Foram coletadas 137 amostras sanguíneas de 132 espécimes diferentes, pertencentes a sete espécies de Strigiformes (n=92) e onze espécies de Falconiformes (n=45), entre os meses de setembro de 2008 e julho de 2009. As amostras foram submetidas a hemograma completo, pesquisa de hemoparasitos através da avaliação do esfregaço sanguíneo, sexagem através da técnica de PCR e mensuração da atividade de colinesterases plasmáticas através do método de Ellman (1961). Apenas sete corujinhas-do-mato (Megascops choliba) apresentaram hemoparasitos (5,3 % do total de rapinantes). Houve diferença significativa conforme o estado de saúde da ave, sendo que a todos os animais clinicamente hígidos não estavam infectados, porém 20,4% dos animais com desordens clínicas apresentavam hemosporídeos. Quantitativamente, a parasitemia mediana dos animais infectados foi de 3,8 hemácias parasitadas por 10.000 hemácias, um índice de infecção relativamente baixo. A grande maioria dos parâmetros hematológicos não apresentou diferenças estatísticas em função da presença ou ausência de hemoparasitos, o que na maioria dos casos caracterizou uma infecção branda e pouco patogênica. A atividade de colinesterases plasmáticas apresentou uma distribuição normal, com uma média geométrica de 0,8333 micromoles/mL e desvio padrão geométrico de 1,7087 µmols/mL. A atividade de colinesterases plasmáticas apresentou diferenças estatísticas significativas entre Falconiformes (0,5705±1,6497 µmols/mL) e Strigiformes (1,0070±1,5620 µmols/mL). Não houve diferenças significativas entre machos ou fêmeas e adultos ou jovens dentre os Falconiformes ou Strigiformes. Falconiformes não hígidos apresentaram valores surpreendentemente mais baixos do que os clinicamente hígidos. Entre os Strigiformes, os espécimes de cativeiro apresentaram valores mais baixos de atividade de colinesterases plasmáticas do que os de vida livre. A atividade de colinesterases plasmáticas foi significativamente diferente entre os animais com hemoparasitos (mediana geométrica = 2,0132 µmols/mL) e sem hemoparasitos (mediana geométrica = 0,8331 µmols/mL). Este estudo contribui com informações relevantes sobre a ocorrência de hemosporídeos em aves de rapina do Estado de São Paulo, alertando as Instituições sobre a necessidade de monitoração destes protozoários sanguíneos. Além disso, contribui com dados inéditos sobre a atividade de colinesterases plasmáticas em rapinantes. / 137 blood samples were collected from 132 different specimen, belonging to seven species of Strigiformes (n=92) and eleven species of Falconiformes (n=45), from September 2008 to July 2009. Samples were submitted to complete hemogram, survey for hemoparasites through evaluation of blood smears, sex identification by PCR and measurement of plasmatic cholinesterases by the method of Ellman (1961). Only seven black-capped-screech-owls (Megascops choliba) were positive for hemoparasites (5,3% of total raptors). There were significative difference according to avian health status; all the clinicaly higid animal were not infected, but 20,4% of animals with clinical desorders presented haemosporidians. Quantitatively, the average parasitemia was 3,8 parasited erythrocytes in 10.000 erythrocytes, a infection rate relatively low. The most of hematologic parameters did not present statistic differences in function of presence or absence of hemoparasites, what in most of the cases characterized a low and not much pathogenic infection. The plasmatic cholinesterase activity presented normal distribution, with geometric average of 0,8333 micromoles/mL and geometric standard deviation of 1,7087 µmols/mL. The plasmatic cholinesterase activity presented statistic differences between Falconiformes (0,5705±1,6497 µmols/mL) and Strigiformes (1,0070±1,5620 µmols/mL). There were not significative differences between males of females and adults or youngs between Falconiformes or Strigiformes. Non-higid Falconiformes presented surprisiling much low values than those clinically higid. Between Strigiformes, the captivity specimens present much lower plasmatic cholinesterase activity values than free ranging animals. The plasmatic cholinesterase activity was signifactively different among animal with hemoparasites (geometric average = 2,0132 µmols/mL) and without hemoparasites (geometric average = 0,8331 µmols/mL). This research contributes with relevant information about the haemosporidian occurence in raptors of State of São Paulo, alerting the Institutions about the necessity of monitorizing haemoprotozoa. Besides that, contributes with unpublished data about the plasmatic cholinesterase activity in raptors.

