Ever since the advent of computer-aided drug design (CADD), in silico simulation methods have greatly accelerated the drug discovery process and lead to the discovery of numerous drug candidates. With the exponential growth of computational power, we nowadays simulate biologic systems at a scale unimaginable a decade ago and thus provides perspectives for drug design. In this dissertation research, combining in silico simulation methods like molecular docking and molecular dynamics (MD) simulation with organic synthesis, in vitro/in vivo experiments and clinical data mining, we developed new drug discovery strategies. These strategies were applied in our drug discovery projects and led to the discovery of inhibitors of microsomal prostaglandin E2 synthase 1 (mPGES-1) and butyrylcholinesterase (BChE) as potential drug candidates. Protein mPGES-1 is known as an ideal target for next generation of anti-inflammatory drugs without the side-effects of currently available anti-inflammatory drugs. Unfortunately, almost all the previously reported human mPGES-1 inhibitors are inactive (or possess very low activity) against mouse or rat mPGES-1 that prevents using well-established mouse/rat models of inflammation, pain, and other diseases for preclinical studies. It would be extremely challenging for the mPGES-1-based drug development to follow traditional drug discovery and development route. In order to solve this problem, we developed and applied Drug Repurposing Effort Applying Integrated Modeling-in vitro/vivo-Clinical Data Mining (DREAM-in-CDM) strategy in this project. With molecular dynamics simulation, we observed the process of how mPGES-1 adopts an alternative conformation to control the access of co-factor GSH (glutathione) and its impact on the function of the protein. Based on the simulation results, we not only found an explanation for the difference between the X-ray and CryoEM (cryogenic electron microscopy) structure of mPGES-1 but also used molecular docking method to identify FDA approved drug, lapatinib, as an mPGES-1 inhibitor by virtual screening and the subsequent in vitro experiments. By mining the available clinical trial data, we found solid evidence that lapatinib can be used to relieve various types of pain in cancer patients. Since lapatinib is very well tolerated, we expect lapatinib to be repurposed as a new treatment for cancer-related pain. BChE has been identified as an ideal drug target for the treatment of Alzheimer’s disease (AD) and heroin overdose. The selectivity of a therapeutically useful inhibitor for BChE over AChE is very important. Unfortunately, there is no good selective BChE inhibitor. With a robust and virtual screening strategy combining with in vitro experiments, we identified a series of compounds from the NCI compound depository as BChE inhibitors with novel scaffolds, high activity and selectivity at the same time. The most potent compound was re-synthesized and the enantiomers of the compound were separated for the first time. The binding mode of the most potent compound was also analyzed and the origin of its high activity and selectivity was revealed that will guide the development of BChE selective inhibitors in the future. In addition, a new tacrine-based BChE affinity chromatography resin was developed. The developed new resin has enabled us to more conveniently and efficiently purify the BChE proteins with improved high purity. In general, we have successfully developed new drug discovery strategies to identify novel inhibitors of different enzymes. With these newly developed strategies, we expect additional drug discoveries to be made in the foreseeable future.

Durant la tesi doctoral s'ha comprovat que a partir de biosensors fabricats mitjançant una tecnología relativament senzilla i económica, com és la formació de matrius i la impressió serigràfica de tintes, es poden realitzar determinacions de diversos analits d'interès en l'àmbit alimentari no tan sols en solucions estàndard sinó també en mostres reals. Els resultats aconseguits suposen una important millora en l'abaratiment dels costos d'anàlisi i la reducció del temps necessari.El treball realitzar s'ha portat a terme en dues parts molt diferenciades; en una primera s'han desenvolupat biosensors screen-printing basats en resines epoxi per a l'anàlisi de pesticides i en una segona biosensors screen-printing basats en matrius de sílice (sol-gel) per a la determinació d'àcid màlic i làctic en vins.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, often leading to functional, biochemical and structural changes, especially in the central nervous system. These changes are usually accompanied by a marked immune-inflammatory response, in immunocompetent hosts. Primary infections with T. gondii stimulate production of high levels of interleukins, such as IL-12 and IFN-γ, by cells of the innate immune system, being central efforts to resistance to the disease. In terms of immune response, the purinergic and cholinergic systems have some well-defined properties, but we still without much information on the interrelation of these properties with toxoplasmosis. Thus, the objectives of this study were to evaluate the activities of the (1) E-NTPDase and E-ADA in lymphocytes; (2) AChE on total blood and lymphocytes, and BChE on serum, using the RH (virulent) strain of T. gondii, with sampling on day 5 and 10 post-infection (pi); and (3) assess the purine levels and E-ADA activity in brain, using strains RH and Me-49 (cystogenic). First assessment´s result