Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T19:24:15Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:15:45Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-21T20:15:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) / Made available in DSpace on 2018-05-21T20:15:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Renata_Zanella.pdf: 575197 bytes, checksum: 59c20b438c7e122081f43156f1b94bf3 (MD5) Previous issue date: 2016-08-26 / Sem bolsa / O sistema calicreínas-cininas (SCC) está envolvido em diversos processos biológicos como modulação de dor, vasodilatação, permeabilidade vascular, edema e inflamação, tendo seus efeitos mediados por dois receptores específicos acoplados a proteína G, o receptor B1 e B2 de cininas. O SCC tem sido recentemente relacionado com a homeostase da glicose e resistência à insulina. O objetivo do presente estudo foi investigar a participação do receptor B1 (B1R) de cininas nos processos metabólicos relacionados à obesidade e ao diabetes mellitus tipo 2 em camundongos obesos. Para isso foram utilizados camundongos deficientes para a leptina e nocautes para o B1R de cininas (obB1). Quando comparados, os animais obB1 mostraram uma redução na ingestão alimentar em todo período avaliado (4° a 26° semana de vida); menor ganho de peso, tanto aos 3 meses (obWT, 63,79 ± 1,59; obB1, 51,22 ± 1,09 g, p < 0,001) quanto aos 6 meses de idade (obWT, 74,61 ± 1,04; obB1, 57,98 ± 1,58 g, p < 0,001); e menor depósito de gordura. No teste de tolerância a glicose, animais obB1 apresentaram maior captação de glicose do que seus controles quando jovens (obWT, 46045 ± 3492; obB1, 30875 ± 2311 (mg/dL).min, p< 0,01). Adicionalmente, foi observado aumento do peso do fígado nestes animais, quando mais velhos (obWT, 3,29 ± 0,24; obB1 4,09 ± 0,06 g, p<0,001). Em conclusão, o silenciamento do gene do B1R oferece uma proteção contra a obesidade com um significativo impacto na homeostase da glicose. Estes resultados sugerem que o SCC pode ser um novo alvo para desenvolvimento de novas estratégias farmacológicas para o tratamento de doenças metabólicas como obesidade e diabetes mellitus tipo 2. / The kallikrein-kinin system (KKS) is involved in many biological processes such as modulating pain, vasodilation, vascular permeability, edema and inflammation, with effects mediated by two specific receptors coupled to G protein, B1 and B2 kinin receptor. The KKS has recently been associated with glucose homeostasis and insulin resistance. The aim of this study was to investigate the participation of B1 receptor (B1R) of kinins in the metabolic processes related to obesity and type 2 diabetes in obese mice. For this we used mice deficient for leptin and knockouts for B1R kinin (obB1). When compared, the animals obB1 showed a reduction in food intake in the whole study period (4 to 26 ° week of life); less weight gain, both at 3 months (obWT, 63.79 ± 1.59; obB1, 51.22 ± 1.09 g, p <0.001) and at 6 months of age (obWT, 74.61 ± 1, 04; obB1, 57.98 ± 1.58 g, p <0.001); and less fat deposit. In the glucose tolerance test, obB1 animals had higher glucose uptake than their controls when young (obWT, 46045 ± 3492; obB1, 30875 ± 2311 (mg / dL) .min, p <0.01). Additionally, there was an increase in liver weight in these animals, while older (obWT, 3.29 ± 0.24, 4.09 ± 0.06 g obB1, p <0.001). In conclusion, the B1R gene silencing offers a protection against obesity have a significant impact on the glucose homeostasis. These results suggest that the KKS may be a new target for the development of new pharmacologic strategies for treating metabolic diseases such as obesity and type 2 diabetes mellitus.