by Zhang Xiang. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 165-174). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Foram coletadas 137 amostras sanguíneas de 132 espécimes diferentes, pertencentes a sete espécies de Strigiformes (n=92) e onze espécies de Falconiformes (n=45), entre os meses de setembro de 2008 e julho de 2009. As amostras foram submetidas a hemograma completo, pesquisa de hemoparasitos através da avaliação do esfregaço sanguíneo, sexagem através da técnica de PCR e mensuração da atividade de colinesterases plasmáticas através do método de Ellman (1961). Apenas sete corujinhas-do-mato (Megascops choliba) apresentaram hemoparasitos (5,3 % do total de rapinantes). Houve diferença significativa conforme o estado de saúde da ave, sendo que a todos os animais clinicamente hígidos não estavam infectados, porém 20,4% dos animais com desordens clínicas apresentavam hemosporídeos. Quantitativamente, a parasitemia mediana dos animais infectados foi de 3,8 hemácias parasitadas por 10.000 hemácias, um índice de infecção relativamente baixo. A grande maioria dos parâmetros hematológicos não apresentou diferenças estatísticas em função da presença ou ausência de hemoparasitos, o que na maioria dos casos caracterizou uma infecção branda e pouco patogênica. A atividade de colinesterases plasmáticas apresentou uma distribuição normal, com uma média geométrica de 0,8333 micromoles/mL e desvio padrão geométrico de 1,7087 µmols/mL. A atividade de colinesterases plasmáticas apresentou diferenças estatísticas significativas entre Falconiformes (0,5705±1,6497 µmols/mL) e Strigiformes (1,0070±1,5620 µmols/mL). Não houve diferenças significativas entre machos ou fêmeas e adultos ou jovens dentre os Falconiformes ou Strigiformes. Falconiformes não hígidos apresentaram valores surpreendentemente mais baixos do que os clinicamente hígidos. Entre os Strigiformes, os espécimes de cativeiro apresentaram valores mais baixos de atividade de colinesterases plasmáticas do que os de vida livre. A atividade de colinesterases plasmáticas foi significativamente diferente entre os animais com hemoparasitos (mediana geométrica = 2,0132 µmols/mL) e sem hemoparasitos (mediana geométrica = 0,8331 µmols/mL). Este estudo contribui com informações relevantes sobre a ocorrência de hemosporídeos em aves de rapina do Estado de São Paulo, alertando as Instituições sobre a necessidade de monitoração destes protozoários sanguíneos. Além disso, contribui com dados inéditos sobre a atividade de colinesterases plasmáticas em rapinantes. / 137 blood samples were collected from 132 different specimen, belonging to seven species of Strigiformes (n=92) and eleven species of Falconiformes (n=45), from September 2008 to July 2009. Samples were submitted to complete hemogram, survey for hemoparasites through evaluation of blood smears, sex identification by PCR and measurement of plasmatic cholinesterases by the method of Ellman (1961). Only seven black-capped-screech-owls (Megascops choliba) were positive for hemoparasites (5,3% of total raptors). There were significative difference according to avian health status; all the clinicaly higid animal were not infected, but 20,4% of animals with clinical desorders presented haemosporidians. Quantitatively, the average parasitemia was 3,8 parasited erythrocytes in 10.000 erythrocytes, a infection rate relatively low. The most of hematologic parameters did not present statistic differences in function of presence or absence of hemoparasites, what in most of the cases characterized a low and not much pathogenic infection. The plasmatic cholinesterase activity presented normal distribution, with geometric average of 0,8333 micromoles/mL and geometric standard deviation of 1,7087 µmols/mL. The plasmatic cholinesterase activity presented statistic differences between Falconiformes (0,5705±1,6497 µmols/mL) and Strigiformes (1,0070±1,5620 µmols/mL). There were not significative differences between males of females and adults or youngs between Falconiformes or Strigiformes. Non-higid Falconiformes presented surprisiling much low values than those clinically higid. Between Strigiformes, the captivity specimens present much lower plasmatic cholinesterase activity values than free ranging animals. The plasmatic cholinesterase activity was signifactively different among animal with hemoparasites (geometric average = 2,0132 µmols/mL) and without hemoparasites (geometric average = 0,8331 µmols/mL). This research contributes with relevant information about the haemosporidian occurence in raptors of State of São Paulo, alerting the Institutions about the necessity of monitorizing haemoprotozoa. Besides that, contributes with unpublished data about the plasmatic cholinesterase activity in raptors.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2017-08-30T17:39:39Z No. of bitstreams: 2 Isabella Calvo Bramatti.pdf: 1175071 bytes, checksum: b4b43f726550d831d384392724e6c260 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-30T17:39:39Z (GMT). No. of bitstreams: 2 Isabella Calvo Bramatti.pdf: 1175071 bytes, checksum: b4b43f726550d831d384392724e6c260 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Tamoxifen is classified as a selective estrogen receptor modulator, with much use in the treatment of breast cancer. Quercetin has aroused interest, mainly for its antioxidant action, which may minimize the damage caused by tamoxifen. Due to the lack of studies about the coadministration of quercetin with tamoxifen, the objective of this study was to elucidate the effect upon the cholinergic activity and antioxidante defense. At the study, ovariectomized rats were separated into groups and treated with tamoxifen (5 mg.Kg-1), quercetin (22,5 mg.Kg-1) and the coadministration of tamoxifen (5 mg.Kg-1) plus quercetin (22,5 mg.Kg-1). The hippocampus was isolated and utilized for the determination of acetylcholinesterase and butyrylcholinesterase activity, superoxide dismutase and catalase