The present work describes the phytochemistry study and of biological activity of the species Scutellaria racemosa Pers (Labiatae) and Pfaffia tuberosa Spreng (Amaranthaceae). Four compounds were isolated of the S. racemosa Pers: lupeol (14), oroxilin A (10), dinatin (12) and oroxyloside (11). The antimicrobial and cytotoxic activities and the capacity of inhibition of enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase IV (DPP IV) and acetylcholinesterase (AChE) of the crude extract (EB), fractions and isolated compounds of the S. racemosa Pers were evaluated. The n-hexane (FH) and ethyl acetate (FA) fractions were the most active against Staphylococcus epidermidis, Bacillus subtilis and Pseudomonas aeruginosa. The evaluation of the crude extract and fractions using the Brine Shrimp Lethality Test indicated that this plant does not present toxicity. About the tests of enzymatic inhibition, the ethyl acetate (FA) and n-butanol (FB) fractions of S. racemosa Pers and the compounds dinatin (12) and oroxyloside (11) demonstrated significant capacity of inhibition of the POP. The inhibition promoted for the dinatina (12) (100 μM) corresponded 43% and for the oroxyloside (11) (100 μM) corresponded 34% of the total enzyme tested. The crude extract (EB) and the respective fractions of the Pfaffia tuberosa Spreng were also evaluated about the antimicrobial and cytotoxic activities and of enzymatic inhibition of the POP, DPP IV and AChE. Through these assays, it was verified that the crude extract (EB) and the fractions of the Pfaffia tuberosa Spreng do not present important antimicrobial and cytotoxic activity. In relation to the tests of inibitory activity of the POP, the dichloromethane (FD) and ethyl acetate (FA) fractions present IC50 of 21.4 and 28.5 μg/mL against of POP, respectively. Dichloromethane, ethyl acetate and n-butanol fractions (FD, FA and FB) presented low activity against DPP IV (< 20%). The ethyl acetate (FA) and n-butanol (FB) fractions showed significant inhibition of the AChE in the amounts, 6.25 and 25 μg, respectively. / O presente trabalho descreve o estudo fitoquímico e a atividade biológica das espécies Scutellaria racemosa Pers (Labiatae) e Pfaffia tuberosa Spreng (Amaranthaceae). Quatro compostos foram isolados da S. racemosa Pers: lupeol (14), oroxilina A (10), dinatina (12) e oroxilosídeo (11). A atividade antimicrobiana, citotóxica e a capacidade de inibição das enzimas prolil oligopeptidase (POP), dipeptidil peptidase IV (DPP IV) e acetilcolinesterase (AChE) do extrato bruto (EB), frações e compostos isolados da S. racemosa Pers foram avaliadas. As frações n-hexano (FH) e acetato de etila (FA) foram as mais ativas contra Staphylococcus epidermidis, Bacillus subtilis e Pseudomonas aeruginosa. A avaliação do extrato bruto (EB) e frações através do teste de letalidade frente a Artemia salina indicou que esta planta não apresenta toxicidade significativa. Quanto aos testes de inibição enzimática, as frações acetato de etila (FA) e n-butanol (FB) de S. racemosa Pers e os compostos dinatina (12) e oroxilosídeo (11) demonstraram significativa capacidade de inibição da POP. A inibição promovida pela dinatina (12) (100 μM) correspondeu a 43% e pelo oroxilosídeo (11) (100 μM) correspondeu a 34% do total de enzima testada. O extrato bruto (EB) e as respectivas frações da Pfaffia tuberosa Spreng também foram avaliadas quanto a atividade antimicrobiana, citotóxica e de inibição enzimática da POP, DPP IV e AChE. Através destes ensaios, verificou-se que o extrato bruto (EB) e a frações da Pfaffia tuberosa Spreng não apresentam importante atividade antimicrobiana e citotóxica. Em relação aos testes de atividade inibitória da POP, as frações diclorometano (FD) e acetato de etila (FA) foram as que apresentaram os melhores resultados, com IC50 de 21,4 e 28,5 μg/mL, respectivamente. As frações diclorometano, acetato de etila e n-butanol (FD, FA e FB) apresentaram baixa capacidade de inibição da DPP IV (< 20%). As frações acetato de etila (FA) e n-butanol (FB) apresentaram significativa inibição da AChE, nas quantidades de 6,25 e 25 μg, respectivamente.

