<p>S. 1-96: sammanfattning, s. 97-134: 5 uppsatser</p> / digitalisering@umu

G protein-coupled receptors (GPCRs) are composed of seven transmembrane domains and play an essential role in regulating physiological functions and mediating responses to environmental stimuli, biogenic amines, neurotransmitters, peptides, lipids, and hormones. The calcitonin-like diuretic hormone 31 (DH31) is known to elicit natriuresis from the Malpighian tubules (MTs) of mosquitoes Anopheles gambiae and Aedes aegypti upon blood feeding. However, the contribution of DH31 cognate receptor, calcitonin receptor 1 (GPRCAL1), has not been evaluated with respect to postprandial fluid regulation or myostimulatory activity in blood feeding insects. Thus, this dissertation has investigated potential roles of AedaeGPRCAL1 in the regulation of fluid homeostasis and hindgut muscle contraction in female A. aegypti mosquito. The full length cDNA encoding AedaeGPRCAL1 was cloned and sequenced. The receptor expression in the MTs and hindgut from female mosquito was analyzed by western blot and immunohistochemistry using anti-AedaeGPRCAL1 affinity purified antibodies, and subsequently its role in fluid transport and hindgut contraction was evaluated by RNA interference (RNAi). The mosquitoes that underwent knock-down of the AedaeGPRcal1 exhibited up to 57% lower rate of MT fluid secretion in presence of Aedae-DH31 in the in vitro assay and a ~30% reduction in the fluid excreted from live females upon blood feeding. The receptor was immunolocalized in principal cells, predominantly towards the distal end of MTs. Analyses of receptor signal probability indicate the receptor is expressed in a gradient-like fashion along the length of the MTs. A striking discovery was the fact that not all principal cells express the receptor, contrary to previous belief. Immunolocalization revealed the AedaeGPRCAL1 is expressed in hindgut circular and longitudinal muscles. The application of DH31 increased the frequency of hindgut contractions in all female mosquitoes, those injected with AedaeGPRcal1 dsRNA and controls, as compared to their basal contraction rate, but the percent change in frequency of hindgut contraction from AedaeGPRcal1 knock-down females was about 2-fold lower than the controls after application of Aedae-DH31. To my knowledge, this is first evidence of RNAi-induced phenotypes in any invertebrate that allowed the quantification of the contribution of single family B GPCR to fluid loss and muscle contractility.

Made available in DSpace on 2014-07-29T16:11:53Z (GMT). No. of bitstreams: 1 DissertacaoLiuba_Final_Corrigida_2009.pdf: 1243448 bytes, checksum: a9a3811f0c73f5c17c672063bf78489f (MD5) Previous issue date: 2009-06-16 / The Rudgea viburnoides (Cham.) Benth. (Rubiaceae), popularly known as "congonha, congonha-de-Bugre or Bugre" is used in folk medicine as diuretic, hypotensive, anti-rheumatic and depurative of blood (leaves tea). This species is being traded as "porangaba" and used in weight loss programs. This work aimed to study the acute toxicity, the diuretic and antioxidant activities of R. viburnoides. The test for acute toxicity in single dose was performed with the ethanolic crude extract, administered orally, at concentrations of 2000mg/kg and 5000mg/kg in mice and rats of both sexes by the testo of Class.To test for diuretic activity in rats, it was used the crude ethanolic extract of the leaves at concentrations of 40mg/kg, 80mg/kg and 160mg/kg, and furosemide 20mg/kg as positive control. The determination of total phenols of the crude extract and fractions (hexane, dichloromethane, ethyl acetate and methanol/water) was performed by the method of Butler and Hargerman and the evaluation of antioxidant activity performed by the method of 2,2-diphenyl-1- picryl -hidrazil (DPPH). There was no acute toxicity of crude ethanolic extract of the leaves and it was classified as non toxic.The results has shown that the ethanolic extract presented dependent dose diuretic effect higher than furosemide, used as positive control. The crude ethanolic extract showed 0.84% of total phenols, the fraction hexanic 0.44%, the dichloromethane fraction 1.91%, the fraction of ethyl acetate fraction 15.43% and the fraction methanol/water 7.92%. The ethyl acetate fraction showed better antioxidant action. The results obtained so far may justify the popular use of Rudgea viburnoides as diuretic / A Rudgea viburnoides (Cham.) Benth. (Rubiaceae), popularmente conhecida como congonha, congonha-de-bugre ou bugre , é utilizada na medicina popular como diurética, hipotensora, anti-reumática e depurativa do sangue (chá das folhas). Essa espécie vem sendo comercializada como porangaba e utilizada em regimes de emagrecimento. Este trabalho teve como objetivos estudar a toxicidade aguda e as atividades diurética e antioxidante da R. viburnoides. O teste de toxicidade aguda, em dose única, foi realizado com o extrato etanólico bruto, administrado por via oral, nas concentrações de 2000 mg/kg e 5000 mg/kg em camundongos e ratos, de ambos os sexos pelo teste de Classe. Para o teste de atividade diurética, em ratas, utilizou-se o extrato etanólico bruto das folhas nas concentrações de 40 mg/kg, 80 mg/kg e 160 mg/kg e a furosemida 20 mg/kg como controle positivo. O doseamento dos fenóis totais do extrato bruto e das frações (hexânico, diclorometano, acetato de etila e metanol/água) foi realizado pelo método de Hagerman e Butler e a avaliação da atividade antioxidante realizada pelo método do 2,2-difenil-1-picril-hidrazil (DPPH). Os resultados possibitaram concluir que o extrato etanólico bruto das folhas não provocou toxicidade aguda nas doses testadas, podendo ser classificado como atóxico; que o extrato etanólico apresentou efeito diurético dose dependente e superior à furosemida utilizada como controle positivo. O extrato etanólico bruto apresentou 0,84% de fenóis totais, a fração hexânica 0,44%, a fração diclorometano 1,91%, a fração acetato de etila 15,43% e a fração metanol/água 7,92%. A fração acetato de etila apresentou melhor ação antioxidante. Os resultados obtidos até o momento podem justificar a utilização popular da Rudgea viburnoides como diurética

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