O desenvolvimento na área agroquímica associado as novas técnicas de plantio, asseguram ao país recordes anuais na produção agrícola. Entretanto, os defensivos agrícolas e fertilizantes utilizados para aumentar a produção das lavouras não são inertes a saúde humana. Exposições contínuas e inadequadas a esses compostos químicos podem causar sua absorção e resultar em intoxicações agudas ou crônicas. Com o objetivo de avaliar essas exposições, dosamos a atividade da enzima acetilcolinesterase, assim como, avaliamos os hemogramas e dosamos os níveis sanguíneos dos metais arsênio (As), cádmio (Cd), chumbo (Pb), manganês (Mn), zinco (Zn), cobre (Cu) e o não metal selênio (Se) em trabalhadores rurais e moradores da área urbana da região de Rio Verde-GO e comparamos esses resultados. A atividade média da enzima colinesterase eritrocitária nos trabalhadores rurais apresentou uma depressão significativa indicando uma exposição inadequada aos inseticidas inibidores das colinesterases. As concentrações sanguíneas médias dos metais As, Cd, Mn, e Zn nos trabalhadores rurais são maiores do que na população urbana, indicando que as exposições inadequadas aos fertilizantes e defensivos agrícolas podem causar absorção desses metais. A concentração sanguínea média de Se na população urbana é maior do que nos trabalhadores agrícolas. Esse fato pode estar ligado a alimentação, sendo que, uma provável causa seria o baixo consumo de alimentos ricos em selênio (castanha-do-pará, salmão, farelo de trigo, ostras e fígado bovino). Os hemogramas não apresentaram alterações, indicando que, sua utilização isolada na monitorização das exposições ocupacionais aos defensivos agrícolas e fertilizantes é inadequada. / Advancements in the agrochemical industry associated with new seeding techniques make it possible for the country to reach annual crop records in production. However, the chemicals and fertilizers used for increasing productions are by no means harmless to human health. Repeated and inadequate exposures to such chemicals may result in their absorption and cause acute and chronic poisoning. In order to assess these exposures, we measured the activity of the acetylcholinesterase enzyme and evaluated hemograms. In addition, we measured blood levels of metals such as arsine (As), cadmium (Cd), lead (Pb), manganese (Mn), zinc (Zn), copper (Cu), and the non metallic selenium in farm workers and urban residents in a region of Rio Verde- GO- Brazil and compared the results. Mean activity of the erythrocyte cholinesterase enzyme in the rural workers presented a significant decrease, indicative of inadequate exposure to cholinesterase inhibiting insecticides. Mean concentrations of the metals As, Cd, Mn, and Zn were higher in rural workers compared to urban residents, which suggests that inadequate exposure to fertilizers and agricultural chemicals may result in their absorption. Mean blood concentration of Se in urban residents was higher compared to rural workers. That can be associated with diet and a possible cause may be a low consumption of high Se foods (Brazils nuts, salmon, oysters, wheat bran, and bovine liver). The hemograms did not present any changes, indicating that its use for monitoring occupational exposures to fertilizers and agricultural chemicals is inadequate.