demonstrated that the hydrolysis of ATP and ADP was increased, as well as the E-ADA activity also was increased. In the second experiment, there was an increase in AChE activity on lymphocytes and on whole blood. Finally, on the third experiment, it was found, in infection with RH, a significant increase of ATP, ADP, AMP, adenosine, hypoxanthine and xanthine levels, along with a reduction of inosine levels. Animals infected with the strain Me-49 showed increased concentrations of ATP and ADP, followed by a reduction of nucleotides and nucleosides levels. Regarding to E-ADA activity, it was observed a reduction on its activity on RH infection, and initially for Me-49 infection; however, E-ADA increased its activity, for this cystogenic at the end of the third experiment. The evaluations of E-NTPDase and E-ADA activities led to the preposition that their hydrolytic activity would be linked to a mechanism of protection against secondary tissue damage, possibly generated by an exacerbated response against T. gondii infection. Additionally, the data of AChE activity, in corresponding samples, confirm the onset of a pro-inflammatory response, corroborating with the hypothesis of necessity of a modulation mechanism. On brain tissue, the experimental model using RH strain repeated its pro-inflammatory pattern, with data of this pattern better observed in a long run, when the results of Me-49 study were evaluated. On Me-49 infection model, it was observed an increase in extracellular ATP, at the beginning of infection, with histopathology data showing inflammatory infiltrate and identification of tissue cysts later on the experiment. Finally, in this last period, it was observed a decrease in purine´s concentration, most likely as a result of the aggravation of local cellular condition, due to the disease´s evolution. Therefore, it was possible to observe that there was a direct involvement of purinergic and cholinergic systems on immunomodulation of experimental toxoplasmosis, with virulent or cystogenic strains, contributing to the establishment of an adequate immune response on the parasite, and also to a mechanism of tissue damage reduction, which is normally associated with direct parasite damage and excessive immune response. / A toxoplasmose é doença zoonótica causada pelo Toxoplasma gondii, podendo levar à alterações funcionais, bioquímicas e estruturais, especialmente no sistema nervoso central. Estas, geralmente, são acompanhadas por uma marcante resposta imunoinflamatória em hospedeiros imunocompetentes. Infecções primárias com T. gondii estimulam a produção de altos níveis de citocinas, tais como IL-12 e IFN-γ, pelas células do sistema imune inato, consistindo em um ponto principal no controle do parasito e resistência a doença. Em se tratando de resposta imune, os sistemas purinérgico e colinérgicos possuem algumas propriedades bem definidas, porém sem muitas informações sobre a inter-relação destes com a toxoplasmose. Desta maneira, os objetivos deste estudo foram avaliar as atividades (1) da E-NTPDase e E-ADA em linfócitos; (2) da AChE no sangue total e linfócitos, e BChE no soro; utilizando a cepa RH (virulenta) do T. gondii; e (3) avaliar os níveis de purinas e atividade da E-ADA no cérebro, utilizando-se as cepas RH e Me-49 (cistogênica). Os resultados da primeira avaliação demonstraram que a hidrólise do ATP e ADP foram aumentadas, bem como a atividade da E-ADA também esteve aumentada. No segundo experimento, observou-se um aumento na atividade da AChE nos linfócitos e no sangue total. Por fim, no terceiro experimento, verificou-se na infecção com a cepa RH um aumento nos níveis de ATP, ADP, AMP, adenosina, hipoxantina e xantina, e redução nos níveis de inosina. Nos animais infectados com a cepa Me-49, observaram-se inicialmente um aumentos nas concentrações de ATP e ADP, seguido de uma redução na concentração dos nucleotídeos e nucleosídeos. Em relação à atividade da E-ADA, verificou-se uma redução na infecção pela cepa RH, e inicialmente para a cepa Me-49, porém a E-ADA aumentou suas atividades para esta cepa cistogênica no final do experimento. A avaliação das atividades da E-NTPDase e E-ADA levou a preposição de que seus comportamentos hidrolíticos estariam ligados a um mecanismo de proteção contra danos teciduais secundários, possivelmente gerados respostas exacerbadas a infecção pelo T. gondii. Adicionalmente, os dados da atividade da AChE, em amostras correspondentes, comprovaram o estabelecimento de uma resposta pró-inflamatória, corroborando com a hipótese da necessidade de um mecanismo de modulação. No tecido cerebral, a cepa RH repetiu o seu comportamento pró-inflamatório, e este comportamento pode ser analisado em longo prazo, no estudo que envolveu a cepa Me-49. Neste modelo, observou-se um aumento do ATP extracelular no início da infecção, como dados de histopatologia mostrando infiltrado inflamatório e a identificação de cistos teciduais tardiamente. Por fim, neste último período, observou-se uma diminuição na concentração das purinas, muito provavelmente em consequência da agravação da condição celular local em decorrência da evolução da infecção. Desta forma, foi possível se observar que existe uma participação direta dos sistemas purinérgico e colinérgico na imunomodulação da toxoplasmose experimental com cepas virulenta ou cistogênica, contribuindo para a instalação de uma resposta imune celular adequada ao parasito, e para um mecanismo de redução de danos teciduais associados a danos parasitários diretos e a resposta imune exacerbada.

