No description available.

The increase in the NTPDase and 5'-nucleotidase enzymatic activities, in patients with diabetes and hypertension, unchained the investigation of the possible mechanisms involved in the alterations in ectonucleotidases activities. The oxidative stress level and also the answer of the antioxidant systems were evaluated in patients with type 2 diabetes, hypertension and type 2 diabetes with associated hypertension. The interference of the glucose concentration was evaluated in the enzymatic activity of the ectonucleotidases, serum cholinesterase and also antioxidant enzymes. The curves in vitro demonstrated that the increase in enzymatic activity was proportional to the elevation in the glucose concentrations, demonstrating a direct interference of the hyperglycemia. The increase in the NTPDase expression demonstrated an important correlation with adenine nucleotide ATP and ADP hydrolyze in patients with diabetes and associated hypertension. The increment in markers of oxidative stress and antioxidant systems levels in patients with diabetes and associated hypertension seems to be related with a compensatory mechanism to prevent oxidative damages. Low serum acid ascorbic levels increase exposes to oxidative damages in patients with diabetes and associated hypertension, resultant of the increase in reactive oxygen species generation. The increment in the serum cholinesterase activity can be potentially related with glycemia levels and lipid metabolism in patients with diabetes and associated hypertension. The medicines administered to the patients did not alter the enzymatic responses in the analyzed groups. Therefore, there were a possible interference of the diabetes and hypertension in the catalytic mechanism of the serum cholinesterase enzyme. Data obtained in the studies permit to suggest that high blood glucose levels constitute one of the principal factors capable to promote alterations in the enzymatic responses in patients with diabetes and associated hypertension. / O aumento na atividade enzimática da NTPDase e 5'-nucleotidase, em pacientes com diabetes e hipertensão, desencadeou a investigação dos possíveis mecanismos envolvidos nas alterações na atividade das ectonucleotidases. O nível de estresse oxidativo e também a resposta dos sistemas antioxidantes foram avaliados em pacientes com diabetes tipo 2, hipertensão e diabetes tipo 2 com hipertensão associada. A interferência da concentração de glicose foi avaliada na atividade enzimática das ectonucleotidases, colinesterase sérica e também das enzimas antioxidantes. As curvas in vitro demonstraram que o aumento na atividade dos sistemas enzimáticos foi proporcional à elevação nas concentrações de glicose, demonstrando uma interferência direta da hiperglicemia. O aumento na expressão da enzima NTPDase demonstrou uma importante correlação com a hidrólise dos nucleotídeos de adenina ATP e ADP em pacientes com diabetes e hipertensão associada. O incremento nos níveis de marcadores de estresse oxidativo e dos sistemas antioxidantes, em pacientes com diabetes e hipertensão associada, parecem estar relacionados com um mecanismo compensatório para prevenir o dano oxidativo. Os baixos níveis de ácido ascórbico sérico aumentam a exposição dos pacientes, com diabetes e hipertensão associada, aos danos oxidativos resultantes do aumento na geração de espécies reativas de oxigênio. O aumento na atividade da enzima colinesterase sérica, em pacientes com diabetes e hipertensão associada, pode estar potencialmente relacionado com os níveis de glicemia e com o metabolismo dos lipídios. Os medicamentos administrados aos pacientes não alteraram as respostas enzimáticas nos grupos analisados. Portanto, houve uma possível interferência do diabetes e da hipertensão no mecanismo catalítico da colinesterase sérica. Os dados obtidos nos estudos permitem sugerir que os elevados níveis de glicose sangüínea constituem um dos principais fatores capazes de promover alterações nas respostas enzimáticas em pacientes diabéticos e com hipertensão associada.

Organophosphate (OP) poisoning is a health issue worldwide with over 200,000 deaths per year. Although not a problem in most developed countries, in some third world countries, one third of a hospital’s population could be patients with OP exposure. Even with the most aggressive therapy, 10-40% of patients admitted to an intensive care unit will die. Research into the best practice for treating OP poisoning is lacking, due somewhat to a lack of detailed understanding of the physiology of OP poisoning. Our research uses animal models of acute OP poisoning to explore the mechanism of OP-induced respiratory failure. Our research shows that animals poisoned with dichlorvos demonstrated a uniformly fatal central apnea that, if prevented, was followed immediately by a variable pulmonary dysfunction. Potential mechanisms for dichlorvos-induced central apnea can be divided into direct effects on the central respiratory oscillator (CRO) and feedback inhibition of the CRO. Two afferent pathways that can induce apnea include vagal feedback pathways and feed-forward pathways from the cerebral hemispheres. In our studies we found that vagal feedback and feed forward inhibition from the cerebral hemispheres were not required for OP-induced central apnea. The pre-Botzinger complex in the brainstem is thought to be the kernel of the CRO, but exposure of the pre-Botzinger complex to dichlorvos was not sufficient for apnea. Although OP induced central apnea was uniformly fatal, partial recovery of the CRO occurred post apnea with mechanical ventilation. Central apnea was ubiquitous in our rat poisoning model, but pulmonary dysfunction was extremely variable, with a range of pulmonary effects from fulminate pulmonary failure with prominent pulmonary secretions to no pulmonary dysfunction at all. Vagal efferent activity is involved in neural control of pulmonary tissue but the vagus was not involved in OP-induced pulmonary dysfunction. Anti-muscarinic medications are the mainstay of clinical therapy and are commonly dosed by their effects on pulmonary secretions. Our studies found that atropine (the most common therapeutic agent for OP poisoning) resulted in a ventilation-perfusion mismatch secondary to effects on the pulmonary vasculature.

