Orientador: Ana Maria Lima de Azeredo-Espin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T11:05:22Z (GMT). No. of bitstreams: 1 Silva_NormaMachadoda_D.pdf: 6129934 bytes, checksum: 5d8a07895afd2e470c9fd5caf8ac9fb8 (MD5) Previous issue date: 2009 / Resumo: Cochliomyia hominivorax é considerada uma importante praga por causar perdas econômicas para a pecuária na região Neotropical. Este ectoparasita tem sido controlado principalmente por inseticidas, entretanto, este método normalmente resulta na seleção de indivíduos resistentes. Nesta tese foram investigados mecanismos associados com resistência a inseticidas organofosforados e piretróides. Os resultados apresentados nesta tese estão divididos em dois artigos. No artigo 1 foram investigados dois mecanismos de resistência a piretróides, um conhecido como kdr (knockdown resistance), associado à mutações no canal de sódio, e a mutação W251S no gene da carboxilesterase E3, a qual têm sido associada à hidrólise de piretróides. Foram investigadas populações da atual distribuição geográfica de C. hominivorax através de PCR-RFLP. Para a mutação kdr (L1014F) nenhum indivíduo mutante foi encontrado. Entretanto, a mutação W251S foi encontrada em todas as populações, com as mais altas freqüências nas amostras da Venezuela (100%) e Colômbia (93.75%). Em algumas populações amostradas, apesar da freqüência do alelo mutante ter sido relativamente baixa, o número de indivíduos portadores de pelo menos um alelo mutante foi alto. No bioensaio com cipermetrina (piretróide tipo II), utilizando amostras de Estiva (MG), os resultados analisados através do teste exato de Fisher indicaram que para a mais baixa concentração (p=0.0003) e para uma concentração intermediária (p= 0.024), a presença da mutação W251S está correlacionada com as chances de sobrevivência. Entretanto, na mais alta concentração (p= 0.221) a presença desta mutação não foi correlacionada com a sobrevivência, o que possivelmente indica dificuldade em hidrolisar este tipo de piretróide em concentrações maiores. No Artigo 2, as mutações G137D no gene da carboxilesterase E3, e as mutações I298V, G401A e F466Y no gene da acetilcolinesterase, associadas com resistência a organofosforados, foram investigadas nas mesmas populações do artigo 1, através de PCR-RFLP e sequenciamento direto de PCR. A região codificante completa da acetilcolinesterase também foi caracterizada e proposto um modelo para a estrutura tridimensional desta proteína com base na estrutura da acetilcolinesterase de Drosophila melanogaster. Possíveis alterações na proteína causadas pelas mutações associadas com resistência a organofosforados foram discutidas. No gene da acetilcolinesterase somente 2 de 135 indivíduos analisados apresentaram uma das mutações investigadas, a F466Y, o que pode estar associado ao alto custo no valor adaptativo do indivíduo, causado por algumas mutações neste gene ou inexistência dos demais alelos mutantes nestas populações. Entretanto, para a mutação G137D no gene da E3 foi encontrado um alto número de indivíduos portadores de pelo menos um alelo mutante na maioria das populações brasileiras e no Uruguai. Em Cuba, Venezuela e Colômbia nenhum indivíduo mutante para G137D foi encontrado, o que pode estar relacionado com os inseticidas usados no controle de C. hominivorax nestas regiões ou com a inexistência do alelo mutante nestas localidades. Os resultados de ambos os trabalhos indicam que mutações no gene da carboxilesterase E3 são um dos principais mecanismos de resistência selecionados nesta espécie, dentre os investigados até o momento. Este estudo representa um importante avanço no entendimento da base molecular da resistência a inseticidas em um importante ectoparasita. / Abstract: Cochliomyia hominivorax is an important ectoparasite, causing considerable economic losses to livestock sector in Neotropical region. This ectoparasite has been controlled mainly by applying insecticides, however, this method usually results in the selection of resistant individuals. In this thesis, mechanisms associated with organophosphate and pyrethroid insecticide resistance were investigated. The results presented in this thesis are divided in two articles. In the Article 1 two mechanisms associated with pyrethroid resistance were investigated, the kdr (knockdown resistance), a generic term for mutations in the sodium channel, and a mutation in the residue 251 of the carboxylesterase E3 gene, which have been associated with pyrethroid hydrolysis. Populations of actual geographical distribution of C. hominivorax were investigated through PCR-RFLP. For the kdr mutation, no mutant individuals were found. However, the W251S mutation was found in all populations investigated, with the highest frequencies in the samples from Colombia (100%) and Venezuela (93.75%). In some populations, despite relatively low mutant allele frequency, the number of individuals having at least one mutant allele was high. A bioassay with cypermethrin (pirethroid type II) was also performed, using samples from Estiva (MG). The results of the Fisher's exact test for the lowest concentration (p=0.0003, a= 0.05) and for an intermediate concentration (p= 0.024, a= 0.05) indicated that the presence of W251S mutation are correlated with the survival. However, at the highest concentration, Fisher's exact test (p= 0.221, a= 0.05) indicated that the presence of this mutation was not correlated with survival. These results indicate that the W251S mutation in C. hominivorax probably presents difficulties in hydrolyzing of this kind of pyrethroid, in high concentrations. In the Article 2, the G137D mutation, in the carboxylesterase E3 gene, and the I298V, G401A and F466Y mutations in the acetylcholinesterase gene, associated with organophosphate resistance, were investigated. The same populations of article 1 were analyzed using PCR-RFLP and PCR direct sequencing. The complete coding region of acetylcholineserase also was characterized and a putative model for the three-dimensional structure of this protein was proposed, based on solved structure of Drosophila melanogaster. Possible alterations in the protein, caused by mutations associated with organophosphate resistance, were discussed. For the acetylcholinesterase gene, only 2 of 135 individuals analyzed presented one of the mutations investigated, the F466Y. This fact may be related with the fitness cost of some mutations in this gene, or the inexistence of mutant alleles in these populations. For the G137D mutation, a high number of individuals having at least one mutant allele were found in most of the Brazilian populations and in Uruguay. In Cuba, Venezuela and Colombia no G137D mutant was found, a finding that may be related to the insecticides used for C. hominivorax control in these regions. The results of these two works indicate that mutations in the carboxylesterase E3 gene are one of the main insecticide resistance mechanisms selected in this species, so far investigated. This study represents a significant advance in the understanding of the molecular basis of insecticide resistance in an important livestock ectoparasite. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Ce travail de recherche est centré sur la valorisation de deux Euphorbiacées : Ricinus communis et Jatropha curcas. La première est une espèce autochtone connue comme plante dont l’huile des graines est utilisée pour ses vertus cosmétiques quant à la deuxième, c’est une espèce allochtone récemment introduite à titre expérimental en Tunisie et connue comme plante bioénergetique. Pour le ricin huit populations Tunisiennes ont été étudiées: Riadh Andalous, Nefza, Béja, Nabeul, Hammamet, Bouficha, Khanguet Hajej et Aouled Amer. Quant au jatropha, le matériel végétal est récolté dans la station expérimentale de Nabeul (Tunisie). Il s’agit de huit populations qui proviennent d’Arusha en Tanzanie, de Mozambique, de Suriname et de Brésil à partir de cinq provenances à savoir : Paranà, Norte de Minas, Mato Grosso, Regiao sudeste et Vale do Jequitinhonha. Le travail comprend une première partie consacrée à une étude bibliographique. Une deuxième partie est consacrée aux matériels et méthodes utilisés et une dernière partie qui montre l’ensemble des résultats obtenues. Ainsi, les résultats montrent que les extraits des feuilles des populations des deux espèces étudiées sont plus riches en composés phénoliques que les extraits des racines. L’étude phytochimique a montré que le ricin contient surtout de l’acide gentisique. Quant au jatropha, il contient surtout de l’épicatechine et de la naringine. L’étude chimique des huiles fixes des deux espèces montrent que l’huile de ricin renferme essentiellement d’acide ricinoléique. Quant à l’huile de jatropha, elle contient deux acides gras en proportions majeures qui sont l’acide oléique et l’acide linoléique. L’évaluation des activités anti-oxydantes des extraits des deux espèces indique une corrélation positive entre ces activités et les teneurs en composés phénoliques. Par ailleurs, l’étude de l’activité anti-acétylcholinestérase des extraits testés montrent que ces extraits sont des inhibiteurs de l’AChE plus puissants que la galanthamine utilisée comme contrôle positif. Notre étude a confirmé que les différents extraits de ricin et de jatropha, autres que les huiles fixes, peuvent donc être exploités pour d’autres activités biologiques, parmi les quelles l’action inhibitrice de l’AChE une des principales cibles des traitements contre la maladie l’Alzheimer et le piégeage des radicaux libres, en raison de leur richesse en composés phénoliques / The aim