activity, and the oxidative damage markers (lipid peroxidation and dosage of thiols groups). It was possible observe the oxidant effect of tamoxifen at the hippocampus, where acted like an antagonist at the estrogen receptors, increasing the lipid peroxidation, levels of thiol groups and attenuating catalase activity; in addition to a cholinergic effect, with the decrease of acetylcholinesterase and butyrylcholinesterase activity. When coadministered the quercetin with tamoxifen, it can be seen that at a situation of oxidative stress, the quercetin acts like pro oxidant in the studied. / Tamoxifeno é classificado como um modulador seletivo dos receptores de estrogênio (SERM), sendo muito utilizado no tratamento contra o câncer de mama. A quercetina tem particularmente despertado interesse, principalmente por sua ação antioxidante a qual supostamente poderia vir a minimizar os danos causados pelo tamoxifeno. Devido à falta de estudos sobre a coadministração da quercetina com o tamoxifeno e a ambiguidade sobre a ação de ambos no cérebro, o objetivo deste estudo foi elucidar o efeito sobre a atividade colinérgica e defesa antioxidante. No estudo, ratas ovariectomizadas foram separadas em grupos e tratadas com tamoxifeno (5 mg.Kg-1), quercetina (22,5 mg.Kg-1) e a coadministração de tamoxifeno (5 mg.Kg-1) mais quercetina (22,5 mg.Kg-1). O hipocampo foi isolado e utilizado para a determinação da atividade da acetilcolinesterase, butirilcolinesterase da superóxido dismutase e catalase e dos marcadores de dano oxidativo (lipoperoxidação e dosagem de grupamentos tióis). Foi possível observar a ação oxidante do tamoxifeno no hipocampo, onde agiu como antagonista nos receptores de estrogênio, aumentando a lipoperoxidação, os níveis de tióis e diminuindo a atividade da catalase; além de um efeito colinérgico com a diminuição da atividade da acetilcolinesterase e butirilcolinesterase. Quando coadministrada a quercetina com o tamoxifeno, pode-se observar que em uma situação de estresse oxidativo, a quercetina age como pró-oxidante na dose estudada.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Type 1 diabetes (T1D) is a disease characterized by the individual's inability to produce the hormone insulin due to destruction of pancreatic β-cells by the immune cells of the organism itself. So far, there is no 100% effective therapy for treating the symptoms of T1D. Current treatments involve constant monitoring of blood glucose levels and regular insulin injections. Inflammatory processes are associated with the development and progression of T1D, and so new therapies involving drugs with anti-inflammatory properties have been the subject of increasing interest in the scientific community. Polyphenols are a class of heterocyclic compounds that include anthocyanins and rosmarinic acid, both known for their antioxidant and anti-inflammatory properties. We studied the effects of two polyphenol rich extracts (cooked potato and rosemary) and a concentrate of anthocyanins in a model of type 1 streptozotocin-induced diabetic rats. The extracts were administered by gavage for 30 days. After this period, the rats were euthanized and we assessed acetylcholinesterase (AChE) activity in lymphocytes and serum butyrylcholinesterase (BuChE) activity, because of the importance of these enzymes as biomarkers of inflammation. The activities of both enzymes were shown to be increased in diabetic rats compared to controls. The cooked potato peel extract and concentrated anthocyanins did not altered AChE activity in lymphocytes, probably due to low bioavailability, while rosemary extract was a potent inhibitor and reverted the activity to normal levels. None of the treatments had any effect on serum BuChE activity. These results support the involvement of inflammatory processes on TD1 pathology and present the rosemary extracts as a promising alternative for future phytochemical therapies for the treatment of inflammatory-related diseases, such as type 1 diabetes. / O diabetes tipo 1 (DT1) é uma doença caracterizada pela incapacidade do indivíduo de produzir o hormônio insulina devido à destruição das células-β pancreáticas pelas células imune do próprio organismo. Ainda não existe uma terapia 100 % eficaz para tratar os sintomas do DT1. Os tratamentos atuais envolvem monitoramento constante dos níveis de glicose do sangue e injeções periódicas de insulina. Os processos inflamatórios estão associados com o desenvolvimento e a progressão do DT1 e, por isso, novas terapias envolvendo fármacos com propriedades anti-inflamatórias têm sido alvo crescente de interesse no meio científico. Os polifenois são uma classe de compostos heterocíclicos dos quais fazem parte as antocianinas e o ácido rosmarínico, conhecidos por suas propriedades antioxidantes e anti-inflamatórias. Neste trabalho, estudamos os efeitos de dois extratos ricos em polifenois batata cozida e alecrim - e de um concentrado de antocianinas em um modelo de ratos com diabetes tipo 1 induzido por estreptozotocina. Os extratos foram administrados por gavagem durante 30 dias. Após, os ratos foram eutanasiados e verificou-se os efeitos do tratamento sobre a atividade da acetilcolinesterase (AChE) de linfócitos e da butirilcolinesterase (BuChE) de soro, devido à importância dessas enzimas como biomarcadores de inflamação. As atividades de ambas as enzimas mostraram-se aumentadas nos ratos diabéticos em relação aos controles. O extrato de casca de batata cozida e as antocianinas concentradas não alteraram significativamente a atividade da AChE em linfócitos, provavelmente devido à sua baixa biodisponibilidade, enquanto o extrato de alecrim foi um potente inibidor dessa enzima. Nenhum dos tratamentos surtiu efeito sobre a atividade da BuChE de soro. Estes resultados reforçam a hipótese inflamatória na patologia do DT1 e apresentam o extrato de alecrim como uma possível terapia secundária para o tratamento de doenças associadas à inflamação, como o diabetes tipo 1.