ColQ est un collagène spécifique qui ancre l’acétylcholinestérase (AChE) dans la fente synaptique de la jonction neuromusculaire (JNM). L'importance du complexe AChE-ColQ dans la physiologie humaine de cette synapse est soulignée par l’identification de mutations dans le gène codant pour ColQ qui conduisent à un syndrome myasthénique congénital (SMC) associé à une déficience en AChE. Le déficit en AChE a, jusqu’à présent, été considéré comme l’unique facteur responsable des symptômes observés chez les patients ainsi que des défauts de la JMN chez le modèle de souris SMC (souris déficiente pour ColQ). Toutefois, ces symptômes sont complexes et l’absence d’AChE ne peut probablement pas expliquer tous les symptômes. Nous avons montré auparavant que ColQ participait à la formation de la synapse ce qui expliquerait les symptômes observés chez les patients et la souris modèle. En effet, nous avons pu montrer que ColQ contrôle l’agrégation du récepteur à l’acétylcholine (RACh) et de l’expression de gènes spécifiques de la synapse. En particulier, nous avons montré in vitro et in vivo, que l’absence de ColQ induit une augmentation du niveau des ARNm codant pour toutes les sous-unités de RACh et une expression réduite du niveau de leurs protéines. Des résultats préliminaires indiquent que cette augmentation de ces ARNm n’est pas transcriptionnelle. L’objectif de cette thèse est d’expliquer les mécanismes qui induisent l’augmentation du niveau des ARNm de AChR en l’absence de ColQ et les voies de signalisation qui relient ColQ au métabolisme des ARN du RACh. Notre hypothèse de travail a été que l'absence de ColQ stimule la stabilisation post-transcriptionnelle des ARNm codant pour les sous-unités du RACh via la protéine HuR. HuR est une protéine qui stabilise les ARNm quand elle se fixe sur les AU-richelement (ARE) dans la séquence 3’UTR. HuR est une protéine clé dans la myogenèse et la formation de la JNM parce qu’elle stabilise de manière post-transcriptionnelle de nombreux transcrits tels que myogénine, MyoD et AChE. Dans cette étude, nous montrons pour la première fois qu’un mécanisme post-transcriptionnel de stabilisation des ARNm est responsable de l’augmentation du niveau des ARNm du RACh via ColQ. De plus, nous constatons qu’en absence de ColQ, il y a une augmentation aux niveaux d’ARNm et de protéine de HuR. HuR est également capable de se lier au domaine ARE dans le 3’UTR des ARNm des sous-unités de AChR. De plus, l’interaction entre HuR et les ARNm du RACh augmente la stabilité et par conséquence les niveaux des transcrits du RACh. Trois conclusions importantes ressortent de ma thèse : nous démontrons que (1) en plus de la régulation transcriptionnelle, il existe des mécanismes de régulation post-transcriptionnlle du RACh (2) ColQ régule la stabilité des ARNm RACh via HuR médiée par MuSK (3) la voie de signalisation p38 contrôle les niveaux de HuR de manière dépendante de ColQ. Ensemble, ces résultats donnent un aperçu des voies de signalisation du muscle qui sont affectées par les mutations de ColQ conduisant à des SMC avec une déficience en AChE. Nos résultats mettront en évidence des nouvelles cibles moléculaires spécifiques qui peuvent conduire au développement des interventions thérapeutiques dans le cadre de myasthénies congénitales. / ColQ is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction (NMJ). The importance of AChE-ColQ complex in the physiology of this synapse has been highlighted by the identification of COLQ mutations in the human gene, leading to a congenital myasthenic syndrome (CMS) with AChE deficiency. The lack of AChE has been incriminated for the symptoms observed in patients along with NMJ defects in the CMS mouse model (ColQ-deficient). However, symptoms observed in the patients and mouse model of CMS with AChE deficiency are complex and AChE deficiency cannot account for all of them. We have demonstrated that ColQ could play a role per se in synapse formation which would explain some of the defects observed in patients and model mice. Indeed, we have shown that ColQ controls the clustering of Acetylcholine Receptors (AChR) and the expression of a number of specific synaptic genes. The most striking effect of the absence of ColQ is an upregulation of all AChR subunit mRNAs correlated by an increase in their protein levels. Preliminary results indicate that AChR mRNA upregulation is not transcriptional. This thesis deciphers the mechanisms that drive AChR mRNA upregulation in the absence of ColQ and the pathways that connect ColQ to the AChR RNA metabolism. Accordingly, we hypothesize that the absence of ColQ induces an upregulation of the stabilization of AChR subunit mRNAs, a post-transcriptional mechanism mediated by HuR. HuR is an RNA binding protein which stabilizes its target transcript by binding AU-rich elements (AREs) in their 3’UTR. HuR is critical during skeletal myogenesis and post-synaptic NMJ formation due to its stabilization of such transcripts as myogenin, MyoD and AChE. In this study, we show for the first time that a post-transcriptional mechanism of AChR mRNA stabilization is responsible for the ColQ mediated increase of AChR mRNAs. In support of these findings, the absence of ColQ also increased HuR mRNA and protein levels. We demonstrate that HuR is capable of binding to conserved ARE elements in the 3’UTR of AChR subunit mRNA. HuR’s interaction with AChR mRNA increased the stability of the transcripts, resulting in an increase in mRNA levels. Three major conclusions emerge from my thesis: we provide evidence that (1) in addition to transcriptional and assembly regulation of AChR, post-transcriptional mechanisms of AChR mRNA exist (2) ColQ regulates HuR mediated AChR stability through MuSK and (3) the p38 signalling pathway controls the levels of HuR in a ColQ dependent manner. Collectively, our data provides insight into the muscle signaling pathways which are affected by ColQ mutations leading to CMS with AChE deficiency. Thus, we have identified specific new molecular targets that may become important for the development of therapeutic interventions for patients with CMS.