of this study is to promote two Euphorbiaceae plants the Ricinus communis and the Jatropha curcas, the first one is known for its oil used in the cosmetic products, whereas the second one is known especially for its seeds used in the production of biodiesel. For the castor plant, eight Tunisian populations are studied: Riadh Andalous, Nefza, Beja, Nabeul, Hammamet, Bouficha, Khanguet Hajej and Aouled Amer. For the jatropha, recently introduced in Tunisia, the plant material is collected from the Nabeul station (Tunisia). Eight populations coming from Arusha in Tanzania, Mozambique, Suriname and Brazil from regions of Paraná, Minas Norte, Mato Grosso, Regiao sudeste and Vale do Jequitinhonha are studied. In first, a bibliographic study is made. In second, the used materials and methods are cited. Fanilly, all the results are mentioned. The study shows that the phenolic compounds are higher in the leaves extracts than in the roots extracts for both species. The phytochemical study shows that the gentisic acid is the major phenolic compound identified in the castor plant extracts. In contrast, the epicatechin and the naringin are the most important phenolic compounds identified in the jatropha extracts. The GC-MS analysis reveals that the castor oil contains mainly the ricinoleic acid. For the jatropha oil, two major fatty acids are identified: the oleic and the linoleic acids. Results of the antioxidant properties of leaves and roots of both species indicate a positive correlation between the leaves and the roots activities and their contents of phenolic compounds. Furthermore, the anti-acetylcholinesterase activity of the tested extracts shows for the first time that some tested extracts are more active than the galantamine used as a positive control. Our study confirmed that, in addition to their oils, different extracts of the castor plant and the jatropha can be used for biological activities such as the scavenging free radicals and the inhibitory action of AChE enzyme, which is a major target for treatments against the Alzheimer's disease due to their high levels of phenolic compounds. Owing to the activities of the leaves and the roots extracts confirmed by this study, the agricultural exploitation of the castor plant and the jatropha can be economically more profitable

Drabbová, A.: Title of Diploma Thesis: Centaurea cyanus L. (Asteraceae) alkaloids and their biological activity related to Alzheimer's disease. Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové 2020. Ethyl acetate and chloroform alkaloids extracts were prepared from Centaurea cyanus L. seeds. A novel alkaloid AD-1 (Adrabbin) was isolated from the ethyl acetate extract by common chromatografic methods (column chromatography, preparative TLC). Its structure was elucidated by mass spektrometry, NMR analysis and determination of optical rotation. The alkaloid AD-1 (Adrabbin) possesses a unique structure which consists of a cyclic indole moiety. Also LC-MS analysis of fractions obtained by flash chromatography was performed. In those fractions were detected molecular ions related to compounds previously isolated from other Centaurea species. Alkaloid AD-1 (Adrabbin) was tested on ability to inhibit human cholinesterases, prolyl oligopeptidase and gylcogen synthase kinase 3β. The compound was considered against human cholinesterases inactive (IC50 values > 100 µM). Interestibgly, the alkaloid inhibited prolyl oligopeptidase the same intensity as a standard berberine (AD-1: IC50 143,0 ± 6,0 µM; berberin: IC50 142,0 ± 21,0 µM). The novel compound...

V. Hojgrová: Alkaloids of Vinca minor L. and their biological activity VIII. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany. Number of pages 69. This diploma thesis deals with the isolation of alkaloids from Vinca minor L. from the family Apocynaceae. Separation of alkaloids from the selected fraction (VM 215-258) or from their subfractions (VM 34-41, VM 86, VM 87-113) was performed by preparative TLC. Two pure alkaloids were isolated from the subfraction (VH 34-41). The first VH-1 alkaloid not yet isolated and the second VH 2 alkaloid: (-)-raucubainin. Alkaloids were identified by EI-MS, LC-MS, NMR and optical rotation and were compared with data in the literature. Isolated alkaloids were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyloligopeptidase (POP) inhibitory activity and for cytotoxicity. Both substances did not show significant cholinesterase inhibitory activity IC50 against AChE after measurement, only (-)-raucubainin showed a slight activity against BuChE (IC50 = 94 ± 7 μM), VH-1 was found to be inactive (IC50 > 100 μM). POP inhibitory activity has so far only been tested for (-)-raucubainin; was found to be inactive (IC50 > 1000 µM). The results of the cytotoxic activity of the alkaloids...