The distribution, absorption, metabolism and tissue binding of radioactivity were studied in hamsters after oral and intraperitoneal administration of alpha-chaconine- (3H). The material was well absorbed from the gastrointestinal tract and nearly 22 percent of the label was excreted via urine and feces in 7 days. The excretion was higher in urine (21 percent) than in feces ( < 1 percent). Tissue concentrations of radioactivity peaked at 12 hours following oral administration, with the highest concentrations found in lungs, liver, spleen, skeletal muscle, kidney and pancreas, with heart and brain containing moderate amounts. Concentrations of radioactivity in tissues following intraperitoneal administration were significantly higher than those observed after oral treatment. Excretion of chloroform-soluble products in the feces was 10 times higher than that of the chloroform-insoluble metabolites after both oral and intraperitoneal administration. In the urine, the activity was predominantly in the chloroform-insoluble form and the chloroform-soluble metabolites were relatively minor in amounts (0.27, 0.85, and 2.45 percent versus 0.005, 0.14 and 0.19 percent of dose for 12, 24 and 72 hours, respectively). After 7 days, the chloroform-soluble metabolites in urine increased to 20 percent of the excreted radioactivity, while the amount of chloroform-insoluble metabolites was less than 1 percent. Subcellular distribution of the labeled compound indicated the highest concentration of radioactivity in the nuclear and microsomal fractions of brain, liver and heart tissues. A small amount of radioactivity, shown by a minor peak, was also observed in the fractions between the mitochondrial and microsomal fractions on a sucrose gradient. Binding of radioactivity was observed in brain, testes, kidney, lung, liver and heart . All of the label in the brain appeared to be in the bound form . The results indicated that alpha-chaconine is slowly absorbed from the gastrointestinal tract after oral administration, and persists in various tissues, much of it in bound (non-extractable) form (in nuclear and microsomal fractions). Excretion of alpha-chaconine- (3H) and its metabolites was investigated after oral and intraperitoneal administration in hamsters. The separation of the glycoalkaloid and its metabolites in feces and urine was accomplished by thin-layer chromatography. An increase in the concentration of excreted alpha-chaconine metabolites in feces and urine was observed. In urine over 50 percent of the eliminated radioactivity during the initial 24 hours was due to the aglycone, solanidine. The fraction of the total dose administered which was excreted represented only 27 percent (26 percent in feces and less than 1 percent in urine) during the 7 day test period. Contrary to the general belief that potato glycoalkaloid absorption is poor following oral administration, only 5 percent or less was excreted in feces during the initial 72 hours, a fact explained by the binding of radioactivity to tissues. Inhibition of acetylcholinesterases by alpha-chaconine was studied. The inhibition of purified erythrocyte acetylcholinesterase and horse serum cholinesterase by alpha-chaconine was found to be a mixed-type with kinetic constants. An inhibition constant (Ki) for both the specific and pseudocholinesterases was 8.3 x 10-6 M and 4.0 x 10-4 M, respectively. Kinetic constants obtained for both enzymes were as follows: Vmax of 7.14 x 10-5 and 3.76 x 10-4 max moles/liter/min, respectively, and Km of 6.2 x 10-5 and 1.33 x 10-4, respectively. The distribution of acetylcholinesterase among the subcellular fractions of rat brain homogenate separated by sucrose density gradient centrifugation was determined, as well as the inhibition pattern of these fractions following in vitro incubation with 0.016 M alpha-chaconine. Enzyme activity was found to be distributed equally between the mitochondrial and microsomal fractions, with the nuclear fraction having the least activity. Percentage inhibition of the various fractions obtained was: whole homogenate 43, nuclear fraction 55, mitochondria 35, and microsomes 33. Brain acetyl cholinesterase activity of animals given intraperitoneal doses (10, 30, 60 mg / Kg) of alpha-chaconine was 79, 55 and 18 percent of the control group. Acetylcholinesterase activity of heart and plasma of animals administered alpha-chaconine did not show the dose-related response observed in the brain. Inhibition of heart acetylcholinesterase was 61 percent, while plasma gave 51 percent for the rats given a dose of 10 mg/Kg and no inhibition for rats given 30 mg/ Kg. Acrylamide gel electrophoretic separation of cholinesterases in aqueous homogenates from whole brain and heart of adult male rats administered alpha-chaconine was investigated. Brain acetylcholinesterase isoenzymes were found to be inhibited by 30 and 60 mg/ Kg dosage levels of alpha-chaconine administered intraperitoneally. Electrophoretic separation of plasma from the treated animals resulted in five anodally migrating zones having properties of cholinesterases. These sites hydrolyze acetylthiocholine and alpha-naphthylacetate, and all were inhibited by alpha-chaconine except the slowest migrating band (band 5). Inhibition of isoenzyme activity of bands 1 and 2 is observed for the groups administered 10 and 30 mg/Kg alpha-chaconine with the percentage inhibition of both bands (l and 2) being 40 and 77 percent for animals given 10 mg/Kg and 100 and 75 percent for the latter group. Isoenzyme bands 3 and 4 were completely absent in the alkaloid treated animals. Inhibition of non-specific cholinesterase isoenzymes (butyrylthiocholine hydrolyzable bands) by alpha-chaconine was clearly demonstrated. In vitro inhibition of plasma, erythrocyte and brain esterase isoenzymes was estimated by incubating gels with 10-4 M alpha-chaconine after the electrophoretic separations. With this concentration of alpha-chaconine, the various isoenzymes in rat plasma, erythrocyte and brain showed some response to the inhibitory potency of alpha-chaconine. The slower-moving isoenzyme bands were inhibited to 100 percent with the different concentrations of inhibitor. The fast migrating isoenzyme bands in plasma and erythrocytes were least affected by alpha-chaconine (10-4 M), with no inhibition. Plasma protein isoenzymes from adult male rats were not affected by alphachaconine.

