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Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-03-16T21:37:33Z No. of bitstreams: 2 Dissertação - Soraia Santana de Moura - 2012.pdf: 3848911 bytes, checksum: 6c2c1f811c32328fc8cdb22d9edaf90a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-20T13:38:54Z (GMT) No. of bitstreams: 2 Dissertação - Soraia Santana de Moura - 2012.pdf: 3848911 bytes, checksum: 6c2c1f811c32328fc8cdb22d9edaf90a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-20T13:38:54Z (GMT). No. of bitstreams: 2 Dissertação - Soraia Santana de Moura - 2012.pdf: 3848911 bytes, checksum: 6c2c1f811c32328fc8cdb22d9edaf90a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-11-28 / Fundação de Apoio à Pesquisa - FUNAPE / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Financiadora de Estudos e Projetos- Finep / Cardiovascular diseases are among the diseases of higher mortality rates in Brazil and worldwide . In contrast, it is reducing deaths of cardiovascular diseases due, in large part, to the production of drugs that can control their risk factors, but the wide reporting of side effects and contraindications persist and interfere negatively in the context of mortality these diseases. In this point, it is necessary to develop new drugs more effective and safer. The process of development of new drug requires numerous preclinical studies that generate information regarding safety profile in vivo determinants for making the decision to start clinical trials. Although it is a conventional practice, the use of animals in scientific research should happen consciously, always considering the ethical issues of animal experimentation. In this study, we investigated the basal cytotoxicity against 3T3 cells of seven pyrazole compounds (LQFM011, LQFM012, LQFM020, LQFM021, LQFM022, and LQFM023 LQFM024) and classified then in GHS system. Whereas the compound LQFM021 proved to be the most effective in the tests performed in parallel pharmacodynamic study, we investigated whether acute oral toxicity, subacute and mutagenicity of this compound. The results showed that the compounds have low cytotoxicity profile in basal cell line (3T3). The estimated LD50 values for compounds LQFM011, LQFM012, LQFM020, LQFM021, LQFM022, and LQFM023 LQFM024 were 548, 551, 568, 533, 457, 482, 565 mg / kg, respectively. The compounds LQFM020, LQFM023 LQFM024 were classified in category 5 GHS system and LQFM021 was classified in category 4. The subacute toxicological research for 28 days LQFM021 the compound showed that this compound did not affect the metabolic, hematological and biochemical animal parameters in any of the doses of exposure However, the histopathology indicated hepatotoxic and nephrotoxic potential of this compound and interference in the process of hematopoiesis, but did not indicate mutagenic potential. . Given the above, we conside / As doenças cardiovasculares estão entre as doenças de maior letalidade no Brasil e no mundo. Em contrapartida, vê-se um quadro redução das mortes atribuídas às enfermidades cardiovasculares devido, em grande parte, à produção de medicamentos capazes de controlar seus fatores de risco, porém, o vasto relato de efeitos colaterais e contraindicações persistem e interferem negativamente no quadro de morbimortalidade dessas doenças. Neste sentindo, é imprescindível buscar novos fármacos mais efetivos e seguros. O processo de desenvolvimento de um novo fármaco exige numerosos estudos pré-clínicos que geram informações quanto ao seu perfil de segurança in vivo, determinantes para a tomada de decisão de se iniciar os estudos clínicos. Embora seja uma prática convencional, o uso de animais em pesquisas científicas deve ocorrer de forma consciente, considerando sempre as questões éticas de experimentação animal. Neste trabalho, investigou-se a citotoxicidade basal frente as células 3T3 de sete compostos pirazolínicos (LQFM011, LQFM012, LQFM020, LQFM021, LQFM022, LQFM023 e LQFM024) e suas classificações no sistema GHS. Considerando que o composto LQFM021 demonstrou ser o mais eficaz nos ensaios realizados em paralelo de farmacodinâmica, investigou-se toxicidade oral aguda, subaguda e mutagenicidade do composto. Os resultados mostraram que os compostos possuem perfil de baixa citotoxicidade em linhagem basal (3T3). Os valores de DL50 estimados para os compostos LQFM011, LQFM012, LQFM020, LQFM021, LQFM022, LQFM023 e LQFM024 foram 548, 551, 568, 533, 457, 482, 565 mg/kg, respectivamente. Os compostos LQFM020, LQFM023 e LQFM024 foram classificadas na categoria 5 do sistema GHS e o composto LQFM021 foi classificado na categoria 4. A investigação toxicológica subaguda por 28 dias do composto LQFM021 mostrou que este composto não interferiu nos parâmetros metabólico, hematológico e bioquímico dos animais em nenhuma das doses de exposição. No entanto, o estudo histopatológico indicou potencial nefrotóxico e hepatotóxico deste composto e interferência sobre o processo de hematopoiese, entretanto, não apresentou potencial mutagênico. Diante do exposto, consideramos que o composto LQFM021 apresenta um baixo perfil de toxicidade e os estudos de continuidade devem ser encorajados.

Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.