Malaria continues to cause significant mortality in sub-Saharan Africa and elsewhere, and existing vector control measures are being threatened by growing resistance to pyrethroid insecticides. With the goal of developing new human-safe, resistance-breaking insecticides we have explored several classes of acetylcholinesterase inhibitors. In vitro assay studies demonstrate that tri- and difluoromethyl ketones can potentially inhibit An. gambiae AChE (AgAChE). These compounds inhibit the enzyme by making a covalent adduct with the catalytic serine of AChE. Trifluoromethyl ketones however are poor inhibitors of the G119S resistant mutant of AgAChE. However difluoromethyl ketones can inhibit G119S AgAChE and compound 3-10g showed an IC₅₀ value of 25.1 nM after 23h incubation time. Despite this potent inhibition of AgAChE, the tri-, di-, and (mono)fluoroketones showed very low toxicity to An. gambiae, perhaps due to hydration and rapid clearance. In an attempt to improve An. gambiae toxicity, oximes and oxime ethers of these compounds were prepared as potential prodrugs. These structures identified trifluoromethyl ketone oxime 3-2d as a potent toxin against both wild-type (G3-strain) and a multiply resistant (Akron) strain of An. gambiae. This compound is within 3-fold of the toxicity of propoxur to wild type An. gambiae (LC₅₀ values of 106 and 39 µg/mL, respectively). Most significantly, 3-2d was much more toxic than propoxur to multiply-resistant (Akron) strain An. gambiae (LC₅₀ = 112 and >5,000 µg/mL, respectively). However, thus far we have not been able to link the toxicity of these compounds to a cholinergic mechanism. Pre-incubation studies suggest that significant hydrolysis of these compounds to TFKs does not occur over 22 h at pH 7.7 or 5.5. The mechanism of action of 3-2d remains unknown. Our enzyme inhibition studies have demonstrated that 3-2d does not hydrolyze to the trifluoromethyl ketone 2-9d at pH 7.7. The high Akron toxicity of 3-2d and poor inhibition of G119S AgAChE by 2-9d argue against enzyme mediated conversion of 3-2d to 2-9d within the mosquito. Thus, we can rule out an AChE inhibition mechanism for toxicity. Additional experiments by our collaborator (Dr. Jeffrey Bloomquist, University of Florida) also rule out inhibition of mitochondrial respiration or agonism of the muscarinic acetylcholine receptor. Future work will address other potential insecticidal modes of action. / Ph. D.

Malaria is one of the deadliest diseases known to mankind. In 2010, 219 million cases were reported, and 666,000 deaths were attributed to this disease. In the past, pyrethroid-treated mosquito nets have shown efficacy in reducing malaria transmission in many malaria endemic regions. However, an upsurge in the mosquito population that is resistant to pyrethroids threatens to compromise the efficacy of pyrethroid-treated bed nets. In an effort to develop another class of insecticide with a different mode of action, we have explored three classes of five membered heterocyclic carbamates (isoxazol-3-yl, pyrazol-5-yl, and pyrazol-4-yl), and 3-oxoisoxazole- 2(3H)-carboxamide as acetylcholinesterase inhibitors (AChE) targeting wild type (G3) and resistant (Akron) malaria mosquito Anopheles gambiae (Ag). Isoxazole carboxamide and carbamates were obtained regioselectively through judicious use of two different protocols. The final products were characterized and identified using ¹H and ¹³C NMR, and mass spectroscopy. In addition, the carboxamide structure was confirmed using X-ray diffraction. Several of the novel carbamates and carboxamides evaluated exhibited excellent toxicity towards susceptible G3 and resistant Akron strain An. gambiae (48f LC₅₀ G3 = 41 μg/mL, LC₅₀ Akron = 58 μg/mL, and 47i LC₅₀ G3 = 38 μg/mL, LC₅₀ Akron = 40 μg/mL). Hence, achieving the resistance- breaking goal. On the contrary, the commercial aryl methylcarbamates currently approved for indoor residual sprays (IRS) showed no potency towards the resistant strain An. gambiae (LC₅₀ G3 = 16-42 μg/mL, and LC₅₀ Akron >5,000 μg/mL). Further, we observed low toxicological cross-resistance ratios (RR) for the toxic isoxazol-3-yl and pyrazol-4-yl carbamates, and 3- oxoisoxazole-2(3H)-carboxamides (RR = 0.5-2.0). Amongst the commercial AChE inhibitors approved for IRS, only aldicarb exhibited such low RR (RR = 0.5), whereas the RR for commercial