O uso de agrotóxicos e a consequente contaminação têm sido motivo de constante preocupação, sendo necessário monitorar esses compostos por metodologias que as quantifiquem em água e alimentos. As técnicas cromatográficas são as mais utilizadas para a este tipo de análise, mas apresentam desvantagens para aplicações \"in situ\" ou em tempo real. As técnicas eletroquímicas, como os biossensores enzimáticos que utilizam a acetilcolinesterase, têm sido estudadas para a determinação quantitativa de pesticidas em diferentes amostras. No entanto, as enzimas apresentam algumas desvantagens relacionadas com sua capacidade de desnaturação e com a inibição por outras espécies. Diante disso, foi proposta neste projeto a síntese de uma molécula mimética [ácido (4-(1E)-etanohidrazonoil) benzóico] para a acetilcolinesterase para catalisar a hidrólise do substrato (acetiltiocolina). A molécula mimética foi caracterizada por RMN e FTIR. Os produtos da reação, acetato e tiocolina, foram identificados pelo método de Ellman e FTIR. A cinética química do processo catalítico foi estudada, verificando-se que a reação era de primeira ordem em relação à concentração de acetiltiocolina e a constante de velocidade foi 0,623 s-1. Os dados experimentais obtidos com a molécula artificial foram aplicados ao modelo cinético de Michaelis-Menten e os parâmetros cinéticos foram determinados, constatando que a constante de velocidade calculada foi 13000 vezes menor que aquela calculada pelo método diferencial, o que mostrou a inconveniência de aplicar tal modelo enzimático aos catalisadores sintéticos. Além disso, verificou-se que o KM é uma constante que não possui significado quando aplicada às moléculas miméticas. / The application of pesticides and the resulting contamination have been a matter of constant concern, being necessary to monitor these compounds by methods that are able to quantify these compounds in water and food. The chromatographic techniques are most often used for this kind of analysis, but present some drawbacks for \"in situ\" or in real time applications. Electrochemical techniques, like biosensors based on the inhibition of acetylcholinesterase, have been studied for the quantitative determination of pesticides in different samples. However, the use of enzymes is complicated due to their ability to denaturation and the possible inhibition by other species. Therefore, in this project the synthesis of [4-(1E)etanehydrazonoylbenzoic acid], a mimetic molecule for acetylcholinesterase, was carried out, aiming the catalysis of acetylcholine hydrolysis. The mimetic molecule was characterized by NMR and FTIR. The products of reaction, such as acetate and thiocholine, were identified by Ellman\'s method and FTIR. The chemical kinetic of the catalytic process was characterized as of first order with respect to the acetylthiocholine concentration with a rate constant of 0.623 s-1. The experimental data obtained with the artificial molecule were applied to the Michaelis-Menten\'s model and the kinetic parameters were determined, noting that the rate constant was calculated as 13000 times smaller than that obtained by the differential method, which indicates the inconvenience of using the enzymatic model to the mimetic catalysts. Moreover, it was found that KM has no meaning when applied to mimetic molecules.

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Agradecemos a compreensão. on 2017-03-22T14:32:46Z (GMT) / Submitted by JOAO PEDRO CORREA DE CAMARGO null (joaoquimica1991@hotmail.com) on 2017-03-22T15:13:54Z No. of bitstreams: 1 Autoarquivamento da dissertação corrigido .pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-03-24T16:41:36Z (GMT) No. of bitstreams: 1 camargo_jpc_me_bot.pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) / Made available in DSpace on 2017-03-24T16:41:36Z (GMT). No. of bitstreams: 1 camargo_jpc_me_bot.pdf: 2162415 bytes, checksum: 64ada6268776f57730601f9cd0ffc35b (MD5) Previous issue date: 2017-02-09 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um novo biossensor foi desenvolvido baseado na simples eletrocodeposição do óxido de grafeno reduzido (rGO), polipirrol (PPy) e da enzima acetilcolinesterase (AChE) na superfície do eletrodo de platina (Pt). No intervalo de potencial -0,2 