Made available in DSpace on 2015-07-22T20:50:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-28. Added 1 bitstream(s) on 2015-08-11T03:25:41Z : No. of bitstreams: 1 Publico-091.pdf: 651136 bytes, checksum: 35401eae81afb55be34c6f47a332e47e (MD5) / Objetivo: Estudar o efeito da obesidade na regulação do sistema calicreína-cininas por meio da expressão dos receptores B1 e B2 de cininas em humanos e camundongos, e as alterações na síntese e funcionalidade dos receptores em tecidos murinos. Métodos: Foram coletados tecido adiposo branco humano e diferentes tecidos de camundongo. Desses tecidos foi extraído o RNA e analisada a expressão dos receptores de cinina por meio da reação em cadeia da polimerase em tempo real. Tecidos como estômago e aorta de camundongos ob/ob e selvagens foram utilizados para extração de proteínas e estudos fisiológicos. Por Western Blotting estudou-se a quantidade de receptor produzida nos animais. O fundus de estômago e aorta abdominal foram utilizados para se obter registros de contrações isométricas para determinação da potência e eficácia dos agonistas em camundongos obesos e magros. Foram aplicadas doses crescentes cumulativas dos agonistas peptídicos dos receptores B1 e B2, bradicinina e des-Arg9-bradicinina. Resultados: Nos experimentos de PCR em tempo real, a expressão gênica dos receptores B1 e B2 de cininas foi mostrada alterada em alguns tecidos dos animais deficientes na produção do hormônio leptina em relação ao controle. Nos tecidos: adiposo branco, aorta, fígado, hipotálamo e estômago, a expressão do receptor B1 apresentou-se aumentada, porém em tecido cardíaco e tecido adiposo marrom, essa estava diminuída. O receptor B2 teve expressão diminuída em tecido adiposo branco e hipotálamo. Nos demais tecidos estudados não houve alteração da expressão do receptor B2. Em humanos, esses receptores apresentaram-se alterados em indivíduos obesos. O estudo foi realizado em tecido adiposo humano de duas regiões de depósito diferentes, visceral e subcutâneo. Foi observada diferença na expressão do mesmo tecido, porém de regiões distintas. Nos tecidos dos camundongos obesos a resposta aos agonistas dos receptores B1 e B2, bradicinina e des-Arg9-bradicinina, respectivamente, foi mostrada diminuida. Em fundus de estômago foi observada diminuição significativa na resposta ao agonista BK em animais obesos e tratados com dieta hiperlipídica. Tais efeitos podem ser devido às conseqüências do aumento excessivo de peso, como inflamação crônica apresentada nesses animais, ou mesmo devido a diabetes tipo II, a qual consiste em uma patologia diretamente relacionada à obesidade, sugerida neste trabalho como fator capaz de alterar a ação do sistema calicreína-cininas em determinados tecidos. Conclusão: Análises de expressão gênica mostraram que a obesidade afeta os receptores de cinina em diversos tecidos de camundongo, assim como em humanos. Análises fisiológicas funcionais mostraram diminuição na resposta ao agonista de B1 em animais obesos. Os dados deste trabalho sugerem que a obesidade afeta a modulação do sistema calicreína-cininas em modelo murino e humano. Dessa forma, uma possível interação entre obesidade e sistema calicreína-cininas poderia estar envolvida nesta patologia, assim como ser um fator para desenvolvimento a sindrome metabólica. / Objectives: To study the effect of obesity on the kallikrein-kinin system through the expression of receptors B1 and B2 on humans and mice, and alterations in the synthesis and functionality of the receptor in murine tissues. Methods: white human adipose tissue and different kinds of mice tissues were collected. RNA was extracted and the kinin receptors expression analyzed through a real-time polymerase chain reaction. Tissues and organs such as stomach and aorta were used for protein extraction and physiological studies. By Western Blotting, receptor quantitation was studied. Stomach fungus and abdominal aorta were used to register isometric contractions to determine the potency and effectiveness of the agonists on obese and control mice. Increasing accumulating doses of bradykinin and des-Arg9-bradykinin, B2 and B1 receptors agonists respectively, were applied. Results: In the real-time PCR experiments, the gene expression of the B1 and B2 receptors were altered in some tissues of the animals deficient for leptin, when compared to the control. In the white adipose tissue, aorta, liver, hypothalamus and stomach, the B1 receptor expression was increased, but in cardiac tissues and brown adipose tissue, it was decreased. The expression of B2 receptor was decreased in white adipose tissue and hypothalamus. In the other studied tissues, no changes was detected in the B2 receptor expression. In humans, these receptors were altered in obese individuals. The study was performed in human adipose tissue from two different regions of depots, visceral and subcutaneous. There was a tendency of different expression in the same tissue, but from different areas. In tissues from obese mice the response to the B2 and B1 agonists, bradykinin and des-Arg9-bradykinin, respectively, had a decreasing tendency. A significant decrease was observed in stomach fundus in response to the BK agonist. Such effects can be due to the increased weight and its consequences, such as chronic inflammation or diabetes type II, which is a pathology directly related to obesity. Conclusion: expression and functional analysis show that obesity affects kinin receptors in many different mouse tissues as well as in humans. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-22T20:49:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-31 / Objetivo: caracterizar farmacológicamente as respostas à bradicinina e outros agonistas no músculo liso não vascular (fundo do estômago) e vascular (aorta abdominal) isolados de animais nocaute B1 e B2 de cininas. Métodos: Foram isolados fundus de estômago e aorta abdominal de animais normais, nocaute B1 e animais nocaute B2; registros de contrações isométricas foram obtidos para determinação da potência e eficácia dos agonistas. Foram aplicadas doses crescentes cumulativas e não-cumulativas dos agonistas peptídicos bradicinina (BK), Des-Arg9 bradicinina, angiotensina II (AII) e endotelina-1 (ET-1). Resultados: O estudo da potência em fundus de estômago isolado de animais nocautes revelou que a afinidade dos agonistas testados, não foi alterada. Quanto ao efeito máximo, verificou-se que a ausência do receptor B1 diminuiu a eficácia da BK, porém não da ET-1 e AII. No tecido vascular, a BK mostrou-se menos eficaz em animais nocaute B1 assim como a ET-1, enquanto a AII teve sua eficácia diminuída, tanto em animais nocaute B1 como B2. As respostas à ET-1 e AII apresentaram o Emax alterado nos animais nocautes o qual foi revertido em nocaute duplo B1 e B2. Conclusão: A verificação de que a afinidade relativa dos agonistas testados, no estômago isolado de camundongos nocautes B1 e B2 não foi alterada significativamente em relação à determinada em animais WT, sugeriu que neste tecido não ocorrem interações entre os receptores que afetariam a ligação dos agonistas ao seu receptor. Entretanto a redução na contração máxima induzida pela BK no animal nocaute B1 poderia estar relacionada com o elevado nível de óxido nítrico (NO) endógeno encontrado no fundus de estômago desse animal. Porém, L-NAME (inibidor da NO sintase) não afetou o Emax da BK, descartando essa hipótese. Considerando-se que heterodimerização entre os receptores B1 e B2 ocorre nas células do estômago, a falta de B1 teria favorecido a homodimerização do B2, que leva à ativação e dessensibilização, induzindo a taquifilaxia mais acentuada para a BK, e a redução do Emax. Para o caso da AII, que mostrou ter a afinidade e a eficácia mantidas nos nocautes, mas a taquifilaxia atenuada no KOB1 e no KOB2 poderia ser atribuída ou a heterodimerização entre os receptores B2 e AT1 no KOB1 ou a homodimerização entre os receptores AT1 no KOB2. Quanto à aorta abdominal, esta se mostrou sensível à BK, mas não a DBK que não induziu resposta no WT e nos nocautes, sugerindo que este receptor não seria expresso. Entretanto aorta isolada de KOB1 teve a sensibilidade drasticamente reduzida à BK que é mediada pelo receptor B2 indicando expressão funcional importante do receptor B1 nesta preparação. Quanto a ET-1, a eficácia diminuída somente no animal KOB1 poderia ser devida à interação cruzada do receptor da ET-1 com o B2, que deixaria de dimerizar com o B1. A AII teve sua eficácia diminuída nos animais KOB1 e KOB2, sugerindo que heterodimerização entre estes receptores e também destes com o AT1 da AII poderia ocorrer, favorecendo a ação da AII. Os resultados obtidos no fundus do estômago e na aorta abdominal sugerem importância na expressão funcional simultânea dos receptores B1 e B2 em ambas as preparações, e interferência nos mecanismos de transdução de sinais, afetando a contração e relaxamento de músculos lisos vascular e não-vascular. / Purpose: to pharmacologically characterize the responses to bradykinin (BK) and other agonists in non-vascular (stomach fundus) and vascular (aorta) smooth muscles isolated from kinin B1 and B2 receptor knockout mice. Methods: stomach fundus and aorta were isolated from normal , B1 knockout and B2 knockout animals; isometric contraction recordings were obtained for potency and efficacy determinations of agonists; cumulatively and non-cumulatively increasing doses of agonists such as BK, desArg9BK (DABK), angiotensin II (AII) and endothelin (ET-1) were applyied. Results: Potency (pD2) values determined in stomach fundus of WT and KO animals revealed that the affinity of agonists was maintained. Concerning the maximal effect induced by agonists, the efficacy of BK, but not AII and ET-1 was reduced in KOB1 animals. In the vascular tissue, BK was shown to be less efficient in KOB1 and KOB2 than in WT. The responses to AII and to ET-1 were reduced in KOB1 animals but it was reverted in KOB2 animals. In addition, stomach from KOB1 was found to be impaired to relax to sodium nitroprusside whereas the relaxant response was inaltered in KOB2, indicating that the deletion of B1 receptor led to important changes in the Ca regulation mechanism. Conclusions: The finding that the relative affinity of the agonists was not affected in stomach from knockout animals suggested that in this tissue there is no cross talking between kinin receptors and other agonist receptors. However the reduction in the maximal response to BK1 receptor might favoured the homodimerization of B2, which lead to activation and dessensitization, inducing an accentuated tachyphylaxis to BK and also the reduced maximal effect. In the case of AII, which