aryl methylcarbamates exceed 130-fold. The low RR observed for these novel heterocyclic inhibitors would certainly be favorable for a new anticholinesterase-based mosquitocide targeting both the susceptible and resistant strain mosquitoes. Although the overall selectivity (Ag vs human) did not exceed 24-fold, the heterocyclic carbamates and carboxamides synthesized by the author showed appreciable inhibition of resistant AChE (G119S) in comparison to commercial aryl carbamates, which showed no inhibition at all. During the course of this project, the isoxazol-3-yl and pyrazol-5-yl methylcarbamates proved to be unstable, and thus could not be isolated. The synthesis of pyrazol-4-yl methylcarbamates using N-methylcarbamoyl chloride proved particularly challenging due to the formation of by-products called allophanates. The similar Rf of the by-product and the desired final product made the isolation laborious and time-consuming. We have successfully overcome this problem by employing a new protocol, where triphosgene served as the carbonylating agent and N-methylamine in THF was used as the amine source. In addition, we have also developed another one-pot protocol for a safer synthesis of pyrazol-4-yl methylcarbamates utilizing 1,1- carbonyldiimidazole (CDI), and N-methylamine hydrogen chloride salt. With the pyrazol-4-yl core, apart from achieving excellent toxicity towards both strains of An. gambiae, we have also achieved excellent AgAChE vs hAChE selectivity (Ag vs h >100-fold). Due to our continued interest in developing this core, we have devised a convenient, scalable, no-column approach for the synthesis an intermediate 103 that can be utilized to synthesize these compounds more efficiently. / Ph. D.

Diseases such as malaria and dengue impose great economic burdens and are a serious threat to public health, with young children being among the worst affected. These diseases are transmitted by mosquitoes, also called disease vectors, which are able to transmit both parasitic and viral infections. One of the most important strategies in the battle against mosquito-borne diseases is vector control by insecticides and the goal is to prevent people from being bitten by mosquitoes. Today’s vector control methods are seriously threatened by the development and spread of insecticide-resistant mosquitos warranting the search for new insecticides. This thesis has investigated the possibilities of vector control using non-covalent inhibitors targeting acetylcholinesterase (AChE); an essential enzyme present in mosquitoes as well as in humans and other mammals. A key requirement for such compounds to be considered safe and suitable for development into new public health insecticides is selectivity towards the mosquito enzyme AChE1. The work presented here is focused on AChE1 from the disease transmitting mosquitoes Anopheles gambiae (AgAChE1) and Aedes aegypti (AaAChE1), and their human (hAChE) and mouse (mAChE) counterparts. By taking a medicinal chemistry approach and utilizing high throughput screening (HTS), new chemical starting points have been identified. Analysis of the combined results of three different HTS campaigns targeting AgAChE1, AaAChE1, and hAChE allowed the identification of several mosquito-selective inhibitors and a number of compound classes were selected for further development. These compounds are non-covalent inhibitors of AChE1 and thereby work via a different mechanism compared to current anti-cholinergic insecticides, whose activity is the result of a covalent modification of the enzyme. The potency and selectivity of two compound classes have been explored in depth using a combination of different tools including design, organic synthesis, biochemical assays, protein X-ray crystallography and homology modeling. Several potent inhibitors with promising selectivity for the mosquito enzymes have been identified and the insecticidal activity of one new compound has been confirmed by in vivo experiments on mosquitoes. The results presented here contribute to the field of public health insecticide discovery by demonstrating the potential of selectively targeting mosquito AChE1 using non-covalent inhibitors. Further, the presented compounds can be used as tools to study mechanisms important in insecticide development, such as exoskeleton penetration and other ADME processes in mosquitoes.