a +0,5 V vs. Ag/AgCl/KCl (3,0 mol L-1), utilizando voltametria de pulso diferencial (DPV), observou-se um processo em +0,1 V e este corresponde a dimerização dos produtos de oxidação eletroquímica da tiolcolina, formando ditio-bis-colina. O biossensor desenvolvido foi avaliado utilizando DPV na análise do pesticida carbaril, o qual inibe a ação da enzima AChE. Os melhores resultados obtidos foram com as seguintes condições otimizadas: 75 mV amplitude de pulso, incremento de potencial de 4 mV, e uma solução tampão fosfato (PBS) 0,2 mol L-1 pH 6,0. Usando tais parâmetros observou-se uma resposta linear para o carbaril no intervalo de 0,1 a 0,5 mol L-1, com um limite de detecção de 11,6 nmolL-1 (2,3 µg/kg), que é um limite adequado para determinar carbaril nas culturas em que este pesticida é aplicado considerando o limite máximo de resíduo permitido pelas legislações brasileiras. O biossensor proposto, Pt/rGO/PPy/AChE, foi aplicado com sucesso na determinação de carbaril em amostras de tomate e repolho. / A new biosensor was developed by a simple electrocodeposition of reduced graphene oxide (rGO), polypyrrole (PPy) and the enzyme acetylcholinesterase (AChE) on surface of platinum (Pt) electrode. In potential range of -0.2 to +0.5 V vs. Ag/AgCl/KCl (3.0 mol L-1), using differential pulse voltammetry (DPV), it was observed a process in + 0.1 V and this corresponds to the dimerization of electrochemical oxidation products of thiocholine, resulting in ditio-bis-choline. The biosensor developed was evaluated using DPV in the analysis of carbaryl, which inhibits the AChE enzyme action. The best results achieved were with the followings optimized conditions: 75 mV pulse amplitude, step potential of 4 mV, and a phosphate buffer solution (PBS) 0.2 mol L-1 and pH 6.0. Using these parameters was observed a linear response to carbaryl in a range of 0.1 to 0.5 µmol L-1, with a detection limit of 11.6 nmol L-1 (2.3 µg/kg), which is an appropriate limit for determination of carbaryl in the cultures which these pesticide is applied, considering the maximum reside limit allowed by Brazilian legislation. The biosensor proposed, Pt/rGO/PPy/AChE, was applied successfully in the determination of carbaryl in samples of cabbage and tomato. / FAPESP: 2015/02136-2

O uso de agrotóxicos e a consequente contaminação têm sido motivo de constante preocupação, sendo necessário monitorar esses compostos por metodologias que as quantifiquem em água e alimentos. As técnicas cromatográficas são as mais utilizadas para a este tipo de análise, mas apresentam desvantagens para aplicações \"in situ\" ou em tempo real. As técnicas eletroquímicas, como os biossensores enzimáticos que utilizam a acetilcolinesterase, têm sido estudadas para a determinação quantitativa de pesticidas em diferentes amostras. No entanto, as enzimas apresentam algumas desvantagens relacionadas com sua capacidade de desnaturação e com a inibição por outras espécies. Diante disso, foi proposta neste projeto a síntese de uma molécula mimética [ácido (4-(1E)-etanohidrazonoil) benzóico] para a acetilcolinesterase para catalisar a hidrólise do substrato (acetiltiocolina). A molécula mimética foi caracterizada por RMN e FTIR. Os produtos da reação, acetato e tiocolina, foram identificados pelo método de Ellman e FTIR. A cinética química do processo catalítico foi estudada, verificando-se que a reação era de primeira ordem em relação à concentração de acetiltiocolina e a constante de velocidade foi 0,623 s-1. Os dados experimentais obtidos com a molécula artificial foram aplicados ao modelo cinético de Michaelis-Menten e os parâmetros cinéticos foram determinados, constatando que a constante de velocidade calculada foi 13000 vezes menor que aquela calculada pelo método diferencial, o que mostrou a inconveniência de aplicar tal modelo enzimático aos catalisadores sintéticos. Além disso, verificou-se que o KM é uma constante que não possui significado quando aplicada às moléculas miméticas. / The application of pesticides and the resulting contamination have been a matter of constant concern, being necessary to monitor these compounds by methods that are able to quantify these compounds in water and food. The chromatographic techniques are most often used for this kind of analysis, but