Effects of developmental exposure to chlorpyrifos (CPS) or methyl parathion (MPS) on visuospatial, adaptive fear response, and passive avoidance memory and the signaling mechanisms responsible for these neurocognitive changes were investigated. Using an incremental low dose regimen, rat pups were orally gavaged daily with either corn oil (vehicle), CPS, or MPS from postnatal day 1 (PND1) -PND21. Cholinesterase activity was significantly inhibited with the highest dosages of CPS and MPS for up to 19 days after the last dosages. OP exposure impaired working and reference memory in males whereas in the females, enhancement occurred following CPS exposure. In addition, the adaptive fear response and passive avoidance retention memory was impaired in males whereas differential changes occurred in females. Accordingly, the behavioral deficits observed in males were persistent whereas the enhancement in females was transient. Males were more sensitive to OPs than females in that the medium and high dosages of CPS and MPS produced greater effects in females whereas all dosages of both compounds produced effects in males. Training in the radial arm maze significantly increased protein kinase C gamma (PKC ) expression and activity in the hippocampal membrane fraction of control rats whereas exposure to OPs exhibited a significant decrease in PKC and PKC immunoreactivity in both untrained and trained rats. However, MPS exposed females exhibited a significant increase in PKC expression in the cytosolic fraction but this was not related to improved memory. Reduction of membrane PKC expression and activity and cytosolic PKC expression and activity seemed to be related to visuospatial learning and memory deficits in exposed males but not exposed females. Brain-derived neurotrophic factor (BDNF) gene expression in the hippocampus was significantly increased (60%) in trained control males as compared to untrained control males. In contrast, trained and untrained females exhibited similar levels of BDNF gene expression. However, exposure of both sexes to either CPS or MPS significantly reduced the expression of BDNF in trained rats. In summary, these data indicate that OP exposure induced gender-specific changes in working memory formation and altered PKC isozyme levels/activity and BDNF expression.

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.

Estudos prévios demonstram que intervenções precoces levam a alterações comportamentais e neuroendócrinas na vida adulta. Nossos achados anteriores demonstram que animais manipulados no período neonatal consomem mais alimento palatável na vida adulta e apresentam um menor aumento do depósito de gordura abdominal, após exposição crônica a dieta palatável (chocolate) em relação aos animais intactos. Neste trabalho de tese, nosso objetivo foi avaliar os efeitos da manipulação neonatal sobre a preferência alimentar bem como sobre a regulação metabólica de ratas adultas. Para isso, investigamos parâmetros metabólicos e neuroquímicos em resposta à exposição crônica a uma dieta hipercalórica e palatável, assim como a um período de abstinência desse tipo de alimento em ratas fêmeas adultas expostas ou não à manipulação neonatal (10 min/dia, 10 primeiros dias de vida). A manipulação neonatal induziu maior ingestão de alimento palatável após um curto período de privação. Entretanto, o consumo durante a exposição crônica a essa dieta não diferiu entre os grupos experimentais. Também observamos que ratas fêmeas manipuladas, quando cronicamente expostas à dieta palatável na vida adulta, têm menor aumento da gordura abdominal e esse efeito persiste após a privação da dieta. Ratas não-manipuladas apresentaram níveis mais elevados de colinesterases no soro após exposição crônica a dieta palatável, entretanto, sem alterações na atividade de colinesterases no córtex cerebral. Foi identificado, após exposição crônica a dieta palatável, uma redução na atividade da enzima Na+,K+-ATPse no hipocampo e na amígdala e um aumento nos níveis plasmáticos de S100B em ratas não-manipuladas no período neonatal. Após as primeiras 24h de privação do alimento palatável, ratas fêmeas não-manipuladas demonstraram maior frequência de sinais de abstinência (tremores de cabeça) em comparação com ratas manipuladas no período neonatal. Assim, esses achados sugerem que a manipulação neonatal determina alterações persistentes no comportamento alimentar e previne algumas alterações periféricas e centrais induzidas pela exposição crônica a uma dieta hiperpalatável, modulando a resposta metabólica de modo a reduzir a vulnerabilidade de dano metabólico e neural. / Previous studies have demonstrated that an intervention early in life leads to behavior and neuroendocrine alterations in adulthood. According to our previous findings neonatallyhandled animals have an increased consumption of palatable food, as well as a lower increase in abdominal fat accumulation after being chronically exposed to a highly palatable diet (chocolate) as compared with intact rats. In the present study, our aim was to evaluate the effects of neonatal handling on food preference and metabolic regulation in adult female rats. Therefore, we investigated metabolic and neurochemical parameters in response to a chronic exposure to a highly palatable diet, and to its withdrawal in adult female rats exposed or not to neonatal handling procedure (10 min/day, 10 first days of life). We observed an effect of neonatal handling inducing an increased palatable food intake after one week of chocolate withdrawal. However, chocolate consumption during long-term exposure to this type of diet did not differ between experimental groups. After a 30-days-period of chocolate exposure, non-handled female rats exhibited an increased abdominal fat deposition in comparison to neonatally-handled rats, and this effect persisted even after chocolate withdrawal. Nonhandled rats had increased serum cholinesterase levels after chronic exposure to palatable diet, without alterations in cerebral cortex cholinesterase activity. We also observed that chocolate consumption lead to a reduced Na+,K+-ATPse activity in hippocampus and amygdala, as well as an increased plasma S100B levels in non-handled females rats. After the first 24h of chocolate withdrawal, non-handled female rats exhibited an increased frequency of head shakes, during the Open Field task, in comparison to handled rats. Therefore, these findings suggest that neonatal handling leads to persistent alterations in feeding behavior, and also prevents some peripheral and central alterations induced by chronic exposure to a highly palatable diet; modulating the metabolic response in order to reduce the vulnerability to metabolic and neuronal damage in adulthood.