Membrane bound AChE from calf brain caudate nucleus was solubilized by use of ion-free media in presence of 10~ M EDTA and 10“5M tetracaine. The irreversible release of AChE was more effectively inhibited by divalent ions compared to monovalent ions added to the medium. EDTA appears to chelate divalent ions released from the tissue while tetracaine competes with the same ions at the membrane. The tetracaine effect is restricted to the procaine series of local anesthetics. Small amounts of soluble AChE are present in the cytosol fraction. In fresh preparations most of the enzyme appeared in a form having a molecular weight of 80.000 daltons as determined by gel filtration. The enzyme seems to be released in this form. It is proposed that this form represents the monomer form of the enzyme. In solution the AChE aggregates seemingly together with a factor that is released from the membrane in amounts stafchio- metric to the enzyme. By treatment with DEAE-Sephadex the enzyme preparation can be made non-aggregating. A highly purified nonaggregating monomeric AChE Specific activity 17150 micromoles acetylthiocholine hydrolyzed per minute at 27° C per mg protein) was obtained by affinity chromatography. Some anomalous binding phenomena was observed during the affinity chromatographic purification. The main observation was that edrophonium eluted crude enzyme preparation adsorbed to the affinity gel in a biphasic manner. It was found that in the crude preparation there is present besides unspecific material competing with the enzyme for the affinity gels a factor that increases the affinity of AChE to certain cholinergic ligands. Since the enzyme could be titrated by the factor it seems to have a very high affinity to the enzyme and the biphasic elution curve is explained by the presence of free as well as factor- bound enzyme in the preparation. In search for compounds having a similar effect it was found that fluoride ion too increased the affinity of AChE to the same ligands as the factor. The affinity of edrophonium to the site defined by the binding of AChE to MTA-CH (65x10“5m) is lower than that defined by the enzyme inhibitory constant (1.8xlO“7M). As an explanation of this finding it is proposed that the substrate induces a conformation having high affinity to edrophonium, a conformation that has a comparatively low relaxation rate. Thus acetylcholinesterase may be added to the list of enzymes that have hystere- tic properties. / <p>S. 1-54: sammanfattning, s. 55-100: 4 uppsatser</p> / digitalisering@umu

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cadmium (Cd) is considered one of the most toxic heavy metals for its ability to affect different tissues, including the brain and the immune system. The molecular mechanisms of toxicity of Cd are not well established, however, it is known that one of the consequences of Cd exposure is the generation of oxidative stress. Conversely, the quercetin, one flavonoid present in various foods performs various therapeutic functions in the body, such as antioxidant activity, anti-inflammatory and neuroprotective action. Thus, the aim of this study was to investigate the effects of quercetin on the behavioral tests, the activity of the enzymes acetylcholinesterase (AChE), Na+, K+-ATPase and the δ-dehydratase aminolevulinic acid (δ-ALA-D), as well as parameters of oxidative stress in the central nervous system of adult male wistar rats exposed to CdCl2. The activities of enzymes AChE, NTPDase and adenosine deaminase (ADA) of peripheral lymphocytes, butyrylcholinesterase (BuChE) in the serum and myeloperoxidase (MPO) in plasma were also measured in the peripheral system of these animals. The rats were exposed to CdCl2 (2.5 mg / kg) and quercetin (5, 25 or 50 mg / kg) by gavage (1 ml/kg) for 45 days. Hence, the animals were divided into eight groups (n = 10-14): saline/control, saline/Querc 5 mg/kg, saline/Querc 25 mg/kg, saline/Querc 50 mg/kg, Cd/ethanol, Cd/Querc 5 mg/kg, Cd/Querc 25 mg/kg and Cd/Querc 50 mg/kg. The groups treated with Cd and quercetin, received the antioxidant quercetin solution after 30 minutes of the administration of Cd solution. At the end of 45 days of the treatment the animals were submitted to training and behavioral tests. After, they were anesthetized by halothane inhalation, and blood collection was performed to set serum, plasma and peripheral lymphocytes apart. Then the animals were euthanized, with part of the brain being removed for analysis of the enzyme δ-ALA-D activity, while the other part of brain was dissected into, cerebral cortex, striatum, cerebellum, hippocampus and hypothalamus, for future enzymatic assays. The results showed that Cd is able to cross the blood brain barrier, therefore, although the amount of Cd accumulated in the different brain structures studied was low, it was significantly higher than in control. Simultaneous treatment of quercetin in Cd exposed animals was ineffective to decrease these levels of Cd. The Cd exposure caused impairment on learning and memory, besides causing an increase in the anxiogenic behavior type. Nevertheless, the treatment with quercetin prevented the undesirable effects caused by exposure to the metal in the anxiety and memory. In relation to enzymatic activities in the brain, it was observed that Cd exposure reduced AChE activity in cerebral cortex and hippocampus, while as activation of the enzyme was observed in hypothalamus. Furthermore, a decrease in the Na+, K+-ATPase enzyme activity in cerebral cortex, hippocampus and hypothalamus was observed, as well as a decrease in the δ-ALA-D activity in total brain of Cd exposed animals. Interestingly, the quercetin co-administration in the Cd exposed animals prevented the changes in the activity of the enzymes AChE and Na+, K+-ATPase in different brain structures, though has not restored the δ-ALA-D enzyme activity. It was also observed an increase in ROS production, in lipid peroxidation, in protein oxidation, the levels of double stranded DNA and changes in the antioxidant system, such as, reduction in the glutathione reductase (GR) activity, levels of total thiols (T-SH) and reduced glutathione (GSH), and an increase in the glutathione S-transferase (GST) enzyme activity in cerebral cortex, hypothalamus and hippocampus of Cd exposed animals. Co-administration of quercetin in Cd exposed rats was able to prevent totally or partially the changes caused by metal exposure in oxidative stress parameters. It is suggested that quercetin is able to reduce the oxidative damage caused by exposure to these metal and subsequently restore the AChE and Na+, K+-ATPase activities, modulating cholinergic neurotransmission and improving cognitive processes. In relation to the peripheral system, there was an increase in the NTPDase, ADA, AChE, BuChE and MPO activities in Cd exposed rats. Based on these results it is possible to infer that the increase in NTPDase activity is a compensatory effect due to the increase in ATP levels in circulation. It is suggested that decreased levels of ACh are available in the circulation