present some drawbacks for \"in situ\" or in real time applications. Electrochemical techniques, like biosensors based on the inhibition of acetylcholinesterase, have been studied for the quantitative determination of pesticides in different samples. However, the use of enzymes is complicated due to their ability to denaturation and the possible inhibition by other species. Therefore, in this project the synthesis of [4-(1E)etanehydrazonoylbenzoic acid], a mimetic molecule for acetylcholinesterase, was carried out, aiming the catalysis of acetylcholine hydrolysis. The mimetic molecule was characterized by NMR and FTIR. The products of reaction, such as acetate and thiocholine, were identified by Ellman\'s method and FTIR. The chemical kinetic of the catalytic process was characterized as of first order with respect to the acetylthiocholine concentration with a rate constant of 0.623 s-1. The experimental data obtained with the artificial molecule were applied to the Michaelis-Menten\'s model and the kinetic parameters were determined, noting that the rate constant was calculated as 13000 times smaller than that obtained by the differential method, which indicates the inconvenience of using the enzymatic model to the mimetic catalysts. Moreover, it was found that KM has no meaning when applied to mimetic molecules.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-02T20:43:30Z No. of bitstreams: 1 LaianeSouto.pdf: 1543926 bytes, checksum: b4812ad4c2c00b039863648640d563e8 (MD5) / Made available in DSpace on 2017-06-02T20:43:30Z (GMT). No. of bitstreams: 1 LaianeSouto.pdf: 1543926 bytes, checksum: b4812ad4c2c00b039863648640d563e8 (MD5) Previous issue date: 2016-12-13 / Microcystin (MC-LR) are a class hepatotoxins produced by cyanobacteria in surface water. scientific records show that the MC-LR, inhibits the action of intracellular proteins, alkaline phosphatases, but has also been proven to increase the enzyme acetylcholinesterase activity (AChE) by the MC-LR action. Therefore, this study aimed to develop biosensors amperometric based AChE enzyme for indirect detection of MC-LR. For construction of the working electrode, a powder graphite paste containing hidroxicetilcelulose (HEC), bovine serum albumin (BSA) and glutaraldehyde (Glu) was prepared. The slurry was incorporated into the AChE enzyme extracted bovine erythrocyte (EB) and electric eel (EE) as well as enzymes derived from Drosophila melanogaster was tested genetically modificadas.Também enzyme butyrylcholinesterase (BChE) obtained from human serum. A portion of the sensitive paste was deposited in the screen-printed sensor working electrode and performed characterization tests involving differential pulse voltammetry and cyclic voltammetry. cronoamperométricas readings were performed and the percentage built on activation curves (% RA) as a function of the concentration of MC-LR. Some operating conditions, such as working potential, electrochemical mediator, medium pH and substrate concentration, were optimized. enzyme activation assays showed that the EE-AChE enzyme showed best results percent relative activation (% RA) (> 10%), these values being directly proportional to the concentration of MC-LR. The biosensor developed proved accurate (CV ~ 8.32%), sensitive (Ld 0.27 μg .L-1 and LQ 0.91 μg .L-1) and accurate (recovery rates varying from 73 to 105%). The initial study proved to be the right biosensor to verify the presence of MC-LR in aquatic environments, this being then used in monitoring pollutant seven points of Bacanga River, an important aquatic ecosystem of San Luis, MA. The results indicated that there was no significant contamination. / Microcistinas (MC-LR) são uma classe de hepatotoxinas produzidas por cianobactérias em águas de superfície. Registros científicos comprovam que a MC-LR inibe a ação de proteínas intracelulares, as fosfatases alcalinas, mas também foi comprovado que sua ação aumenta a atividade da enzima acetilcolinesterase (AChE). Portanto, esse trabalho objetivou desenvolver biossensores amperométricos à base da enzima AChE para detecção indireta da MC-LR. Para construção do eletrodo de trabalho, foi preparada uma pasta de grafite em pó contendo hidroxicetilcelulose (HEC), soroalbumina bovina (BSA) e glutaraldeído (Glu). A pasta foi incorporada às enzimas AChE extraídas do eritrócito bovino (EB) e da enguia elétrica (EE), bem como enzimas geneticamente modificadas extraídas da Drosophila melanogaster. Também foi testada a enzima butirilcolinesterase (BChE) obtida de soro humano. Uma porção da pasta sensivel foi depositada no eletrodo de trabalho de sensores serigrafados, e realizados testes de caracterização envolvendo voltametria de pulso diferencial e voltametria cíclica. Leituras cronoamperométricas foram realizadas, em seguida, construídas curvas da ativação relativa percentual (AR%) em função da concentração da MC-LR. As seguintes condições operacionais: potencial de trabalho, mediador eletroquímico, pH do meio e concentração do substrato, foram otimizadas. Os ensaios de ativação enzimática revelaram que a enzima AChE (EE) apresentou melhores resultados de ativação relativa percentual (AR %) (>10%), sendo esses valores diretamente proporcionais à concentração da MC-LR. O biossensor desenvolvido mostrou-se preciso Coeficiente de Variação (CV ~ 8,32%), sensível Limite de detecção e Quantificação (LD 0,27 μgL-1 e LQ 0,91 μgL-1) e exato (índices de recuperação variando de 73 a 105%). O estudo revelou ser o biossensor adequado à verificação da presença de MC-LR em ambientes aquáticos, tendo sido este então utilizado no monitoramento do poluente em sete pontos do Rio Bacanga, um importante ecossistema aquático de São Luis, MA. Os resultados indicaram não haver uma importante contaminação desse poluente nas áreas investigadas.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2017-08-29T18:48:40Z No. of bitstreams: 2 DISSERTAÇÃO VERSÃO FINAL - FABIANA SARI FERREIRA.pdf: 3287035 bytes, checksum: 4366071c4629680884533f5c945e6d0e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-29T18:48:40Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO VERSÃO FINAL - FABIANA SARI FERREIRA.pdf: 3287035 bytes, checksum: 4366071c4629680884533f5c945e6d0e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Carbamates are essential products for pesticide formulation, mainly used in agriculture for the elimination of insects. Although the production and trade of carbamates are for purpose insects elimination, it is common used to suicide attempts in our environment, in addition to accidental poisonings involving rural workers and children. The toxicological property is inhibit the acetylcholinesterase and buthyrylcholinesterase enzymes which leads a severe poisoning related to cholinergic crisis. The enzymes activity get back to normal only spontaneously, so the study of new substances that can intervene directly on the active site of these enzymes when it is inhibited and reverse this toxic action is necessity for the health. Researchs with Melia azedarach L. (Meliaceae) has been made searching for new strategies for treatment of enzime inhibity by methyl carbamate poisoning. Different concentrations of Melia azedarach L. buffered extract were tested in vitro and in vivo for acetylcholinesterase activity inhibited by methylcarbamate. The results showed that the extract of Melia azedarach L. has a potential reversion effect on the enzyme inhibited by methyl carbamate, becoming an alternative to treatment of carbamate intoxication / Os carbamatos são produtos fundamentais para a formulação de praguicidas. Embora sua produção e a comercialização sejam com a finalidade de eliminação de insetos, a sua utilização em tentativas de suicídio é comum em nosso meio, além das intoxicações acidentais envolvendo trabalhadores rurais e crianças, devido sua propriedade toxicológica em inibir as enzimas acetilcolinesterase e butirilcolinesterase. A atividade destas enzimas só volta ao normal espontaneamente, assim, o objetivo deste trabalho foi verificar a possível ação do extrato de folhas de Melia azedarach L. sobre o sistema colinérgico inibido por metilcarbamato. Foram testadas in vitro e in vivo, diferentes concentrações de extrato tamponado de Melia azedarach L., quanto a atividade da acetilcolinesterase inibida por metilcarbamato. Os resultados mostraram que o extrato de Melia azedarach L. tem potencial efeito de reversão sobre a enzima inibida pelo metilcarbamato, se tornando uma alternativa na terapia do tratamento de intoxicações com carbamatos.