A poluição ambiental por metais pesados tem aumentado muito devido às ações antropogênicas tais como as atividades industriais e o uso de fertilizantes fosfatados na agricultura. Entre os metais tóxicos encontrados no meio ambiente, o cádmio (Cd) é um dos que apresenta maior interesse clínico, uma vez que as intoxicações por esse metal são de difícil tratamento. O Cd pode afetar vários órgãos como o fígado, rins, pulmões, ovários, ossos, testículos e cérebro. A toxicologia do Cd é extremamente complexa e tem sido amplamente estudada, mas ainda não está completamente esclarecida. Sendo assim, o objetivo da presente tese foi investigar os efeitos da intoxicação por Cd sobre parâmetros comportamentais e memória, bioquímicos, hematológicos e a bioacumulação desse metal em diferentes tipos celulares de ratos expostos ao cloreto de Cd (CdCl2) ou ao Cd proveniente de batatas contaminadas, e ainda utilizar o antioxidante N-acetilcisteína (NAC) no tratamento deste tipo de intoxicação buscandose avaliar o seu possível efeito protetor. Para tal, foram realizados dois experimentos: a) ratos machos Wistar adultos foram expostos oralmente a 2 mg/kg de Cd e/ou 150 mg/kg de NAC, um dia sim e outro não, durante um período experimental de 30 dias; b) ratos machos Wistar recém desmamados ingeriram, durante 5 meses, dieta a base de bolo contendo 1, 5 ou 25 mg/kg de CdCl2 ou Cd presente em tubérculos de batatas crescidas na presença de 10 μM de CdCl2. A partir desses experimentos pode-se concluir que: a) A intoxicação por CdCl2 causa aumento na concentração de Cd nas estruturas cerebrais (cerebelo, estriado, hipotálamo, hipocampo e córtex cerebral), nos níveis de peroxidação lipídica e na atividade da enzima AChE nas diferentes estruturas cerebrais estudadas ocasionando prejuízos à memória dos ratos. Além disso, a NAC é capaz de diminuir os níveis de peroxidação lipídica e subsequentemente restaurar a atividade da AChE modulando assim, a neurotransmissão colinérgica e melhorando os processos cognitivos. Sendo assim, sugere-se que a NAC possa ser um fármaco promissor em terapias alternativas contra a neurotoxicidade induzida pelo Cd; b) A exposição ao CdCl2 promove um aumento na concentração de Cd no plasma, baço e timo, causa danos hematológicos, não altera a atividade da enzima NTPDase em linfócitos, diminui a atividade da AChE em linfócitos e sangue total bem como da BChE em soro de ratos. Além disso, a NAC foi eficaz em diminuir os efeitos danosos do Cd provavelmente por diminuir os níveis de Cd nos órgãos linfóides, reverter ou amenizar os danos hematológicos e relacionados aos leucócitos mesmo sem alterar a atividade das enzimas colinesterases; c) O CdCl2 aumenta a hidrólise de nucleotídeos de adenina em sinaptossomas de córtex cerebral e diminui em plaquetas de ratos. Provavelmente, o aumento na atividade das enzimas NTPDase e 5’-nucleotidase no SNC causado pelo Cd seja uma resposta compensatória do organismo uma vez que a rápida hidrólise de ATP e ADP favorece a produção de adenosina, uma molécula neuroprotetora. Por outro lado, em plaquetas o Cd causou uma diminuição na atividade da NTPDase e, não alterou a atividade da 5’-nucleotidase sugerindo que a possível elevação no nível de ATP e ADP conduza a um estado hipercoagulável nos ratos intoxicados por esse metal. Além disso, a NAC restaura a atividade dessas enzimas no SNC, mas não apresenta interferência sobre elas em plaquetas; d) A dieta prolongada com CdCl2 ou com Cd proveniente de batatas contaminadas ocasiona aumento no comportamento de ansiedade e prejuízo à memória de ratos o que provavelmente, seja resultado de uma concentração aumentada de Cd e de uma atividade aumentada da AChE e diminuída da Na+,K+-ATPase em diferentes estruturas cerebrais devido a esse metal. Em linhas gerais, os resultados obtidos na presente tese demonstram que apesar de todos os mecanismos propostos pelos quais o Cd poderia afetar o comportamento continuarem a gerar controvérsia, é evidente que a exposição ao CdCl2 tanto a curto quanto a longo prazo, bem como ao Cd proveniente de batatas contaminadas interfere nas funções cerebrais aumentando a concentração de Cd, diminuindo ou aumentando a atividade da AChE, aumentando a lipoperoxidação bem como diminuindo a atividade da Na+,K+-ATPase em diferentes estruturas cerebrais, consequentemente sendo prejudicial ao comportamento animal, como memória e ansiedade. O Cd afeta sistematicamente o organismo dos animais estando presente tanto no plasma, quanto nos órgãos linfóides e no encéfalo alterando, assim, a atividade das enzimas NTPDase, 5’-nucleotidase, AChE e BChE de diferentes tipos celulares. Além disso, a NAC é capaz de reverter ou amenizar