Le système cholinergique joue un rôle important dans de nombreuses fonctions cognitives telles que l'attention et l'apprentissage perceptuel. La stimulation pharmacologique du système cholinergique par le donépézil, un inhibiteur de l’acétylcholinestérase, est un moyen efficace pour améliorer les fonctions cognitives et le traitement cortical via les récepteurs muscariniques et nicotiniques. En effet, le donépézil permet l'accumulation d'acétylcholine dans la fente synaptique. Toutefois, l’effet de la stimulation pharmacologique du système cholinergique sur le traitement visuel complexe et l’apprentissage perceptuel n’est pas encore bien défini. L'objectif de cette thèse est d'étudier, d'une part, l'effet de la combinaison d’un entrainement visuel répétitif avec une stimulation cholinergique sur les capacités visuelles chez le rat et l’humain et, d'autre part, l’effet de la stimulation pharmacologique du système cholinergique sur la restauration des capacités visuelles dans un modèle de déficit visuel chez les rats. Nos résultats ont montré qu’un entrainement visuel/cholinergique entraînait : 1) une potentialisation à long terme de la réponse visuelle corticale chez le rat, 2) une récupération plus rapide des capacités visuelles chez la rat suite un écrasement du nerf optique 3) une amélioration de la performance dans une tâche perceptivo-cognitive de haut niveau plus rapide et conservée dans le temps chez les jeunes sujets sains. Le patron d’électroencéphalographie chez le sujet humain pratiquant une tâche d’attention visuelle n’est cependant pas modifié par l’administration d’une dose unique de donépézil. Ensembles, ces résultats soulignent le bénéfice considérable de la combinaison d’une stimulation du système cholinergique lors de l’entrainement visuel répétitif afin d'obtenir des améliorations de la perception visuelle. Cela présente une avenue très intéressante pour la réhabilitation chez les humains. / The cholinergic system plays an important role in many cognitive functions such as attention and perceptual learning. Pharmacological stimulation of the cholinergic system via donepezil, an acetylcholinesterase inhibitor, is an efficient tool for enhancing cognitive functions and cortical processing via muscarinic and nicotinic receptors. In fact, donepezil allows the build-up of acetylcholine in the synaptic cleft. However, whether pharmacological manipulation of the cholinergic system has an effect on complex visual processing and perceptual learning remains unclear. The goal of this thesis is to investigate on the one hand the effect of combining repetitive visual training with cholinergic enhancement on visual capacities in rats and humans and on the other hand the effect of the pharmacological stimulation of the cholinergic system on visual restoration in a model of visual deficit in rats. Our results showed that cholinergic potentiation induces 1) a long-term potentiation of visual cortical response following repetitive visual stimulation, 2) a faster recovery of brightness discrimination in rats with an optic nerve crush, 3) a faster progression of and a sustained performance in a highly demanding perceptual-cognitive task for healthy young humans. However, the EEG pattern for subjects performing a visual attention task is not modified by a single administration of donepezil. Together these results underline the substantial benefice of combining cholinergic enhancement with visual training in order to obtain visual perception improvements, which presents an interesting avenue for visual rehabilitation paradigm in humans.

Les trois projets que j’ai développés au cours de ma thèse s’inscrivent dans un même contexte, celui de l’étude de l’acétylcholinestérase (AChE) et des molécules apparentées à l’AChE au plan structural ou fonctionnel. Existence, identification et caractérisation du fonctionnement d’une « porte arrière » dans l’AChE. Caractérisation de la « protéine X », un récepteur non AChE de la fasciculine. Recherche d’un partenaire protéique endogène de l’AChE dans le cerveau de rat. / The three projects I developed during my thesis are related to the study of acetylcholinesterase (AChE) and of molecules related to AChE in their function or structure. Existence, identification and characterization of the functioning of a back door in AChE. Characterization of “protein X”, a non-AChE receptor for fasciculin. Search for an endogenous proteic partner of AChE in rat brain.

Adamcová, M.: Study of biological activity of alkaloids isolated from Argemone grandiflora (Papaveraceae) I. Diploma thesis, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany and Ecology, Hradec Králové 2015. The aim of this study was isolation of substances from total diethyl ether alkaloid extract of Argemone grandiflora Sweet, their identification and assessment of their inhibition activity towards acetylcholinesterase, butyrylcholinesterase and prolyl oligopeptidase. Using common chromatografic methods, four alkaloids were isolated, that was identified as (+)-laudanosine, protopine, (-)-argemonine a (-)-platycerine. These substances was tested for their inhibition activity IC50: (+)-laudanosine (IC50 AChE = 617,00 ± 46,55 μM, IC50 BuChE = 644,77 ± 55,52 μM, IC50 POP = not mesured yet); protopine (IC50 AChE = 229,98 ± 21,02 μM, IC50 BuChE = 208,87 ± 17,67 μM, IC50 POP ˃ 1000 μM); (-)-argemonine (IC50 AChE = 4677,75 ± 1241,08 μM, IC50 BuChE = 885,45 ± 119,50 μM, IC50 POP = 337 ± 83,1 μM); (-)-platycerine (IC50 AChE = 223,65 ± 19,61 μM, IC50 BuChE = 1651,25 ± 327,7 μM, IC50 POP = 687 ± 74 μM). In comparison with the standards galanthamine (IC50 AChE = 1,710 ± 0,065 μM, IC50 BuChE = 42,30 ± 1,30 μM) and huperzine A (IC50 AChE = 0,033 ± 0,001...