Introduction: Des résultats récents indiquent que l’acétylcholine joue un rôle protecteur contre les insuffisances cardiaques et les fibrillations atriales ou ventriculaires. Les cholinestérases (ChE) contrôlent le niveau d’acétylcholine et jouent donc un rôle important pour le système cholinergique du cœur. Cependant, ces enzymes dans le cœur sont peu connues. L'objectif de cette thèse est d’étudier l'acétylcholinestérase (AChE) et la butyrylcholinestérase (BChE) dans le cœur, de quantifier ces enzymes et leurs formes moléculaires et localiser au niveau cellulaire. Méthodes : Nous avons utilisé des souris sans AChE ou BChE et sans ColQ et PRiMA leur protéines d’ancrage. Les activités de l’AChE et de la BChE ont été déterminées par la méthode d’Ellman, que nous avons adaptée pour la faible quantité de ChE dans un extrait brut. Les formes moléculaires des ChE ont été séparées en gradient de saccharose 5-20% et localisées dans des cœurs gonflés par la gélatine et sur coupe à froid. La morphologie globale du cœur a été étudiée en coupes transversales colorées avec l’hématoxyline et l’éosine. Résultats et conclusions : La plus forte activité en AChE a été mesurée dans les oreillettes, la plus faible activité dans le ventricule gauche et le septum. Dans tous les compartiments, PRiMA AChE et ColQ AChE ont été observés. Chacune des formes ancrées est distribuée sur l’épicarde à la base du cœur, et sont co-localisées avec des neurones intracardiaques. PRiMA AChE forme des branches fines à proximité des neurones intracardiaques. L’absence d’ancrage de l’AChE aboutit à une diminution significative du diamètre des cardiomyocytes. L’activité de la BChE est plus élevée que celle de l’AChE. La plus haute activité en BChE a été détectée dans le ventricule gauche et le septum. Le monomère amphiphilic constitue la forme prédominante dans le cœur. Dans le myocarde, le marquage de l’activité BChE est diffus, alors que dans l’épicarde il colocalise avec un seul neurone intracardiaque. Dans ce travail, nous avons réalisé une étude complète des ChE dans le cœur. Nos résultats peuvent aider à définir de nouvelles thérapies pharmacologiques plus efficaces. / Introduction: The results of current research suggest that acetylcholine has a protective role during heart failure and atrial or ventricular fibrillation. Cholinesterases (ChE) control the level of acetylcholine and thus play an important role in the cholinergic system of the heart. However, detail information about these enzymes in the heart is still missing. The aim of this thesis was to provide a complex study of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the heart, specifically of their activities, molecular forms and precise localization. Methods: Mutant mice with lack of ChE or their anchoring proteins, ColQ and PRiMA, were used. AChE and BChE activities in heart compartments were determined by 2-step Ellman’s method, developed by us. Molecular forms of ChE were determined in 5-20 % sucrose gradients and localized in the hearts filled with gelatin and in the cryosections by activity staining method and by immunohistochemistry. Nerve and endothelial cells were identified using specific markers. Basic heart morphology was studied in the transversal sections stained with hematoxyline and eosine. Results and conclusion: The highest AChE activity was determined in the atria, the lowest activity in the left ventricle and septum. In all compartments, PRiMA AChE and ColQ AChE were observed. Both anchored forms were distributed epicardially on the heart base, co-localized with intracardiac neurons. PRiMA AChE formed a subtle branching in the proximity of intracardiac neurons. The lack of AChE anchored forms led to significantly lower cardiomyocyte diameter. The BChE activity was higher than that of AChE. The highest BChE activity was detected in the left ventricle and septum. Amphiphilic monomers were the predominant form of BChE in the heart. In myocardium, the staining of BChE activity was diffused, while in epicardium it co-localized with a single intracardiac neuron. In this work, we have provided a complex study of ChE in heart. Our results could help in the design of new, more effective pharmacotherapy, which may reduce morbidity and mortality of the patients with various heart diseases. / Úvod: Výsledky súčasného výskumu nasvedčujú protektívnemu vplyvu acetylcholínu pri srdcovom zlyhávaní, či pri predsieňovej a komorovej fibrilácií. Cholínesterázy (ChE) regulujú hladinu acetylcholínu a tak zohrávajú dôležitú úlohu v cholinergickom systéme srdca. Avšak, hlbšie informácie o týchto enzýmoch v srdci chýbajú. Cieľom tejto práce bolo komplexné štúdium acetylcholínesterázy (AChE) a butyrylcholínesterázy (BChE) v srdci, konkrétne ich aktivít, molekulových foriem a presnej lokalizácie. Metódy: V projekte sme používali mutantné myši s chýbaním ChE alebo ich kotviacich proteínov, ColQ a PRiMA. Aktivity AChE a BChE sme v jednotlivých častiach srdca stanovili nami vyvinutou modifikovanou dvoj-krokovou Ellmanovou metódou. Molekulové formy ChE sme študovali v 5-20 % sacharózových gradientoch. ChE sme lokalizovali v srdciach vyplnených želatínou a v kryostatických rezoch, a to pomocou aktivitného farbenia a imunohistochémiou. Nervové a endotelové bunky sme identifikovali na základe špecifických markerov. Základnú morfológiu tkaniva sme študovali v priečnych rezoch srdca nafarbených hematoxylínom a eozínom. Výsledky a záver: V predsieňach sme pozorovali najvyššiu a v ľavej komore a septe najnižšiu aktivitu AChE, kotvenú pomocou PRiMA aj ColQ proteínov. PRiMA AChE aj ColQ AChE boli v epikarde distribuované na báze srdca a ko-lokalizované s intrakardiálnymi neurónmi. PRiMA AChE vytvárala jemnú spleť v blízkosti intrakardiálnych neurónov. Chýbanie kotvených foriem AChE viedlo ku signifikantne menším priemerom kardiomyocytov. Aktivita BChE v srdci bola vyššia ako aktivita AChE, s najvyššou aktivitou v ľavej komore a septe. Predominatnou formou BChE v srdci boli amfifilné monoméry. BChE aktivita bola v myokarde lokalizovaná difúzne a v epikarde ko-lokalizovala s jedným intrakardiálnym neurónom. V predloženej práci sme poskytli komplexný obraz o ChE v srdci. Naše výsledky môžu vo významnej miere napomôcť v dizajne novej, efektívnejšej farmakoterapie, ktorá by mohla znížiť morbiditu a mortalitu pacientov s vybranými chorobami srdca.