vários efeitos danosos causados pelo Cd sugerindo que este fármaco possa ser considerado após estudos adicionais um importante aliado em terapias contra a intoxicação por esse metal. / The environmental pollution by heavy metals has increased greatly due to anthropogenic activities such as industrial activities and the use of phosphate fertilizers in agriculture. Among the toxic metals found in the environment, cadmium (Cd) is a metal that presents the greatest clinical interest, since poisoning by this metal is difficult to treat. Cd may affect various organs such as liver, kidneys, lungs, ovaries, bones, testes and brain. The toxicology of Cd is extremely complex and has been widely studied, but it is not yet fully elucidated. Therefore, the objective of this thesis was to investigate the effects of Cd intoxication on memory as well as behavioral, biochemical, hematological and bioaccumulation parameters of this metal in different cell types of rats exposed to Cd chloride (CdCl2) or Cd from contaminated potatoes. Also, we investigated the antioxidant N-acetylcysteine (NAC) in the treatment of this type of poisoning seeking to evaluate its possible protective effect. Two experiments were performed: i) adult male Wistar rats were exposed orally to 2 mg/kg Cd and/or 150 mg/kg NAC, every other day for 30 days; ii) weaned male Wistar rats fed for 5 months, cake-based diet containing 1, 5 or 25 mg/kg CdCl2 or Cd present in tubers of potatoes grown in the presence of 10 μM of CdCl2. From these experiments we conclude: Firstly, CdCl2 intoxication caused an increase in the Cd concentration in brain structures (cerebellum, striatum, hypothalamus, hippocampus and cerebral cortex), in the levels of lipid peroxidation, and in the AChE activity of different brain structures studied causing damage to memory of rats. NAC was able to reduce the levels of lipid peroxidation and subsequently restore the AChE activity, modulating thereby the cholinergic transmission and improving the cognitive processes. Thus, NAC may be a promising drug for alternative therapies against neurotoxicity induced by Cd. Secondly, the exposure to CdCl2 increased the Cd concentration in plasma, spleen and thymus, caused damages in the hematological parameters, did not alter the NTPDase activity in lymphocytes as well as decreased the AChE activity in lymphocytes and whole blood and the BChE activity in serum of rats. NAC was effective in reducing the harmful effects of Cd probably by decreasing the levels of Cd in the lymphoid organs, reversing or minimizing the hematological damage and related to leukocytes even without changing the activity of cholinesterases. Thirdly, CdCl2 increases the hydrolysis of adenine nucleotides in synaptosomes of cerebral cortex and decreases in platelets of rats. The increase in the NTPDase and 5'-nucleotidase activities in CNS caused by Cd could be a compensatory response of the organism since the rapid hydrolysis of ATP and ADP favors the production of adenosine, a neuroprotective molecule. On the other hand, Cd decreased the NTPDase activity and did not alter 5'-nucleotidase activity in platelets suggesting that the possible increase in the ATP and ADP levels could lead to a hypercoagulable state in rats intoxicated with this metal. NAC restores the activity of these enzymes in the CNS, but had no interference in platelets. Finally, the prolonged diet with CdCl2 or Cd from contaminated potatoes resulted in an increased anxiety-like behavior and damaged memory of rats which was likely to be a result of the increased concentration of Cd, the increased AChE activity and decreased Na+,K+-ATPase activity in the different brain structures studied. In general, the results obtained in this thesis show that despite all the proposed mechanisms by which Cd could affect the behavior still generate controversy, it is clear that the exposure to CdCl2 at both short and long terms as well as to Cd from contaminated potatoes interferes the brain function by increasing Cd concentration, decreasing or increasing the AChE activity, increasing lipid peroxidation and decreasing Na+,K+-ATPase activity in several brain structures, thus being detrimental to the animal behavior influencing memory and anxiety. Cd affected systematically the animal body since it is present in plasma, lymphoid organs and brain, changing the activity of the enzymes NTPDase, 5'-nucleotidase, AChE and BChE of different cell types. In addition, NAC was able to reverse or ameliorate several deleterious effects caused by Cd suggesting that this drug may be, after additional studies, considered an important factor in therapies against intoxication by this metal.