Die vorliegende Arbeit befasst sich mit der Synthese und biologischen Testung von Anti-Alzheimer-Substanzen, die nicht nur auf einen Angriffspunkt der Krankheit abzielen, sondern an mehreren krankheitsauslösenden bzw. krankheitsfördernden Punkten inhibierend wirken können. Als Leitstruktur dienten bereits bekannte Acetylcholinesteraseinhibitoren der DUO-Reihe, welche sukzessive abgewandelt wurden, bis die entscheidenden Strukturmerkmale für eine Acetylcholinesterasehemmung herausgefiltert waren. Wichtig für die Interaktion mit der Acetylcholinesterase ist ein quartärer Stickstoff sowie aromatische Reste in seiner näheren Umgebung. Durch die Pyridylen-Hydrazone ist dies gewährleistet. Aufgrund dessen wurde eine Substanzbibliothek synthetisiert, die nun die Acetylcholinesterasehemmung mit einer Inhibition der Fibrillenbildung verbindet. Synthese: Aus 4-Chlorpyridin-Hydrochlorid wurde zuerst die freie Base freigesetzt und diese mit Hydrazin-Hydrochlorid zu 4 Hydrazinylpyridin-Hydrochlorid umgesetzt. Dieses reagierte im nächsten Schritt mit dem entsprechenden Aldehyd in einer SN2-Reaktion zu den Zwischenstufen 2-14, die nun einen variablen Rest an der Hydrazinylgruppe tragen. Im letzten Schritt erfolgte die Quarternisierung mit Hilfe des entsprechenden Alkylbromids, wodurch am Pyridylrest derivatisiert wurde und schlussendlich die Verbindungen 2A-14O erhalten wurden. Dieser letzte Schritt, welcher anfangs durch klassische Erhitzung zum Rückfluss in DMF durchgeführt wurde, konnte zuerst durch die Durchführung im Bombenrohr zeitlich verkürzt werden und schließlich in der Mikrowelle optimiert werden, was zu einer Reduktion der Reaktionszeit von 500 h auf 3 h führte. Die Testung der Substanzen bezüglich ihrer AChE- und BuChE-Hemmung erfolgte mittels Ellman’s Test. Für die Testung der hemmenden Wirkung bezüglich der Fibrillen wurde zuerst das Testsystem mit Aβ (1-42) aufgebaut und anschließend auf ein verkürztes, elf Aminosäuren langes Peptid (HHQKLVFFAED), das mit Hilfe eine Peptidsynthesizers hergestellt wurde, übertragen. Nimmt man nun wieder Bezug auf den „Multi-target-Ansatz“, so lassen sich folgende Ergebnisse festhalten: Die Verbindung eines elektronen-ziehenden Substituenten am Phenylring an der Hydrazonseite mit einem Propylrest zwischen dem Phenylring und dem Pyridinrest ist vorteilhaft für die Kombination der Hemmung der Acetylcholinesterase und der Inhibiton der Aβ-Fibrillen (vgl. 13C). Während viele Substanzen selektiv gegenüber der Acetylcholinesterase sind, gibt es nur wenige, die selektiv die Butyrylcholinesterase hemmen. Eine Hemmung aller drei Angriffspunkte im niedrigen mikromolaren Bereich zeigten nur wenige Substanzen, und zwar die Verbindung mit jeweils einem Naphthylrest sowohl an der Hydrazon- als auch der Pyridin-Seite (14K: IC50 (AChE) = 1.12 µM; IC50 (BuChE) = 2.32 µM; IC50 (Fibrillen) = 2.5 µM). Zusätzlich wurden die Substanzen auf eine mögliche ROS-Hemmung von Kooperationspartnern getestet. Sie zeigten einen kleinen aber signifikanten Beitrag zur Reduzierung der ROS. Um erfolgreiche Wirkstoffe gegen die Alzheimer-Krankheit zu entwickeln, ist deren Blut-Hirn-Schrankengängigkeit von entscheidender Bedeutung. Deshalb wurde von den Substanzen mittels HPLC der logP-Wert bestimmt. Die logP-Werte der Substanzen liegen in einem Bereich von 3.2 bis 4.4. Aufgrund dieser Ergebnisse und der berechneten pKS-Werte, welche im Bereich von 8.3 bis 10.2 liegen, kann davon ausgegangen werden, dass die Substanzen auf Grund der „sink“-Bedingungen die Blut-Hirn-Schranke überqueren können. Um eine definitive Aussage zur Blut-Hirn-Schrankengängigkeit treffen zu können, wurde ein Transwell-System mit cerebralen Endothelzellen entwickelt, die die Blut-Hirn-Schranke simulieren. Die