due to increase in the peripheral cholinesterase activity. When rats were treated with the quercetin, flavonoid was able to modulate the activities of these enzymes probably due to the anti-inflammatory property of the compound. Accordingly, it is suggested that quercetin prevents or eases the toxicity caused by exposure to metal due to its antioxidant and anti-inflammatory activities. Therefore, it is believed that the flavonoid may be a promising drug in alternative therapies against toxicity induced by the metal in the central nervous system and peripheral system. / O cádmio (Cd) é considerado um dos metais pesados de maior toxicidade devido a sua capacidade de afetar diferentes tecidos, incluindo o encéfalo, bem como o sistema imunológico. Os mecanismos moleculares de toxicidade do Cd ainda não estão bem estabelecidos, contudo, sabe-se que uma das consequências da exposição ao Cd é a geração de estresse oxidativo. Por outro lado, a quercetina, um flavonoide presente em vários alimentos, exerce diversas funções terapêuticas no organismo, como atividade antioxidante, anti-inflamatória e ação neuroprotetora. Sendo assim, o objetivo do presente estudo foi investigar os efeitos da quercetina sobre os testes comportamentais, a atividade das enzimas acetilcolinesterase (AChE), a Na+,K+-ATPase e a δ-desidratase aminolevulinato (δ-ALA-D), bem como os parâmetros de estresse oxidativo no sistema nervoso central de ratos machos wistar adultos expostos ao CdCl2. Também foi avaliada, no sistema periférico destes animais, a atividade das enzimas AChE, NTPDases e adenosina desaminase (ADA) de linfócitos periféricos, butirilcolinesterase (BuChE) do soro e a mieloperoxidase (MPO) do plasma. Os ratos foram expostos ao CdCl2 (2,5 mg/kg) e quercetina (5, 25 ou 50 mg/kg) por gavagem (1ml/kg) durante 45 dias. Para isso, os animais foram distribuídos em oito grupos (n=10-14): salina/controle, salina/Querc 5mg/kg, salina/Querc 25 mg/kg, salina/Querc 50 mg/kg, Cd/etanol, Cd/ Querc 5mg/kg, Cd/Querc 25mg/kg e Cd/Querc 50 mg/kg. Os grupos tratados com Cd e quercetina receberam a solução antioxidante após 30 minutos da administração da solução de Cd. No final do período de 45 dias de tratamento os animais foram submetidos aos treinos e aos testes comportamentais. Posteriormente, foram anestesiados, através da inalação de halotano, e foi realizada a coleta de sangue e separação de soro, plasma e linfócitos periféricos. Em seguida os animais foram submetidos à eutanásia, com parte do encéfalo sendo retirada para a análise da atividade da enzima δ-ALA-D, enquanto que outra parte foi dissecada em córtex cerebral, estriado, cerebelo, hipocampo e hipotálamo, para posteriores ensaios enzimáticos. Os resultados obtidos mostraram que o Cd é capaz de atravessar a barreira hematoencefálica, pois, embora a quantidade de Cd acumulada nas diferentes estruturas encefálicas estudadas tenha sido baixa, ainda assim, foi significativamente maior que o controle. O tratamento concomitante da quercetina nos animais expostos ao Cd foi ineficiente em diminuir estes níveis de Cd. A exposição ao Cd causou prejuízos na aprendizagem e memória, além de causar um aumento no comportamento do tipo ansiogênico. Por outro lado, o tratamento com a quercetina preveniu os efeitos indesejáveis causados pela exposição ao metal na ansiedade e memória. Em relação às atividades enzimáticas no encéfalo, verificou-se que a exposição ao Cd reduziu a atividade da enzima AChE no córtex cerebral e no hipocampo, enquanto que uma ativação da enzima foi observada no hipotálamo. Além disso, observou-se uma diminuição na atividade da enzima Na+, K+-ATPase no córtex cerebral, hipocampo e hipotálamo, bem como uma diminuição na atividade da δ-ALA-D no encéfalo total de animais expostos ao Cd. Interessantemente, a co-administração com a quercetina em animais expostos ao Cd impediu as alterações na atividade das enzimas AChE e Na+, K+-ATPase em diferentes estruturas encefálicas, embora não tenha restaurado a a atividade da enzima δ-ALA-D. Verificou-se, também, um aumento na produção de ROS, na lipoperoxidação, na oxidação de proteínas, nos níveis de DNA dupla fita e alterações no sistema antioxidante, como a diminuição na atividade da enzima glutationa redutase (GR), nos níveis de tióis totais (T-SH) e glutationa reduzida (GSH), e um aumento na atividade da enzima glutationa S-transferase (GST) no córtex cerebral, hipocampo e hipotálamo dos animais expostos ao Cd. A co-administração da quercetina nos ratos expostos ao Cd foi capaz de impedir totalmente ou parcialmente as alterações causadas pela exposição ao metal nos parâmetros do estresse oxidativo. Sugere-se que a quercetina é capaz de diminuir o dano oxidativo causado pela exposição ao metal e, subsequentemente, restaurar a atividade da AChE e Na+, K+-ATPase, modulando, assim, a neurotransmissão colinérgica e melhorando os processos cognitivos. Em relação ao sistema periférico, verificou-se um aumento na atividade das enzimas NTPDase, ADA, AChE, BuChE e MPO nos ratos expostos ao Cd. A partir desse resultado pode-se inferir que o aumento na atividade da NTPDase seja um efeito compensatório devido ao aumento dos níveis de ATP na circulação. Sugere-se que níveis diminuídos de ACh estão disponíveis na circulação devido ao aumento na atividade das colinesterases periféricas. Quando os ratos foram tratados com quercetina o flavonoide foi capaz de modular a atividade dessas enzimas provavelmente devido à propriedade anti-inflamatória do composto. Deste modo, propõe-se que a quercetina previne ou ameniza a toxicidade causada pela exposição ao metal devido a sua atividade antioxidante e anti-inflamatória. Logo, acredita-se que este flavonoide possa ser um fármaco promissor em terapias alternativas contra a toxicidade induzida pelo metal no sistema nervoso central e periférico.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Mgr. Kateřina Breiterová Supervisor: Assoc. prof. Ing. Lucie Cahlíková PhD. Title of Doctoral Thesis: Alkaloids of the Amaryllidaceae family as potential drugs in therapy of diseases of affluence Key words: alkaloids, Amaryllidaceae, analogues, AChE, BuChE, POP, GSK-3β, cell cycle progression, apoptosis Narcissus cv. Professor Einstein was chosen based on results of previous screening studies for detailed phytochemical work for the purpose of isolation of the widest range of AmA. From 34,3 kg of fresh bulbs was obtained 31,7 g of purified alkaloidal extract, which was processed using column chromatography with stepwise elution by light petrol, chloroform and ethanol in different ratios to almost 500 fractions. These fractions were fused into 27 subfractions, which were processed by preparative TLC, vacuum column chromatography and crystallization. Finally, 25 pure alkaloids were isolated. All compounds were identified by GC-MS, ESI-MS, NMR, optical rotation and literature. One compound was identified as a new unpublished alkaloid of lycorine structure type. All alkaloids isolated in sufficient amount were tested for their biological activities associated with Alzheimer's disease (inhibition of...

The enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are inhibited by nerve agents such as sarin and tabun. In general, the inhibited enzymes are reactivated by bisquaternary ammonium compounds (oximes). The binding free energies of the oximes; 2-PAM, MMB-4, HI-6, and obidoxime bound to human AChE (hAChE) and human BChE (hBChE) inhibited by sarin and tabun and also to the uninhibited enzymes were calculated using various computational methods. Using thermodynamic integration, the binding free energies of all the inhibited and uninhibited systems of MMB-4 and obidoxime were evaluated. The standard binding free energies (dA) were more negative than the experimental values due to limitations of the ff99 forcefield. The RMS error of dA for the inhibited systems of MMB-4 was 2.1 kcal/mol, and for obidoxime systems it was 4.8 kcal/mol with respect to the experimental free energies. The binding enthalpies calculated using MM-GBSA and MM-PBSA methods for 2-PAM, MMB-4, HI-6, and obidoxime systems were negative, except for hBChE-sarin-MMB-4 and hBChE-sarin-obidoxime. For all the systems the TdS values calculated using normal mode analysis were equal to or lower in magnitude than their corresponding binding enthalpies. As a result, the estimated free energies were positive for most of the systems. Clearly, the present algorithms cannot effectively estimate the binding entropies for a protein-ligand system. Met81 has commonly shown favorable interactions, and lysine or arginine exhibited unfavorable interactions with the reactivator in all the systems. Second, the interactions between chloropyrifos-oxon (Cpo) and experimentally tested neutral and monopyridinium oximes bound to the Q192 or R192 polymorphs of human paraoxonase1 (hPON1) were studied. The equilibrated Q192 and R192 hPON1 were structurally different than the crystal structure of recombinant PON1. The neutral oximes have shown more favorable interactions with Cpo in Q192 hPON1 + Cpo system compared to R192 hPON1 + Cpo. Whereas the monopyridinium oximes interacted more affectively with Cpo in R192 hPON1 than Q192 hPON1. The relative deprotonation energy of the monopyridinium oxime was lower than the neutral oxime. Hence, the monopyridinium oxime can hydrolyze an organophosphate at a higher rate than a neutral oxime.

In this thesis, we have employed two computational methods, molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) MD simulations with umbrella sampling (US), to gain insights into the molecular mechanism governing the molecular recognition and reactivity in several protein-ligand complexes. Three systems involving protein-ligand interactions are examined in this dissertation utilizing well-established computational methodologies and mathematical modeling. The three proteins studied here are acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1). These enzymes are known to interact with a variety of ligands. AChE dysfunction caused by organophosphorus (OP) chemicals is a severe hazard since AChE is a critical enzyme in neurotransmission. Oximes are chemical compounds that can reactivate inhibited AChE; hence in the development of better oximes, it is critical to understand the mechanism through which OPs block AChE. We have described the covalent inhibition mechanism between AChE and the OP insecticide phorate oxon and its more potent metabolites and established their free energy profiles using QM/MM MD-US for the first time. Our results suggest a concerted mechanism and provide insights into the challenges in reactivating phorate oxon inhibited AChE. Reactivating BChE is another therapeutic approach to detoxifying circulating OP molecules before reaching the target AChE. We explored the covalent modification of BChE with phorate oxon and its metabolites using hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling simulations (PM6/ff14SB) for the inhibition process. Our results reveal that the mechanism is distinct between the inhibitors. The PM6 methodology is a good predictor of these compounds' potency, which may efficiently help study OPs like phorate oxon with larger leaving groups. Finally, we investigated the interactions between Peptidyl-prolyl isomerase (PPIase), which consists of a peptidyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) protein-binding domain, and chimeric peptides based on the human histone H1.4 sequence (KATGAApTPKKSAKW), as well as the effects on inter-domain dynamics. Using explicit solvent MD simulations, simulated annealing, and native contact analysis, our modeling sugget that the residues in the N-terminal immediate to the pSer/Thr Pro site connect the PPIase and WW domains via a series of hydrogen bonds and native contacts.