Estudos prévios demonstram que intervenções precoces levam a alterações comportamentais e neuroendócrinas na vida adulta. Nossos achados anteriores demonstram que animais manipulados no período neonatal consomem mais alimento palatável na vida adulta e apresentam um menor aumento do depósito de gordura abdominal, após exposição crônica a dieta palatável (chocolate) em relação aos animais intactos. Neste trabalho de tese, nosso objetivo foi avaliar os efeitos da manipulação neonatal sobre a preferência alimentar bem como sobre a regulação metabólica de ratas adultas. Para isso, investigamos parâmetros metabólicos e neuroquímicos em resposta à exposição crônica a uma dieta hipercalórica e palatável, assim como a um período de abstinência desse tipo de alimento em ratas fêmeas adultas expostas ou não à manipulação neonatal (10 min/dia, 10 primeiros dias de vida). A manipulação neonatal induziu maior ingestão de alimento palatável após um curto período de privação. Entretanto, o consumo durante a exposição crônica a essa dieta não diferiu entre os grupos experimentais. Também observamos que ratas fêmeas manipuladas, quando cronicamente expostas à dieta palatável na vida adulta, têm menor aumento da gordura abdominal e esse efeito persiste após a privação da dieta. Ratas não-manipuladas apresentaram níveis mais elevados de colinesterases no soro após exposição crônica a dieta palatável, entretanto, sem alterações na atividade de colinesterases no córtex cerebral. Foi identificado, após exposição crônica a dieta palatável, uma redução na atividade da enzima Na+,K+-ATPse no hipocampo e na amígdala e um aumento nos níveis plasmáticos de S100B em ratas não-manipuladas no período neonatal. Após as primeiras 24h de privação do alimento palatável, ratas fêmeas não-manipuladas demonstraram maior frequência de sinais de abstinência (tremores de cabeça) em comparação com ratas manipuladas no período neonatal. Assim, esses achados sugerem que a manipulação neonatal determina alterações persistentes no comportamento alimentar e previne algumas alterações periféricas e centrais induzidas pela exposição crônica a uma dieta hiperpalatável, modulando a resposta metabólica de modo a reduzir a vulnerabilidade de dano metabólico e neural. / Previous studies have demonstrated that an intervention early in life leads to behavior and neuroendocrine alterations in adulthood. According to our previous findings neonatallyhandled animals have an increased consumption of palatable food, as well as a lower increase in abdominal fat accumulation after being chronically exposed to a highly palatable diet (chocolate) as compared with intact rats. In the present study, our aim was to evaluate the effects of neonatal handling on food preference and metabolic regulation in adult female rats. Therefore, we investigated metabolic and neurochemical parameters in response to a chronic exposure to a highly palatable diet, and to its withdrawal in adult female rats exposed or not to neonatal handling procedure (10 min/day, 10 first days of life). We observed an effect of neonatal handling inducing an increased palatable food intake after one week of chocolate withdrawal. However, chocolate consumption during long-term exposure to this type of diet did not differ between experimental groups. After a 30-days-period of chocolate exposure, non-handled female rats exhibited an increased abdominal fat deposition in comparison to neonatally-handled rats, and this effect persisted even after chocolate withdrawal. Nonhandled rats had increased serum cholinesterase levels after chronic exposure to palatable diet, without alterations in cerebral cortex cholinesterase activity. We also observed that chocolate consumption lead to a reduced Na+,K+-ATPse activity in hippocampus and amygdala, as well as an increased plasma S100B levels in non-handled females rats. After the first 24h of chocolate withdrawal, non-handled female rats exhibited an increased frequency of head shakes, during the Open Field task, in comparison to handled rats. Therefore, these findings suggest that neonatal handling leads to persistent alterations in feeding behavior, and also prevents some peripheral and central alterations induced by chronic exposure to a highly palatable diet; modulating the metabolic response in order to reduce the vulnerability to metabolic and neuronal damage in adulthood.