Introduction: Des résultats récents indiquent que l’acétylcholine joue un rôle protecteur contre les insuffisances cardiaques et les fibrillations atriales ou ventriculaires. Les cholinestérases (ChE) contrôlent le niveau d’acétylcholine et jouent donc un rôle important pour le système cholinergique du cœur. Cependant, ces enzymes dans le cœur sont peu connues. L'objectif de cette thèse est d’étudier l'acétylcholinestérase (AChE) et la butyrylcholinestérase (BChE) dans le cœur, de quantifier ces enzymes et leurs formes moléculaires et localiser au niveau cellulaire. Méthodes : Nous avons utilisé des souris sans AChE ou BChE et sans ColQ et PRiMA leur protéines d’ancrage. Les activités de l’AChE et de la BChE ont été déterminées par la méthode d’Ellman, que nous avons adaptée pour la faible quantité de ChE dans un extrait brut. Les formes moléculaires des ChE ont été séparées en gradient de saccharose 5-20% et localisées dans des cœurs gonflés par la gélatine et sur coupe à froid. La morphologie globale du cœur a été étudiée en coupes transversales colorées avec l’hématoxyline et l’éosine. Résultats et conclusions : La plus forte activité en AChE a été mesurée dans les oreillettes, la plus faible activité dans le ventricule gauche et le septum. Dans tous les compartiments, PRiMA AChE et ColQ AChE ont été observés. Chacune des formes ancrées est distribuée sur l’épicarde à la base du cœur, et sont co-localisées avec des neurones intracardiaques. PRiMA AChE forme des branches fines à proximité des neurones intracardiaques. L’absence d’ancrage de l’AChE aboutit à une diminution significative du diamètre des cardiomyocytes. L’activité de la BChE est plus élevée que celle de l’AChE. La plus haute activité en BChE a été détectée dans le ventricule gauche et le septum. Le monomère amphiphilic constitue la forme prédominante dans le cœur. Dans le myocarde, le marquage de l’activité BChE est diffus, alors que dans l’épicarde il colocalise avec un seul neurone intracardiaque. Dans ce travail, nous avons réalisé une étude complète des ChE dans le cœur. Nos résultats peuvent aider à définir de nouvelles thérapies pharmacologiques plus efficaces. / Introduction: The results of current research suggest that acetylcholine has a protective role during heart failure and atrial or ventricular fibrillation. Cholinesterases (ChE) control the level of acetylcholine and thus play an important role in the cholinergic system of the heart. However, detail information about these enzymes in the heart is still missing. The aim of this thesis was to provide a complex study of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the heart, specifically of their activities, molecular forms and precise localization. Methods: Mutant mice with lack of ChE or their anchoring proteins, ColQ and PRiMA, were used. AChE and BChE activities in heart compartments were determined by 2-step Ellman’s method, developed by us. Molecular forms of ChE were determined in 5-20 % sucrose gradients and localized in the hearts filled with gelatin and in the cryosections by activity staining method and by immunohistochemistry. Nerve and endothelial cells were identified using specific markers. Basic heart morphology was studied in the transversal sections stained with hematoxyline and eosine. Results and conclusion: The highest AChE activity was determined in the atria, the lowest activity in the left ventricle and septum. In all compartments, PRiMA AChE and ColQ AChE were observed. Both anchored forms were distributed epicardially on the heart base, co-localized with intracardiac neurons. PRiMA AChE formed a subtle branching in the proximity of intracardiac neurons. The lack of AChE anchored forms led to significantly lower cardiomyocyte diameter. The BChE activity was higher than that of AChE. The highest BChE activity was detected in the left ventricle and septum. Amphiphilic monomers were the predominant form of BChE in the heart. In myocardium, the staining of BChE activity was diffused, while in epicardium it co-localized with a single intracardiac neuron. In this work, we have provided a complex study of ChE in heart. Our results could help in the design of new, more effective pharmacotherapy, which may reduce morbidity and mortality of the patients with various heart diseases. / Úvod: Výsledky súčasného výskumu nasvedčujú protektívnemu vplyvu acetylcholínu pri srdcovom zlyhávaní, či pri predsieňovej a komorovej fibrilácií. Cholínesterázy (ChE) regulujú hladinu acetylcholínu a tak zohrávajú dôležitú úlohu v cholinergickom systéme srdca. Avšak, hlbšie informácie o týchto enzýmoch v srdci chýbajú. Cieľom tejto práce bolo komplexné štúdium acetylcholínesterázy (AChE) a butyrylcholínesterázy (BChE) v srdci, konkrétne ich aktivít, molekulových foriem a presnej lokalizácie. Metódy: V projekte sme používali mutantné myši s chýbaním ChE alebo ich kotviacich proteínov, ColQ a PRiMA. Aktivity AChE a BChE sme v jednotlivých častiach srdca stanovili nami vyvinutou modifikovanou dvoj-krokovou Ellmanovou metódou. Molekulové formy ChE sme študovali v 5-20 % sacharózových gradientoch. ChE sme lokalizovali v srdciach vyplnených želatínou a v kryostatických rezoch, a to pomocou aktivitného farbenia a imunohistochémiou. Nervové a endotelové bunky sme identifikovali na základe špecifických markerov. Základnú morfológiu tkaniva sme študovali v priečnych rezoch srdca nafarbených hematoxylínom a eozínom. Výsledky a záver: V predsieňach sme pozorovali najvyššiu a v ľavej komore a septe najnižšiu aktivitu AChE, kotvenú pomocou PRiMA aj ColQ proteínov. PRiMA AChE aj ColQ AChE boli v epikarde distribuované na báze srdca a ko-lokalizované s intrakardiálnymi neurónmi. PRiMA AChE vytvárala jemnú spleť v blízkosti intrakardiálnych neurónov. Chýbanie kotvených foriem AChE viedlo ku signifikantne menším priemerom kardiomyocytov. Aktivita BChE v srdci bola vyššia ako aktivita AChE, s najvyššou aktivitou v ľavej komore a septe. Predominatnou formou BChE v srdci boli amfifilné monoméry. BChE aktivita bola v myokarde lokalizovaná difúzne a v epikarde ko-lokalizovala s jedným intrakardiálnym neurónom. V predloženej práci sme poskytli komplexný obraz o ChE v srdci. Naše výsledky môžu vo významnej miere napomôcť v dizajne novej, efektívnejšej farmakoterapie, ktorá by mohla znížiť morbiditu a mortalitu pacientov s vybranými chorobami srdca.