Extensive use of the organophosphate diazinon has led to its accumulation in aquatic environments and negative effects on fish health. Most studies focus on the effects of short term exposure to high levels of organophosphate pesticides. This research was conducted to assess the effect of chronic sub-lethal exposure to 0, 0.01, and 0.1 mg/L diazinon for 15 and 30 days on blood parameters and histopathological damage in alligator gar, Atractosteus spatula. Fish exposed to either concentration were motionless, produced excess mucous, had lighter skin color, and had skin lesions. Blood indices of red blood cells, leukocytes and hematocrit values significantly decreased but there was no significant change in mean cell volume. Hemoglobin values significantly increased in fish exposed to the low dose for 15 days, but significantly decreased for the other exposure doses and times. On the contrary, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration values significantly increased in fish exposed to both concentrations and duration times. Leukocyte differentials showed significant decreases in lymphocytes with significant increases of monocytes in fish exposed to the high dose in both exposure times. The most frequent biomarker for exposure to organophosphate pesticides is the inhibition of cholinesterase (ChE). Diazinon significantly reduced plasma ChE activity in a dose-dependent manner, with 62% and 72% in the low and high concentrations for 30 days, respectively. Plasma ChE could be determined from peripheral blood samples and did not require sacrifice of the fish. Fish exposed to either diazinon concentration or duration time developed histopathological changes in skin, gills, liver, and kidney tissues. The skin lesions were on the head and body, and progressed into deep ulcerations. The histopathological changes in the liver included hepatic vacuoles, swollen hepatocytes, steatosis, aggregation of macrophages, necrosis, and hepatic fibrosis. Also, exposed fish demonstrated vacuolar degeneration in the hematopoietic tissues of the kidney. Gills showed epithelial hyperplasia in the secondary lamellae. In conclusion, long term exposure to sub-lethal concentrations of diazinon induced significant changes in hematological indices and histopathological alteration in various tissues. Plasma ChE can be used to monitor diazinon exposure in wild gar populations.

O envelhecimento é um processo no qual ocorrem alterações estruturais e funcionais da maioria dos órgãos, que podem levar ao aumento da susceptibilidade a várias doenças associadas à idade. Assim, várias estratégias têm sido investigadas a fim de se reduzir os sintomas relacionados à idade e o exercício físico tem demonstrado efeito neuroprotetor em diferentes modelos experimentais. Nesta tese, investigamos os efeitos do exercício físico moderado sobre a memória e sobre parâmetros bioquímicos no hipocampo e no músculo sóleo de ratos Wistar de 3, 6 e 22 meses de idade. Para isso, foram realizados três experimentos distintos que deram origem aos três capítulos apresentados na tese. No primeiro experimento, estudamos o efeito do exercício físico em ratos de 3 e 22 meses de idade. Neste experimento, o exercício preveniu o déficit de aquisição da memória de referência relacionado à idade. Além disso, preveniu o aumento do estresse oxidativo no hipocampo de ratos envelhecidos e também promoveu o aumento da expressão dos fatores neurotróficos BDNF, NT-3 e IGF-1 no hipocampo destes animais. É importante ressaltar que houve uma correlação positiva entre a redução do estresse oxidativo e a latência para encontrar a plataforma no 5º dia de treino na tarefa de memória de referência, ou seja, a redução do conteúdo de espécies reativas e da lipoperoxidação pelo exercício está correlacionada com a melhora do desempenho de memória dos ratos envelhecidos. No segundo experimento, avaliamos o efeito do exercício físico em ratos de 3, 6 e 22 meses de idade. Corroborando com os resultados apresentados no experimento anterior, foi demonstrado que o exercício físico moderado preveniu os déficits de memória espacial de referência e de trabalho relacionados à idade. O treinamento cognitivo no Water maze aumentou a atividade das enzimas Na+,K+- ATPase e AChE no hipocampo de ratos adultos e envelhecidos. O aumento na atividade da Na+,K+-ATPase foi ainda maior nos ratos envelhecidos submetidos ao exercício físico combinado com o treinamento cognitivo. Além disso, foi observada uma correlação positiva entre a atividade da Na+,K+-ATPase no hipocampo dos ratos envelhecidos exercitados e a latência para encontrar a plataforma no 5º dia de treino na tarefa de memória de referência, ou seja, o aumento da atividade da Na+,K+-ATPase está associado com a melhora do desempenho de memória relacionado ao exercício físico. De acordo com esses dados, também foi observada uma correlação negativa entre a atividade da Na+,K+-ATPase e a diferença (delta) entre a média das latências entre os trials 1 e 4 na tarefa de memória de trabalho, o que demonstra que os ratos envelhecidos exercitados apresentaram um melhor desempenho na tarefa de memória de trabalho associado com o aumento na atividade da Na+,K+-ATPase. No terceiro experimento, investigamos o efeito do exercício físico em ratos de 3 e 22 meses de idade. O exercício aumentou o conteúdo de espécies reativas e a lipoperoxidação no músculo sóleo de ratos jovens. Ratos envelhecidos apresentaram um aumento da lipoperoxidação e uma redução na atividade da enzima catalase. O exercício induziu um aumento dos níveis de espécies reativas, uma redução no conteúdo de sulfidrilas e o aumento de proteínas carboniladas; contudo, promoveu o aumento da atividade das enzimas superóxido dismutase e catalase no sóleo dos ratos envelhecidos. Assim, os resultados do primeiro e do segundo experimento demonstram que o exercício físico preveniu o declínio da memória espacial relacionado à idade e que esse efeito pode ser mediado por fatores que incluem a redução do estresse oxidativo, o aumento da expressão de fatores neurotróficos e o aumento da atividade da enzima Na+,K+-ATPase no hipocampo de ratos envelhecidos. Os resultados do músculo demonstram que o sóleo dos ratos jovens, embora susceptível ao aumento das espécies reativas e lipoperoxidação, não apresentou dano às proteínas, sugerindo que outros mecanismos, como o sistema de defesa antioxidante não enzimático, possam estar atuando para compensar os efeitos do exercício. Além disso, o músculo dos ratos envelhecidos parece ser mais sensível que o dos ratos jovens às alterações do estado oxidativo celular