé à un complexe homogène. L'indoxyle a été obtenu avec un système semi-hétérogène {[Pd]@SBA/PPh3}, l'utilisation de la phosphine greffée conduisant à la transformation de l'indoxyle vers le 2-benzylindole. La formation d'α-céto-amides en catalyse hétérogène via double carbonylation a aussi été étudiée / The one-pot selective synthesis of 4-quinolones and indoxyls was achieved through a carbonylative Sonogashira coupling followed by cyclization. A study in homogeneous catalysis revealed that the nature of catalysts in presence controlled the selectivity toward each compounds. Whereas the first coupling step is palladium catalyzed, the cyclizations require organic nucleophilic species. Thus, 4-quinolones were obtained by one-pot multi-catalysis {[Pd]+amine} and indoxyls by one-pot tandem catalysis {[Pd]/phosphine}. The catalytic systems were heterogenized by functionalizing mesostructured SBA silicas. Two strategies were employed aiming at a control of the localization of the functionality. Different Pd complexes were integrated in the pores of the material by post-synthesis grafting or incorporated into the walls via direct synthesis. Different amines and a phosphine were immobilized by post-synthesis grafting affording hybrid materials containing amine or phosphine catalysts in their pores. Catalytic activities of those materials were evaluated in the reaction affording 2-phenyl-4-quinolone and 2-benzylidene-indoxyl. The former was obtained in good yields and the heterogeneous catalytic system {[Pd]@SBA+amine@SBA} was recyclable over 3 runs. All hybrid materials showed Pd leaching but their uses still enables to decrease the Pd contamination of the final product compared to homogeneous complexes. The indoxyle compound was obtained in a semi-heterogeneous system {[Pd]@SBA/PPh3}, the use of the grafted phosphine providing transformation of the indoxyle toward 2-benzylindole. Formation of α-keto-amids by heterogeneously catalyzed double carbonylation was also studied