Introduction: Des résultats récents indiquent que l’acétylcholine joue un rôle protecteur contre les insuffisances cardiaques et les fibrillations atriales ou ventriculaires. Les cholinestérases (ChE) contrôlent le niveau d’acétylcholine et jouent donc un rôle important pour le système cholinergique du cœur. Cependant, ces enzymes dans le cœur sont peu connues. L'objectif de cette thèse est d’étudier l'acétylcholinestérase (AChE) et la butyrylcholinestérase (BChE) dans le cœur, de quantifier ces enzymes et leurs formes moléculaires et localiser au niveau cellulaire. Méthodes : Nous avons utilisé des souris sans AChE ou BChE et sans ColQ et PRiMA leur protéines d’ancrage. Les activités de l’AChE et de la BChE ont été déterminées par la méthode d’Ellman, que nous avons adaptée pour la faible quantité de ChE dans un extrait brut. Les formes moléculaires des ChE ont été séparées en gradient de saccharose 5-20% et localisées dans des cœurs gonflés par la gélatine et sur coupe à froid. La morphologie globale du cœur a été étudiée en coupes transversales colorées avec l’hématoxyline et l’éosine. Résultats et conclusions : La plus forte activité en AChE a été mesurée dans les oreillettes, la plus faible activité dans le ventricule gauche et le septum. Dans tous les compartiments, PRiMA AChE et ColQ AChE ont été observés. Chacune des formes ancrées est distribuée sur l’épicarde à la base du cœur, et sont co-localisées avec des neurones intracardiaques. PRiMA AChE forme des branches fines à proximité des neurones intracardiaques. L’absence d’ancrage de l’AChE aboutit à une diminution significative du diamètre des cardiomyocytes. L’activité de la BChE est plus élevée que celle de l’AChE. La plus haute activité en BChE a été détectée dans le ventricule gauche et le septum. Le monomère amphiphilic constitue la forme prédominante dans le cœur. Dans le myocarde, le marquage de l’activité BChE est diffus, alors que dans l’épicarde il colocalise avec un seul neurone intracardiaque. Dans ce travail, nous avons réalisé une étude complète des ChE dans le cœur. Nos résultats peuvent aider à définir de nouvelles thérapies pharmacologiques plus efficaces. / Introduction: The results of current research suggest that acetylcholine has a protective role during heart failure and atrial or ventricular fibrillation. Cholinesterases (ChE) control the level of acetylcholine and thus play an important role in the cholinergic system of the heart. However, detail information about these enzymes in the heart is still missing. The aim of this thesis was to provide a complex study of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the heart, specifically of their activities, molecular forms and precise localization. Methods: Mutant mice with lack of ChE or their anchoring proteins, ColQ and PRiMA, were used. AChE and BChE activities in heart compartments were determined by 2-step Ellman’s method, developed by us. Molecular forms of ChE were determined in 5-20 % sucrose gradients and localized in the hearts filled with gelatin and in the cryosections by activity staining method and by immunohistochemistry. Nerve and endothelial cells were identified using specific markers. Basic heart morphology was studied in the transversal sections stained with hematoxyline and eosine. Results and conclusion: The highest AChE activity was determined in the atria, the lowest activity in the left ventricle and septum. In all compartments, PRiMA AChE and ColQ AChE were observed. Both anchored forms were distributed epicardially on the heart base, co-localized with intracardiac neurons. PRiMA AChE formed a subtle branching in the proximity of intracardiac neurons. The lack of AChE anchored forms led to significantly lower cardiomyocyte diameter. The BChE activity was higher than that of AChE. The highest BChE activity was detected in the left ventricle and septum. Amphiphilic monomers were the predominant form of BChE in the heart. In myocardium, the staining of BChE activity was diffused, while in epicardium it co-localized with a single intracardiac neuron. In this work, we have provided a complex study of ChE in heart. Our results could help in the design of new, more effective pharmacotherapy, which may reduce morbidity and mortality of the patients with various heart diseases. / Úvod: Výsledky súčasného výskumu nasvedčujú protektívnemu vplyvu acetylcholínu pri srdcovom zlyhávaní, či pri predsieňovej a komorovej fibrilácií. Cholínesterázy (ChE) regulujú hladinu acetylcholínu a tak zohrávajú dôležitú úlohu v cholinergickom systéme srdca. Avšak, hlbšie informácie o týchto enzýmoch v srdci chýbajú. Cieľom tejto práce bolo komplexné štúdium acetylcholínesterázy (AChE) a butyrylcholínesterázy (BChE) v srdci, konkrétne ich aktivít, molekulových foriem a presnej lokalizácie. Metódy: V projekte sme používali mutantné myši s chýbaním ChE alebo ich kotviacich proteínov, ColQ a PRiMA. Aktivity AChE a BChE sme v jednotlivých častiach srdca stanovili nami vyvinutou modifikovanou dvoj-krokovou Ellmanovou metódou. Molekulové formy ChE sme študovali v 5-20 % sacharózových gradientoch. ChE sme lokalizovali v srdciach vyplnených želatínou a v kryostatických rezoch, a to pomocou aktivitného farbenia a imunohistochémiou. Nervové a endotelové bunky sme identifikovali na základe špecifických markerov. Základnú morfológiu tkaniva sme študovali v priečnych rezoch srdca nafarbených hematoxylínom a eozínom. Výsledky a záver: V predsieňach sme pozorovali najvyššiu a v ľavej komore a septe najnižšiu aktivitu AChE, kotvenú pomocou PRiMA aj ColQ proteínov. PRiMA AChE aj ColQ AChE boli v epikarde distribuované na báze srdca a ko-lokalizované s intrakardiálnymi neurónmi. PRiMA AChE vytvárala jemnú spleť v blízkosti intrakardiálnych neurónov. Chýbanie kotvených foriem AChE viedlo ku signifikantne menším priemerom kardiomyocytov. Aktivita BChE v srdci bola vyššia ako aktivita AChE, s najvyššou aktivitou v ľavej komore a septe. Predominatnou formou BChE v srdci boli amfifilné monoméry. BChE aktivita bola v myokarde lokalizovaná difúzne a v epikarde ko-lokalizovala s jedným intrakardiálnym neurónom. V predloženej práci sme poskytli komplexný obraz o ChE v srdci. Naše výsledky môžu vo významnej miere napomôcť v dizajne novej, efektívnejšej farmakoterapie, ktorá by mohla znížiť morbiditu a mortalitu pacientov s vybranými chorobami srdca.