induzidas pelo exercício físico, porque apesar dos animais envelhecidos exercitados apresentarem um aumento na atividade das enzimas antioxidantes, não houve uma redução do dano oxidativo. / Aging is a process in which structural and functional changes occur in most organs and may lead to increased susceptibility to various age-related diseases. Several approaches have been investigated with the aim of reducing age-related symptoms and physical exercise is a therapeutic strategy that has presented neuroprotective action in different experimental models. In this context, some studies show that regular physical exercise is related to the improvement of quality of life and to the prevention of age-related cognitive decline. In the present thesis, we investigated the effect of moderate physical exercise on memory and on biochemical parameters in the hippocampus and soleus muscle in 3, 6 and 22 months-old rats. For that, three different experiments were carried out, which gave rise to the three chapters presented in this thesis. In the first experiment, we studied the effect of physical exercise in 3 and 22 months-old rats. In this experiment, the exercise prevented the age-related acquisition deficit of reference memory. In addition, exercise prevented the increased in oxidative stress and also was able to increase the expression of neurotrophic factors BDNF, NT-3 and IGF-1 in the hippocampus of aged rats. It is important to note that there was a positive correlation between the reduction of oxidative stress and latency to find the platform on the 5th day of training in the reference memory task, i.e., reduction of reactive species levels and lipid peroxidation, might be associated with the exercise-related memory improvement. In the second experiment, we evaluated the effect of physical exercise in 3, 6 and 22 months-old rats. Corroborating with the results presented in the previous experiment, it was demonstrated that moderate physical exercise prevented age-related spatial reference and working memory deficits. It has also been shown that the cognitive training in Water maze increased the activity of the Na+,K+-ATPase and AChE enzymes in the hippocampus of adult and aged rats. The increase in Na+,K+-ATPase activity was even further increased in aged rats that were submitted to physical exercise combined with cognitive training. In addition, a positive correlation was observed between the Na+,K+-ATPase activity in the hippocampus of aged exercised rats and the latency to find the platform on the 5th day of training in the reference memory task, i.e., the increase in Na+,K+-ATPase activity is associated with the exercise-related memory improvement in aged rats. Consistently, a negative correlation between the Na+,K+-ATPase activity and the difference (delta) between the mean latencies of trials 1 and 4 in the working memory task was also found, i.e., the exercised aged rats showed better performance in the working memory task associated with the increase in Na+,K+-ATPase activity. In the third experiment, we investigated the effect of physical exercise in 3 and 22 months-old rats. Exercise increased the reactive species content and lipid peroxidation in soleus muscle of young rats. Aged rats showed an increase in lipid peroxidation and a reduction in the catalase activity. Exercise induced an increase in reactive species levels, a reduction in sulfhydryl content and an increase in carbonyl proteins; however, the exercise was able to increase the superoxide dismutase and catalase activities in the soleus of aged rats. Thus, the results of first and second experiments demonstrate that physical exercise prevents the age-related decline of spatial memory and this effect might be related to the reduction of oxidative stress, increased expression of neurotrophic factors and the increase in the Na+,K+-ATPase activity in the hippocampus of aged rats. The muscle results demonstrate that soleus of young rats, although susceptible to the increased in reactive species and lipid peroxidation, showed no damage to proteins, suggesting that other mechanisms, such as the non-enzymatic antioxidant defense system, may be acting to compensate the effects of exercise. In addition, the muscle of the aged rats seems to be more sensitive than the young rats to changes in the cellular oxidative state induced by exercise, since aged exercised animals showed an increase in the activity of antioxidant enzymes, but there was no reduction of oxidative damage.

O envelhecimento é um processo no qual ocorrem alterações estruturais e funcionais da maioria dos órgãos, que podem levar ao aumento da susceptibilidade a várias doenças associadas à idade. Assim, várias estratégias têm sido investigadas a fim de se reduzir os sintomas relacionados à idade e o exercício físico tem demonstrado efeito neuroprotetor em diferentes modelos experimentais. Nesta tese, investigamos os efeitos do exercício físico moderado sobre a memória e sobre parâmetros bioquímicos no hipocampo e no músculo sóleo de ratos Wistar de 3, 6 e 22 meses de idade. Para isso, foram realizados três experimentos distintos que deram origem aos três capítulos apresentados na tese. No primeiro experimento, estudamos o efeito do exercício físico em ratos de 3 e 22 meses de idade. Neste experimento, o exercício preveniu o déficit de aquisição da memória de referência relacionado à idade. Além disso, preveniu o aumento do estresse oxidativo no hipocampo de ratos envelhecidos e também promoveu o aumento da expressão dos fatores neurotróficos BDNF, NT-3 e IGF-1 no hipocampo destes animais. É importante ressaltar que houve uma correlação positiva entre a redução do estresse oxidativo e a latência para encontrar a plataforma no 5º dia de treino na tarefa de memória de referência, ou seja, a redução do conteúdo de espécies reativas e da lipoperoxidação pelo exercício está correlacionada com a melhora do desempenho de memória dos ratos envelhecidos. No segundo experimento, avaliamos o efeito do exercício físico em ratos de 3, 6 e 22 meses de idade. Corroborando com os resultados apresentados no experimento anterior, foi demonstrado que o exercício físico moderado preveniu os déficits de memória espacial de referência e de trabalho relacionados à idade. O treinamento cognitivo no Water maze aumentou a atividade das enzimas Na+,K+- ATPase e AChE no hipocampo de ratos adultos e envelhecidos. O aumento na atividade da Na+,K+-ATPase foi ainda maior nos ratos envelhecidos submetidos ao exercício físico combinado com o treinamento cognitivo. Além disso, foi observada uma correlação positiva