showed a high affinity and efficacy, but the tachyphylaxis was attenuated in knockout animals, it can be suggested that there was formation of homodimers of B2 receptors rather than heterodimers between kinin B2 and AII AT1 receptors due to the absence of B1 receptors. Concerning BK- but not DABK induced effect in abdominal aorta in normal and knockout animals, indicated that B1 receptors would not express in this tissue. But the drastic reduction in change in sensitivity of the aorta towards BK-induced contraction suggested that B1 receptor should be mediating kinin mediated effect. The finding that the efficacy of ET-1 was reduced in KOB1 whereas that of AII was affected in aorta of the two knockout animals suggest that there must be cross talk between kinin receptors and of ET-1 and AII or between signal transduction activateed by these agonists. It is concluded that B1 and B2 receptors in stomach fundus and in abdominal aorta both receptors play important role for functional expression of kinins and that they are involved in the mechanisms of cell signaling, affecting the vascular and non-vascular smooth muscle contraction and relaxation. / TEDE / BV UNIFESP: Teses e dissertações

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En janvier 2008, une éclosion de réactions anaphylactoïdes (RA) potentiellement mortelles associées à l’injection intraveineuse d’héparine manufacturées en Chine et contaminée par le chondroïtine sulfate hypersulfaté (CSHS) a forcé le rappel de ces dernières par la U.S. Food and Drug Administration. Ces RA ont rapidement été attribuées à la libération de la bradykinine (BK) suite à l’activation du système de contact par le CSHS. Cependant, aucune évidence expérimentale définitive n’est à ce jour venue appuyer directement cette hypothèse. En se basant sur le nombre de morts déclaré et associé à la contamination (>150 morts au niveau mondial) ainsi qu’aux données épidémiologiques, qui stipulent que 25% des patients ayant développés une RA aux États-Unis étaient essentiellement des insuffisant rénaux en dialyse traités au moyen d’un inhibiteur de l’enzyme de conversion de l’angiotensine (iECA), nous avons émis l’hypothèse suivante : les RA causées par l’injection intraveineuse d’héparine contaminée au CSHS sont de nature multifactorielle et complexe. Le but de notre travail est donc, dans un premier temps, d’évaluer le pouvoir kininoformateur du CSHS en présence d’un iECA et de le comparer à celui du sulfate de dextran, un activateur de référence du système de contact. Comme les RA associées à l’injection intraveineuse d’héparine contaminée par le CSHS se produisent généralement dans les premières minutes des séances de dialyse, nous allons étudier l’effet de la dilution du plasma sur la quantité de BK libérée en présence ou en absence d’un iECA. Nous allons également mesurer les profils cinétiques de la libération de la BK sur un plasma stimulé par différents lots d’héparine contaminée, et associée à des RA, et nous comparerons cette cinétique avec celles d’une héparine de référence complémentée ou non avec différentes concentrations de CSHS synthétique. Enfin, nous allons caractériser le profil de libération de la BK et de son métabolite actif, la des-Arg9-BK, dans le plasma de patients dialysé ayant présenté une RA associée à une membrane de dialyse chargée négativement. L’application de méthodes expérimentales développées dans notre laboratoire nous a permis de montrer, pour la première fois, que l’héparine contaminée au CSHS a la capacité de libérer la BK à des concentrations susceptibles d’expliquer le rôle de ce peptide inflammatoire dans la physiopathologie des RA causées par l’injection intraveineuse d’héparine d’origine chinoise contaminée au CSHS. / In January 2008, fatal anaphylactoid reaction (AR) has been associated to oversulfated chondroitin sulphate (OSCS) contaminated heparin. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. While looking at the number of death associated with OSCS (>150 worldwide) and at the epidemiologic fact, who state that 25% of the cases of AR associated to OSCS in United-States were treated with an angiotensin converting enzyme inhibitor (ACEi), we hypothesis that: AR associated with bolus injection of OSCS contaminated heparin are bind to a complex and multi-factorial aspect. The first objective of our study is to measure the kinetics of BK release in human plasma incubated with OSCS in presence of an ACEi and to compare it to the kinetics profile of the reference activator dextran sulfate. As the AR associated with OSCS contaminated heparin occurred mainly in the first minutes of dialysis session, we also studied the effect of the plasma dilution on the amount of BK released when treated or not with an ACEi. We also quantify the BK forming capacity of different batches of OSCS contaminated heparin responsible for AR and we compare this effect with reference heparin spiked or not with increasing concentrations of synthetic OSCS. Finally, we measure the kinetics of BK and des- Arg9-BK, its active metabolite, release in human plasma collected from patients who developed an AR associated to negatively charged dialysis membrane. The application of experimental method developed in our laboratory show, for the first time, that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated Chinese heparins.