A poluição ambiental por metais pesados tem aumentado muito devido às ações antropogênicas tais como as atividades industriais e o uso de fertilizantes fosfatados na agricultura. Entre os metais tóxicos encontrados no meio ambiente, o cádmio (Cd) é um dos que apresenta maior interesse clínico, uma vez que as intoxicações por esse metal são de difícil tratamento. O Cd pode afetar vários órgãos como o fígado, rins, pulmões, ovários, ossos, testículos e cérebro. A toxicologia do Cd é extremamente complexa e tem sido amplamente estudada, mas ainda não está completamente esclarecida. Sendo assim, o objetivo da presente tese foi investigar os efeitos da intoxicação por Cd sobre parâmetros comportamentais e memória, bioquímicos, hematológicos e a bioacumulação desse metal em diferentes tipos celulares de ratos expostos ao cloreto de Cd (CdCl2) ou ao Cd proveniente de batatas contaminadas, e ainda utilizar o antioxidante N-acetilcisteína (NAC) no tratamento deste tipo de intoxicação buscandose avaliar o seu possível efeito protetor. Para tal, foram realizados dois experimentos: a) ratos machos Wistar adultos foram expostos oralmente a 2 mg/kg de Cd e/ou 150 mg/kg de NAC, um dia sim e outro não, durante um período experimental de 30 dias; b) ratos machos Wistar recém desmamados ingeriram, durante 5 meses, dieta a base de bolo contendo 1, 5 ou 25 mg/kg de CdCl2 ou Cd presente em tubérculos de batatas crescidas na presença de 10 μM de CdCl2. A partir desses experimentos pode-se concluir que: a) A intoxicação por CdCl2 causa aumento na concentração de Cd nas estruturas cerebrais (cerebelo, estriado, hipotálamo, hipocampo e córtex cerebral), nos níveis de peroxidação lipídica e na atividade da enzima AChE nas diferentes estruturas cerebrais estudadas ocasionando prejuízos à memória dos ratos. Além disso, a NAC é capaz de diminuir os níveis de peroxidação lipídica e subsequentemente restaurar a atividade da AChE modulando assim, a neurotransmissão colinérgica e melhorando os processos cognitivos. Sendo assim, sugere-se que a NAC possa ser um fármaco promissor em terapias alternativas contra a neurotoxicidade induzida pelo Cd; b) A exposição ao CdCl2 promove um aumento na concentração de Cd no plasma, baço e timo, causa danos hematológicos, não altera a atividade da enzima NTPDase em linfócitos, diminui a atividade da AChE em linfócitos e sangue total bem como da BChE em soro de ratos. Além disso, a NAC foi eficaz em diminuir os efeitos danosos do Cd provavelmente por diminuir os níveis de Cd nos órgãos linfóides, reverter ou amenizar os danos hematológicos e relacionados aos leucócitos mesmo sem alterar a atividade das enzimas colinesterases; c) O CdCl2 aumenta a hidrólise de nucleotídeos de adenina em sinaptossomas de córtex cerebral e diminui em plaquetas de ratos. Provavelmente, o aumento na atividade das enzimas NTPDase e 5’-nucleotidase no SNC causado pelo Cd seja uma resposta compensatória do organismo uma vez que a rápida hidrólise de ATP e ADP favorece a produção de adenosina, uma molécula neuroprotetora. Por outro lado, em plaquetas o Cd causou uma diminuição na atividade da NTPDase e, não alterou a atividade da 5’-nucleotidase sugerindo que a possível elevação no nível de ATP e ADP conduza a um estado hipercoagulável nos ratos intoxicados por esse metal. Além disso, a NAC restaura a atividade dessas enzimas no SNC, mas não apresenta interferência sobre elas em plaquetas; d) A dieta prolongada com CdCl2 ou com Cd proveniente de batatas contaminadas ocasiona aumento no comportamento de ansiedade e prejuízo à memória de ratos o que provavelmente, seja resultado de uma concentração aumentada de Cd e de uma atividade aumentada da AChE e diminuída da Na+,K+-ATPase em diferentes estruturas cerebrais devido a esse metal. Em linhas gerais, os resultados obtidos na presente tese demonstram que apesar de todos os mecanismos propostos pelos quais o Cd poderia afetar o comportamento continuarem a gerar controvérsia, é evidente que a exposição ao CdCl2 tanto a curto quanto a longo prazo, bem como ao Cd proveniente de batatas contaminadas interfere nas funções cerebrais aumentando a concentração de Cd, diminuindo ou aumentando a atividade da AChE, aumentando a lipoperoxidação bem como diminuindo a atividade da Na+,K+-ATPase em diferentes estruturas cerebrais, consequentemente sendo prejudicial ao comportamento animal, como memória e ansiedade. O Cd afeta sistematicamente o organismo dos animais estando presente tanto no plasma, quanto nos órgãos linfóides e no encéfalo alterando, assim, a atividade das enzimas NTPDase, 5’-nucleotidase, AChE e BChE de diferentes tipos celulares. Além disso, a NAC é capaz de reverter ou amenizar