Endothelzellen bilden hierbei eine dichte Monoschicht auf dem Filter aus, wobei die Substanzen nicht zwischen den Zellen hindurch diffundieren können, sondern den Weg durch die Zelle nehmen müssen. Die Messungen ergaben, dass die Substanzen zu ca. 90 % durch die Zellen diffundieren können und somit erfolgreich die Blut-Hirn-Schranke überqueren können. / The thesis is dealing with the synthesis and biological evaluation of substances combating Alzheimer’s disease by using a multi-target approach. Starting point was the lead structure of the known acetylcholinesterase inhibitors of the DUO-series, which was successively modified to filter the crucial structural characteristics of the inhibition of acetylcholinesterase. Important for the interaction with acetylcholinesterase is a quaternary nitrogen as well as an aromatic moiety nearby. This is combined in the pyridylene-hydrazones. Therefore a library of substances has been synthesized and evaluated for the inhibition of acetylcholinesterase and fibril formation. Synthesis: The free base of 4-chloropyridine-hydrochloride was produced and reacted with hydrazine-hydrochloride to give 4 hydrazinylpyridine-hydrochloride. Next, a corresponding aldehyde was added and the intermediates 2-14 including a variable moiety on the side of the hydrazinyl-group, were formed by a SN2-reaction. Finally the quaternization reaction with the corresponding alkylbromide was performed in order to introduce the residues at the side of the pyridyl moiety to achieve the final compounds 2A-14O. This step was carried out by classical heating to reflux in DMF, but could be improved by carrying out the reaction in a pressure vessel and even better in the microwave; here the reaction time could be reduced from 500 h to 3 h. The inhibitory activity of the compounds towards AChE and BuChE were performed with Ellman’s test. In order to determine the inhibition of fibril formation, the Thioflavin T assay was first established with Aβ (1-42) and afterwards transferred to a shortened peptide containing only eleven amino acids (HHQKLVFFAED). This peptide was synthesized with a peptide synthesizer. In summary, combining an electron-withdrawing substituent at the phenyl ring on the site of the hydrazon with a propylene spacer between the phenyl ring an the pyridyl rest is of advantage when both targets – acetylcholinesterase and Aβ fibril formation – should be equally inhibited (see 13C). While many substances are selective towards acetylcholinesterase, only a few inhibit the butyrylcholinesterase selectively. An inhibition of all of the three targets in a low micromolar range of concentration could only be revealed for few compounds, e. g. for compound 14K with naphthyl-substitution on both sides: IC50 (AChE) = 1.12 µM; IC50 (BuChE) = 2.32 µM; IC50 (fibrils) = 2.5 µM). Additionally the substances were tested on a possible ROS-inhibition, however, they showed only little, but significant influence on a reduced production of ROS. In order to develop successfully new compounds against Alzheimer’s disease, their blood-brain-penetration is very important. For this reason the logP-value of the substances has been determined by a HPLC method. The logP-values range from 3.2 to 4.4. Due to these results and the calculated pKA-values, which ranged between 8.3 and 10.2, it can be assumed that the substances are able to cross the blood-brain-barrier because of sink-conditions. In order to verify this statement, a transwell-assay with cerebral endothelial cell monolayer has been developed to simulate the blood-brain-barrier. The measurements of some representative compounds revealed a diffusion rate through the cells of about 90 %, indicating the blood-brain-penetration of the compounds.