En janvier 2008, une éclosion de réactions anaphylactoïdes (RA) potentiellement mortelles associées à l’injection intraveineuse d’héparine manufacturées en Chine et contaminée par le chondroïtine sulfate hypersulfaté (CSHS) a forcé le rappel de ces dernières par la U.S. Food and Drug Administration. Ces RA ont rapidement été attribuées à la libération de la bradykinine (BK) suite à l’activation du système de contact par le CSHS. Cependant, aucune évidence expérimentale définitive n’est à ce jour venue appuyer directement cette hypothèse. En se basant sur le nombre de morts déclaré et associé à la contamination (>150 morts au niveau mondial) ainsi qu’aux données épidémiologiques, qui stipulent que 25% des patients ayant développés une RA aux États-Unis étaient essentiellement des insuffisant rénaux en dialyse traités au moyen d’un inhibiteur de l’enzyme de conversion de l’angiotensine (iECA), nous avons émis l’hypothèse suivante : les RA causées par l’injection intraveineuse d’héparine contaminée au CSHS sont de nature multifactorielle et complexe. Le but de notre travail est donc, dans un premier temps, d’évaluer le pouvoir kininoformateur du CSHS en présence d’un iECA et de le comparer à celui du sulfate de dextran, un activateur de référence du système de contact. Comme les RA associées à l’injection intraveineuse d’héparine contaminée par le CSHS se produisent généralement dans les premières minutes des séances de dialyse, nous allons étudier l’effet de la dilution du plasma sur la quantité de BK libérée en présence ou en absence d’un iECA. Nous allons également mesurer les profils cinétiques de la libération de la BK sur un plasma stimulé par différents lots d’héparine contaminée, et associée à des RA, et nous comparerons cette cinétique avec celles d’une héparine de référence complémentée ou non avec différentes concentrations de CSHS synthétique. Enfin, nous allons caractériser le profil de libération de la BK et de son métabolite actif, la des-Arg9-BK, dans le plasma de patients dialysé ayant présenté une RA associée à une membrane de dialyse chargée négativement. L’application de méthodes expérimentales développées dans notre laboratoire nous a permis de montrer, pour la première fois, que l’héparine contaminée au CSHS a la capacité de libérer la BK à des concentrations susceptibles d’expliquer le rôle de ce peptide inflammatoire dans la physiopathologie des RA causées par l’injection intraveineuse d’héparine d’origine chinoise contaminée au CSHS. / In January 2008, fatal anaphylactoid reaction (AR) has been associated to oversulfated chondroitin sulphate (OSCS) contaminated heparin. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. While looking at the number of death associated with OSCS (>150 worldwide) and at the epidemiologic fact, who state that 25% of the cases of AR associated to OSCS in United-States were treated with an angiotensin converting enzyme inhibitor (ACEi), we hypothesis that: AR associated with bolus injection of OSCS contaminated heparin are bind to a complex and multi-factorial aspect. The first objective of our study is to measure the kinetics of BK release in human plasma incubated with OSCS in presence of an ACEi and to compare it to the kinetics profile of the reference activator dextran sulfate. As the AR associated with OSCS contaminated heparin occurred mainly in the first minutes of dialysis session, we also studied the effect of the plasma dilution on the amount of BK released when treated or not with an ACEi. We also quantify the BK forming capacity of different batches of OSCS contaminated heparin responsible for AR and we compare this effect with reference heparin spiked or not with increasing concentrations of synthetic OSCS. Finally, we measure the kinetics of BK and des- Arg9-BK, its active metabolite, release in human plasma collected from patients who developed an AR associated to negatively charged dialysis membrane. The application of experimental method developed in our laboratory show, for the first time, that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated Chinese heparins.