vários efeitos danosos causados pelo Cd sugerindo que este fármaco possa ser considerado após estudos adicionais um importante aliado em terapias contra a intoxicação por esse metal. / The environmental pollution by heavy metals has increased greatly due to anthropogenic activities such as industrial activities and the use of phosphate fertilizers in agriculture. Among the toxic metals found in the environment, cadmium (Cd) is a metal that presents the greatest clinical interest, since poisoning by this metal is difficult to treat. Cd may affect various organs such as liver, kidneys, lungs, ovaries, bones, testes and brain. The toxicology of Cd is extremely complex and has been widely studied, but it is not yet fully elucidated. Therefore, the objective of this thesis was to investigate the effects of Cd intoxication on memory as well as behavioral, biochemical, hematological and bioaccumulation parameters of this metal in different cell types of rats exposed to Cd chloride (CdCl2) or Cd from contaminated potatoes. Also, we investigated the antioxidant N-acetylcysteine (NAC) in the treatment of this type of poisoning seeking to evaluate its possible protective effect. Two experiments were performed: i) adult male Wistar rats were exposed orally to 2 mg/kg Cd and/or 150 mg/kg NAC, every other day for 30 days; ii) weaned male Wistar rats fed for 5 months, cake-based diet containing 1, 5 or 25 mg/kg CdCl2 or Cd present in tubers of potatoes grown in the presence of 10 μM of CdCl2. From these experiments we conclude: Firstly, CdCl2 intoxication caused an increase in the Cd concentration in brain structures (cerebellum, striatum, hypothalamus, hippocampus and cerebral cortex), in the levels of lipid peroxidation, and in the AChE activity of different brain structures studied causing damage to memory of rats. NAC was able to reduce the levels of lipid peroxidation and subsequently restore the AChE activity, modulating thereby the cholinergic transmission and improving the cognitive processes. Thus, NAC may be a promising drug for alternative therapies against neurotoxicity induced by Cd. Secondly, the exposure to CdCl2 increased the Cd concentration in plasma, spleen and thymus, caused damages in the hematological parameters, did not alter the NTPDase activity in lymphocytes as well as decreased the AChE activity in lymphocytes and whole blood and the BChE activity in serum of rats. NAC was effective in reducing the harmful effects of Cd probably by decreasing the levels of Cd in the lymphoid organs, reversing or minimizing the hematological damage and related to leukocytes even without changing the activity of cholinesterases. Thirdly, CdCl2 increases the hydrolysis of adenine nucleotides in synaptosomes of cerebral cortex and decreases in platelets of rats. The increase in the NTPDase and 5'-nucleotidase activities in CNS caused by Cd could be a compensatory response of the organism since the rapid hydrolysis of ATP and ADP favors the production of adenosine, a neuroprotective molecule. On the other hand, Cd decreased the NTPDase activity and did not alter 5'-nucleotidase activity in platelets suggesting that the possible increase in the ATP and ADP levels could lead to a hypercoagulable state in rats intoxicated with this metal. NAC restores the activity of these enzymes in the CNS, but had no interference in platelets. Finally, the prolonged diet with CdCl2 or Cd from contaminated potatoes resulted in an increased anxiety-like behavior and damaged memory of rats which was likely to be a result of the increased concentration of Cd, the increased AChE activity and decreased Na+,K+-ATPase activity in the different brain structures studied. In general, the results obtained in this thesis show that despite all the proposed mechanisms by which Cd could affect the behavior still generate controversy, it is clear that the exposure to CdCl2 at both short and long terms as well as to Cd from contaminated potatoes interferes the brain function by increasing Cd concentration, decreasing or increasing the AChE activity, increasing lipid peroxidation and decreasing Na+,K+-ATPase activity in several brain structures, thus being detrimental to the animal behavior influencing memory and anxiety. Cd affected systematically the animal body since it is present in plasma, lymphoid organs and brain, changing the activity of the enzymes NTPDase, 5'-nucleotidase, AChE and BChE of different cell types. In addition, NAC was able to reverse or ameliorate several deleterious effects caused by Cd suggesting that this drug may be, after additional studies, considered an important factor in therapies against intoxication by this metal.