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Trypanosomiasis is a disease of worldwide distribution which in Brazil is called "mal das cadeiras", owing the clinical signs shown by horses, which are the major species affected. The disease is caused by the protozoan Trypanosoma evansi, characterized by clinical signs such as weight loss, pale mucous membranes, swelling of the eyelid and vulva. Rabbits have been reported as a resistant species to T. evansi, however, is not yet known how immune aspects, such as innate immune and cholinergic system, behave in this species, not even as this inflammations pathways influence in rabbits's trypanotolerance. Thus, the aim of this study was to investigate the inflammatory response and cholinergic anti-inflammatory pathway and its relation to trypanotolerance in rabbits infected with T. evansi. For this, twelve female adult New Zealand rabbits, weighing 3.6 4.5 kg were used in this study. The animals were divided into two groups, a control group and an infected group, both with six animals. The rabbits belonging to the infected group received, intraperitoneally, 0.5 ml of rat blood containing 108 parasites per animal, while the control group received physiological solution by the same route. The experimental period reached 118 days. Blood was collected on days 0, 2, 7, 12, 27, 42, 57, 87, 102, and 118, for to determine the cholinesterases, and days 0, 5, 20, 35, 65, 95 and 118, for to evaluate blood count, total protein, serum proteinogram, immunoglobulins and acute phase proteins. There was an increase in the activity of butyrylcholinesterase (BChE), at 7th day PI, and in the activity of acetylcholinesterase (AChE), in 27th day PI. Furthermore, the infected group showed an increase in total protein and the fractions alpha, beta and gamma globulins along of experimental period. The reduction in albumin and hematocrit were observed in precise periods of experimental infection, as well as the increase in immunoglobulin G. Infection with T. evansi stimulated the production of acute phase proteins, such as C-reactive protein, haptoglobin, α-2 macroglobulin, being observed increase in immunoglobulin M (IgM) throughout the experimental period (118 days post-infection). From these results, it is concluded that the cholinergic pathway had influence the inflammatory response, through the action of AChE and BChE in the regulation of concentrations of acetylcholine, resulting in increased concentrations of cytokines and, consequently, in the production phase protein acute. The increase in IgM, associated with increased C-reactive protein and haptoglobin, suggests the involvement of these proteins in host defense against flagellated, with possible participation in trypanotolerance of rabbits infected with T. evansi. / A tripanossomose é uma enfermidade de distribuição mundial que, no Brasil, é denominada mal das cadeiras , em função dos sinais clínicos apresentados pelos equinos, principal espécie afetada. A doença é causada pelo protozoário Trypanosoma evansi, sendo caracterizada por sinais clínicos como perda de peso, mucosas pálidas, edema de pálpebra e vulva. Coelhos têm sido relatados como uma espécie resistente ao T. evansi, contudo, ainda não se sabe de que modo aspectos imunitários, como a imunidade inata e o sistema colinérgico, comportam-se nessa espécie, nem mesmo como essas vias da inflamação influenciam a tripanotolerância de coelhos. Assim, o objetivo deste trabalho foi investigar a resposta inflamatória e a via anti-inflamatória colinérgica e sua relação na tripanotolerância em coelhos infectados experimentalmente pelo T. evansi. Para tanto, foram utilizados 12 coelhos adultos, fêmeas, da raça Nova Zelândia, com peso corporal entre 3,6 a 4,5 Kg, divididos em dois grupos, um grupo controle e um grupo infectado, ambos com seis animais. Os coelhos pertencentes ao grupo infectado receberam, pela via intraperitoneal, 0,5 mL de sangue de rato contendo 108 tripanossomas por animal, enquanto que o grupo controle recebeu, pela mesma via, solução fisiológica. O período experimental foi de 118 dias, sendo o sangue coletado nos dias 0, 2, 7, 12, 27, 42, 57, 87, 102, e 118, para determinar as colinesterases, e nos dias 0, 5, 20, 35, 65, 95 e 118, para avaliar hemograma, proteínas totais, proteinograma sérico, imunoglobulinas e proteínas de fase aguda. Foi observado aumento na atividade da butirilcolinesterase (BChE) no 7° dia PI e da atividade da acetilcolinesterase (AChE) no 27° dia PI. Além disso, o grupo infectado apresentou aumento nas proteínas totais, bem como nas frações alfa, beta e gama globulinas ao longo do período experimental. A redução na albumina e no hematócrito foram observados em períodos pontuais da infecção experimental, bem como o aumento na imunoglobulina G. A infecção pelo T. evansi estimulou a produção de proteínas de fase aguda como a proteína C-reativa, haptoglobina, α-2 macroglobulina, sendo observado um aumento na imunoglobulina M (IgM) em todo o período experimental (118 dias pós-infecção). A partir desses resultados, conclui-se que a via colinérgica pode ter influência na resposta inflamatória, através da ação da BChE e AChE na regulação das concentrações de acetilcolina, acarretando em aumento nas concentrações de citocinas e, consequentemente, na produção de proteínas de fase aguda. A elevação na IgM, relacionada ao aumento da proteína C-reativa e haptoglobina, sugere o envolvimento dessas proteínas na defesa do hospedeiro contra o flagelado, e, possivelmente, na tripanotolerância de coelhos infectados com T. evansi.