Estudos prévios demonstram que intervenções precoces levam a alterações comportamentais e neuroendócrinas na vida adulta. Nossos achados anteriores demonstram que animais manipulados no período neonatal consomem mais alimento palatável na vida adulta e apresentam um menor aumento do depósito de gordura abdominal, após exposição crônica a dieta palatável (chocolate) em relação aos animais intactos. Neste trabalho de tese, nosso objetivo foi avaliar os efeitos da manipulação neonatal sobre a preferência alimentar bem como sobre a regulação metabólica de ratas adultas. Para isso, investigamos parâmetros metabólicos e neuroquímicos em resposta à exposição crônica a uma dieta hipercalórica e palatável, assim como a um período de abstinência desse tipo de alimento em ratas fêmeas adultas expostas ou não à manipulação neonatal (10 min/dia, 10 primeiros dias de vida). A manipulação neonatal induziu maior ingestão de alimento palatável após um curto período de privação. Entretanto, o consumo durante a exposição crônica a essa dieta não diferiu entre os grupos experimentais. Também observamos que ratas fêmeas manipuladas, quando cronicamente expostas à dieta palatável na vida adulta, têm menor aumento da gordura abdominal e esse efeito persiste após a privação da dieta. Ratas não-manipuladas apresentaram níveis mais elevados de colinesterases no soro após exposição crônica a dieta palatável, entretanto, sem alterações na atividade de colinesterases no córtex cerebral. Foi identificado, após exposição crônica a dieta palatável, uma redução na atividade da enzima Na+,K+-ATPse no hipocampo e na amígdala e um aumento nos níveis plasmáticos de S100B em ratas não-manipuladas no período neonatal. Após as primeiras 24h de privação do alimento palatável, ratas fêmeas não-manipuladas demonstraram maior frequência de sinais de abstinência (tremores de cabeça) em comparação com ratas manipuladas no período neonatal. Assim, esses achados sugerem que a manipulação neonatal determina alterações persistentes no comportamento alimentar e previne algumas alterações periféricas e centrais induzidas pela exposição crônica a uma dieta hiperpalatável, modulando a resposta metabólica de modo a reduzir a vulnerabilidade de dano metabólico e neural. / Previous studies have demonstrated that an intervention early in life leads to behavior and neuroendocrine alterations in adulthood. According to our previous findings neonatallyhandled animals have an increased consumption of palatable food, as well as a lower increase in abdominal fat accumulation after being chronically exposed to a highly palatable diet (chocolate) as compared with intact rats. In the present study, our aim was to evaluate the effects of neonatal handling on food preference and metabolic regulation in adult female rats. Therefore, we investigated metabolic and neurochemical parameters in response to a chronic exposure to a highly palatable diet, and to its withdrawal in adult female rats exposed or not to neonatal handling procedure (10 min/day, 10 first days of life). We observed an effect of neonatal handling inducing an increased palatable food intake after one week of chocolate withdrawal. However, chocolate consumption during long-term exposure to this type of diet did not differ between experimental groups. After a 30-days-period of chocolate exposure, non-handled female rats exhibited an increased abdominal fat deposition in comparison to neonatally-handled rats, and this effect persisted even after chocolate withdrawal. Nonhandled rats had increased serum cholinesterase levels after chronic exposure to palatable diet, without alterations in cerebral cortex cholinesterase activity. We also observed that chocolate consumption lead to a reduced Na+,K+-ATPse activity in hippocampus and amygdala, as well as an increased plasma S100B levels in non-handled females rats. After the first 24h of chocolate withdrawal, non-handled female rats exhibited an increased frequency of head shakes, during the Open Field task, in comparison to handled rats. Therefore, these findings suggest that neonatal handling leads to persistent alterations in feeding behavior, and also prevents some peripheral and central alterations induced by chronic exposure to a highly palatable diet; modulating the metabolic response in order to reduce the vulnerability to metabolic and neuronal damage in adulthood.