A poluição ambiental por metais pesados tem aumentado muito devido às ações antropogênicas tais como as atividades industriais e o uso de fertilizantes fosfatados na agricultura. Entre os metais tóxicos encontrados no meio ambiente, o cádmio (Cd) é um dos que apresenta maior interesse clínico, uma vez que as intoxicações por esse metal são de difícil tratamento. O Cd pode afetar vários órgãos como o fígado, rins, pulmões, ovários, ossos, testículos e cérebro. A toxicologia do Cd é extremamente complexa e tem sido amplamente estudada, mas ainda não está completamente esclarecida. Sendo assim, o objetivo da presente tese foi investigar os efeitos da intoxicação por Cd sobre parâmetros comportamentais e memória, bioquímicos, hematológicos e a bioacumulação desse metal em diferentes tipos celulares de ratos expostos ao cloreto de Cd (CdCl2) ou ao Cd proveniente de batatas contaminadas, e ainda utilizar o antioxidante N-acetilcisteína (NAC) no tratamento deste tipo de intoxicação buscandose avaliar o seu possível efeito protetor. Para tal, foram realizados dois experimentos: a) ratos machos Wistar adultos foram expostos oralmente a 2 mg/kg de Cd e/ou 150 mg/kg de NAC, um dia sim e outro não, durante um período experimental de 30 dias; b) ratos machos Wistar recém desmamados ingeriram, durante 5 meses, dieta a base de bolo contendo 1, 5 ou 25 mg/kg de CdCl2 ou Cd presente em tubérculos de batatas crescidas na presença de 10 μM de CdCl2. A partir desses experimentos pode-se concluir que: a) A intoxicação por CdCl2 causa aumento na concentração de Cd nas estruturas cerebrais (cerebelo, estriado, hipotálamo, hipocampo e córtex cerebral), nos níveis de peroxidação lipídica e na atividade da enzima AChE nas diferentes estruturas cerebrais estudadas ocasionando prejuízos à memória dos ratos. Além disso, a NAC é capaz de diminuir os níveis de peroxidação lipídica e subsequentemente restaurar a atividade da AChE modulando assim, a neurotransmissão colinérgica e melhorando os processos cognitivos. Sendo assim, sugere-se que a NAC possa ser um fármaco promissor em terapias alternativas contra a neurotoxicidade induzida pelo Cd; b) A exposição ao CdCl2 promove um aumento na concentração de Cd no plasma, baço e timo, causa danos hematológicos, não altera a atividade da enzima NTPDase em linfócitos, diminui a atividade da AChE em linfócitos e sangue total bem como da BChE em soro de ratos. Além disso, a NAC foi eficaz em diminuir os efeitos danosos do Cd provavelmente por diminuir os níveis de Cd nos órgãos linfóides, reverter ou amenizar os danos hematológicos e relacionados aos leucócitos mesmo sem alterar a atividade das enzimas colinesterases; c) O CdCl2 aumenta a hidrólise de nucleotídeos de adenina em sinaptossomas de córtex cerebral e diminui em plaquetas de ratos. Provavelmente, o aumento na atividade das enzimas NTPDase e 5’-nucleotidase no SNC causado pelo Cd seja uma resposta compensatória do organismo uma vez que a rápida hidrólise de ATP e ADP favorece a produção de adenosina, uma molécula neuroprotetora. Por outro lado, em plaquetas o Cd causou uma diminuição na atividade da NTPDase e, não alterou a atividade da 5’-nucleotidase sugerindo que a possível elevação no nível de ATP e ADP conduza a um estado hipercoagulável nos ratos intoxicados por esse metal. Além disso, a NAC restaura a atividade dessas enzimas no SNC, mas não apresenta interferência sobre elas em plaquetas; d) A dieta prolongada com CdCl2 ou com Cd proveniente de batatas contaminadas ocasiona aumento no comportamento de ansiedade e prejuízo à memória de ratos o que provavelmente, seja resultado de uma concentração aumentada de Cd e de uma atividade aumentada da AChE e diminuída da Na+,K+-ATPase em diferentes estruturas cerebrais devido a esse metal. Em linhas gerais, os resultados obtidos na presente tese demonstram que apesar de todos os mecanismos propostos pelos quais o Cd poderia afetar o comportamento continuarem a gerar controvérsia, é evidente que a exposição ao CdCl2 tanto a curto quanto a longo prazo, bem como ao Cd proveniente de batatas contaminadas interfere nas funções cerebrais aumentando a concentração de Cd, diminuindo ou aumentando a atividade da AChE, aumentando a lipoperoxidação bem como diminuindo a atividade da Na+,K+-ATPase em diferentes estruturas cerebrais, consequentemente sendo prejudicial ao comportamento animal, como memória e ansiedade. O Cd afeta sistematicamente o organismo dos animais estando presente tanto no plasma, quanto nos órgãos linfóides e no encéfalo alterando, assim, a atividade das enzimas NTPDase, 5’-nucleotidase, AChE e BChE de diferentes tipos celulares. Além disso, a NAC é capaz de reverter ou amenizar vários efeitos danosos