In skeletal muscle acetylcholinesterase AChE is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Scientists have elucidated many aspects of synaptic AChE structure, function, and localization during the past 80 years. However our understanding of the molecular mechanisms underlying its regulation is incomplete, but it appears to involve both translational and post-translational events as well. We found that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates and that PUM2 binds to the AChE transcripts when immoprecipitation studies were performed. A direct binding between a recombinant PUM2-HD and the Pumilio Binding Site (PBE) in a segment of the AChE 3’UTR was demonstrated by Gel shift assays. Transfecting skeletal muscle cells with shRNAs specific for PUM2 upregulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. We found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific synaptic proteins involved in modulating synaptic transmission, analogous to CNS synapses. PUM2 expression is critically important in many cell types, virtually nothing is known about the regulation of PUM2 expression itself. Analyzing the PUM2 mRNA 3’UTR we found fifteen possible PBEs in the 3 Kb 3’ UTR. We show that PUM2 binds in vivo to its own mRNA. Overexpression of PUM2 in several cell types transfected with a green fluorescent protein (GFP) reporter construct linked to the full length PUM2 3’UTR (GFP-PUM2-3’UTRFL) suppresses GFP expression suggesting that PUM2 downregulates its own expression by binding to its own 3’UTR. Mutations of the first five PBEs yield the expression of the reporter gene indicating that at least one PBE is functional in the autoregulation of PUM2. These observations suggest a novel model for the localized regulation of protein translation through a negative feedback loop. Much is known about PUM2 as a translational regulative protein but little is known about PUM2 cell localization and possible mechanism of translational regulation. In this work we found PUM2 to be highly localized to the cell rough endoplasmic reticulum and that PUM2 is associated with ribosomal RNA. In addition, we found that the GFP protein itself, together with its mRNA and ribosomal RNA (rRNA), were localized in the PUM2 positive complexes when GFP-PUM2-3’UTRFL was transfected into muscle cells. These observations further suggest a mechanism of regulation where translation of the protein occurs but the protein remains associated with the ribonucleoprotein complex, possibly to be transported together with its mRNA to specific domains inside the cell. Thus when needed, more protein is produced in those specific cell regions.

Pesticides are natural or artificial molecules aimed to kill, or mitigate any harmful organism. Although their use in agriculture provides us with an increased crop yield, remains of chemicals on the products creates health concerns in society. Organophosphates and carbamates are two groups of insecticides. Although they are far more lethal against insects and small animals, they can also cause poisoning in humans through the inhibition of acetylcholinesterase enzyme (AChE) that plays an important role in human nervous system. Therefore, the detection of these compounds is crucial. The conventional methods for the detection of these compounds are expensive, time-consuming and need expertise. In this study, a fast, disposable, cheap and accurate acetylcholinesterase biosensor was developed to detect organophosphate and carbamate-based pesticide residues. By means of adsorption method, AChE, the chromophore 5,5&#039 / -Dithio-bis(2-nitrobenzoic acid) (DTNB) and artificial substrate acetylthiocholine (ATCh) were immobilized on the supporting material. In optimization studies / from 3 to 15U/mL concentrations were experimented for AChE, 1 to 5mM DTNB and 1 to 5mM ATCh concentration gradients were used. v As a result of the optimization studies 12U/mL ACHE concentration, 5mM DTNB concentration and 5mM ATCh concentration were determined for constructing a pesticide biosensor. Detection limit of malathion, an organophosphate-based insecticide was found as 2.5ppm in 5% methanol solution. The biosensor conserved its integrity between pH 4 and 8, and gave false positive results after pH 10. Stability studies showed that, biosensor retained its activity for at least 60 days at 4&deg / C to discrimnate between positive and negative controls.

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