entre a atividade da Na+,K+-ATPase no hipocampo dos ratos envelhecidos exercitados e a latência para encontrar a plataforma no 5º dia de treino na tarefa de memória de referência, ou seja, o aumento da atividade da Na+,K+-ATPase está associado com a melhora do desempenho de memória relacionado ao exercício físico. De acordo com esses dados, também foi observada uma correlação negativa entre a atividade da Na+,K+-ATPase e a diferença (delta) entre a média das latências entre os trials 1 e 4 na tarefa de memória de trabalho, o que demonstra que os ratos envelhecidos exercitados apresentaram um melhor desempenho na tarefa de memória de trabalho associado com o aumento na atividade da Na+,K+-ATPase. No terceiro experimento, investigamos o efeito do exercício físico em ratos de 3 e 22 meses de idade. O exercício aumentou o conteúdo de espécies reativas e a lipoperoxidação no músculo sóleo de ratos jovens. Ratos envelhecidos apresentaram um aumento da lipoperoxidação e uma redução na atividade da enzima catalase. O exercício induziu um aumento dos níveis de espécies reativas, uma redução no conteúdo de sulfidrilas e o aumento de proteínas carboniladas; contudo, promoveu o aumento da atividade das enzimas superóxido dismutase e catalase no sóleo dos ratos envelhecidos. Assim, os resultados do primeiro e do segundo experimento demonstram que o exercício físico preveniu o declínio da memória espacial relacionado à idade e que esse efeito pode ser mediado por fatores que incluem a redução do estresse oxidativo, o aumento da expressão de fatores neurotróficos e o aumento da atividade da enzima Na+,K+-ATPase no hipocampo de ratos envelhecidos. Os resultados do músculo demonstram que o sóleo dos ratos jovens, embora susceptível ao aumento das espécies reativas e lipoperoxidação, não apresentou dano às proteínas, sugerindo que outros mecanismos, como o sistema de defesa antioxidante não enzimático, possam estar atuando para compensar os efeitos do exercício. Além disso, o músculo dos ratos envelhecidos parece ser mais sensível que o dos ratos jovens às alterações do estado oxidativo celular induzidas pelo exercício físico, porque apesar dos animais envelhecidos exercitados apresentarem um aumento na atividade das enzimas antioxidantes, não houve uma redução do dano oxidativo. / Aging is a process in which structural and functional changes occur in most organs and may lead to increased susceptibility to various age-related diseases. Several approaches have been investigated with the aim of reducing age-related symptoms and physical exercise is a therapeutic strategy that has presented neuroprotective action in different experimental models. In this context, some studies show that regular physical exercise is related to the improvement of quality of life and to the prevention of age-related cognitive decline. In the present thesis, we investigated the effect of moderate physical exercise on memory and on biochemical parameters in the hippocampus and soleus muscle in 3, 6 and 22 months-old rats. For that, three different experiments were carried out, which gave rise to the three chapters presented in this thesis. In the first experiment, we studied the effect of physical exercise in 3 and 22 months-old rats. In this experiment, the exercise prevented the age-related acquisition deficit of reference memory. In addition, exercise prevented the increased in oxidative stress and also was able to increase the expression of neurotrophic factors BDNF, NT-3 and IGF-1 in the hippocampus of aged rats. It is important to note that there was a positive correlation between the reduction of oxidative stress and latency to find the platform on the 5th day of training in the reference memory task, i.e., reduction of reactive species levels and lipid peroxidation, might be associated with the exercise-related memory improvement. In the second experiment, we evaluated the effect of physical exercise in 3, 6 and 22 months-old rats. Corroborating with the results presented in the previous experiment, it was demonstrated that moderate physical exercise prevented age-related spatial reference and working memory deficits. It has also been shown that the cognitive training in Water maze increased the activity of the Na+,K+-ATPase and AChE enzymes in the hippocampus of adult and aged rats. The increase in Na+,K+-ATPase activity was even further increased in aged rats that were submitted to physical exercise combined with cognitive training. In addition, a positive correlation was observed between the Na+,K+-ATPase activity in the hippocampus of aged exercised rats and the latency to find the platform on the 5th day of training in the reference memory task, i.e., the increase in Na+,K+-ATPase activity is associated with the exercise-related memory improvement in aged rats. Consistently, a negative correlation between the Na+,K+-ATPase activity and the difference (delta) between the mean latencies of trials 1 and 4 in the working memory task was also found, i.e., the exercised aged rats showed better performance in the working memory task associated with the increase in Na+,K+-ATPase activity. In the third experiment, we investigated the effect of physical exercise in 3 and 22 months-old rats. Exercise increased the reactive species content and lipid peroxidation in soleus muscle of young rats. Aged rats showed an increase in lipid peroxidation and a reduction in the catalase activity. Exercise induced an increase in reactive species levels, a reduction in sulfhydryl content and an increase in carbonyl proteins; however, the exercise was able to increase the superoxide dismutase and catalase activities in the soleus of aged rats. Thus, the results of first and second experiments demonstrate that physical exercise prevents the age-related decline of spatial memory and this effect might be related to the reduction of oxidative stress, increased expression of neurotrophic factors and the increase in the Na+,K+-ATPase activity in the hippocampus of aged rats. The muscle results demonstrate that soleus of young rats, although susceptible to the increased in reactive species and lipid peroxidation, showed no damage to proteins, suggesting that other mechanisms, such as the non-enzymatic antioxidant defense system, may be acting to compensate the effects of exercise. In addition, the muscle of the aged rats seems to be more sensitive than the young rats to changes in the cellular oxidative state induced by exercise, since aged exercised animals showed an increase in the activity of antioxidant enzymes, but there was no reduction of oxidative damage.