causados pelo Cd sugerindo que este fármaco possa ser considerado após estudos adicionais um importante aliado em terapias contra a intoxicação por esse metal. / The environmental pollution by heavy metals has increased greatly due to anthropogenic activities such as industrial activities and the use of phosphate fertilizers in agriculture. Among the toxic metals found in the environment, cadmium (Cd) is a metal that presents the greatest clinical interest, since poisoning by this metal is difficult to treat. Cd may affect various organs such as liver, kidneys, lungs, ovaries, bones, testes and brain. The toxicology of Cd is extremely complex and has been widely studied, but it is not yet fully elucidated. Therefore, the objective of this thesis was to investigate the effects of Cd intoxication on memory as well as behavioral, biochemical, hematological and bioaccumulation parameters of this metal in different cell types of rats exposed to Cd chloride (CdCl2) or Cd from contaminated potatoes. Also, we investigated the antioxidant N-acetylcysteine (NAC) in the treatment of this type of poisoning seeking to evaluate its possible protective effect. Two experiments were performed: i) adult male Wistar rats were exposed orally to 2 mg/kg Cd and/or 150 mg/kg NAC, every other day for 30 days; ii) weaned male Wistar rats fed for 5 months, cake-based diet containing 1, 5 or 25 mg/kg CdCl2 or Cd present in tubers of potatoes grown in the presence of 10 μM of CdCl2. From these experiments we conclude: Firstly, CdCl2 intoxication caused an increase in the Cd concentration in brain structures (cerebellum, striatum, hypothalamus, hippocampus and cerebral cortex), in the levels of lipid peroxidation, and in the AChE activity of different brain structures studied causing damage to memory of rats. NAC was able to reduce the levels of lipid peroxidation and subsequently restore the AChE activity, modulating thereby the cholinergic transmission and improving the cognitive processes. Thus, NAC may be a promising drug for alternative therapies against neurotoxicity induced by Cd. Secondly, the exposure to CdCl2 increased the Cd concentration in plasma, spleen and thymus, caused damages in the hematological parameters, did not alter the NTPDase activity in lymphocytes as well as decreased the AChE activity in lymphocytes and whole blood and the BChE activity in serum of rats. NAC was effective in reducing the harmful effects of Cd probably by decreasing the levels of Cd in the lymphoid organs, reversing or minimizing the hematological damage and related to leukocytes even without changing the activity of cholinesterases. Thirdly, CdCl2 increases the hydrolysis of adenine nucleotides in synaptosomes of cerebral cortex and decreases in platelets of rats. The increase in the NTPDase and 5'-nucleotidase activities in CNS caused by Cd could be a compensatory response of the organism since the rapid hydrolysis of ATP and ADP favors the production of adenosine, a neuroprotective molecule. On the other hand, Cd decreased the NTPDase activity and did not alter 5'-nucleotidase activity in platelets suggesting that the possible increase in the ATP and ADP levels could lead to a hypercoagulable state in rats intoxicated with this metal. NAC restores the activity of these enzymes in the CNS, but had no interference in platelets. Finally, the prolonged diet with CdCl2 or Cd from contaminated potatoes resulted in an increased anxiety-like behavior and damaged memory of rats which was likely to be a result of the increased concentration of Cd, the increased AChE activity and decreased Na+,K+-ATPase activity in the different brain structures studied. In general, the results obtained in this thesis show that despite all the proposed mechanisms by which Cd could affect the behavior still generate controversy, it is clear that the exposure to CdCl2 at both short and long terms as well as to Cd from contaminated potatoes interferes the brain function by increasing Cd concentration, decreasing or increasing the AChE activity, increasing lipid peroxidation and decreasing Na+,K+-ATPase activity in several brain structures, thus being detrimental to the animal behavior influencing memory and anxiety. Cd affected systematically the animal body since it is present in plasma, lymphoid organs and brain, changing the activity of the enzymes NTPDase, 5'-nucleotidase, AChE and BChE of different cell types. In addition, NAC was able to reverse or ameliorate several deleterious effects caused by Cd suggesting that this drug may be, after additional studies, considered an important factor in therapies against intoxication by this metal.

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.