Orientador: Ana Maria Lima de Azeredo-Espin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T11:52:20Z (GMT). No. of bitstreams: 1 Carvalho_RenatoAssisde_M.pdf: 3247018 bytes, checksum: a5b7660e4e89972955d72629cfd7e2ca (MD5) Previous issue date: 2007 / Resumo: A pecuária bovina tem significativa importância no contexto sócio-econômico para o país, sendo responsável pela produção anual de mais de 2 milhões de toneladas de carne, uma das principais fontes de proteína animal da dieta do consumidor brasileiro. A alta produtividade deve estar sempre acompanhada de um controle sanitário eficiente desses animais. Os prejuízos econômicos provocados por ecto e endoparasitas estão entre os principais fatores limitantes da produção animal. Dentre os ectoparasitas de maior importância na pecuária nacional e considerada uma das principais pragas, está a Cochliomyia hominivorax (Coquerel), conhecida no Brasil como a mosca da bicheira. Os prejuízos causados pelas infestações dessa espécie vêm através da redução da qualidade do couro e da produção de leite, na perda de peso e fertilidade do hospedeiro, e mortalidade dos animais infestados. No Brasil, o seu controle tem sido realizado principalmente através da aplicação de inseticidas organofosforados (OP). Porém, o uso indevido e/ou prolongado desses agentes químicos pode provocar a seleção de indivíduos resistentes. Com a recém introdução de outros princípios ativos, como os piretróides, nas formulações dos inseticidas para controle da C. hominivorax, associada ao fato de que a espécie L. cuprina (Calliphoridae), causadora de miíases em ovelhas, adquiriu resistência ao inseticida OP em menos de 10 anos de uso de tal inseticida, torna-se evidente a importância da investigação da resistência a inseticidas OP em C. hominivorax. Através da amplificação heteróloga com 'primers¿ previamente construídos para L. cuprina, e com 'primers¿ construídos especificamente para C. hominivorax, o gene da esterase E3 foi amplificado e seqüenciado nesta espécie. Foram identificadas as mesmas mutações (Gly137/Asp e Trp251/Ser) responsáveis por conferir resistência a inseticidas organofosforados em L. cuprina e Musca domestica. Além da caracterização dessas mutações, foi desenvolvido um método rápido e eficiente para identificação da principal mutação relacionada com resistência a OP. Toda a região codificante da carboxilesterase E3 foi amplificada e seqüenciada em C. hominivorax, permitindo sua caracterização nesta espécie. Através da realização de um bioensaio utilizando inseticidas organofosforados, a principal mutação (Gly137/Asp) foi associada com a resistência a tal classe de inseticidas em C. hominivorax. A caracterização deste gene nas populações naturais da mosca da bicheira permitirá a realização de uma análise da freqüência de indivíduos resistentes nas diferentes regiões do Brasil, o que possibilitará o uso mais apropriado e eficiente dos agentes químicos nas propriedades com atividade pecuária, contribuindo para uma maior produtividade animal e menor dano ambiental / Abstract: The cattle breeding is very important to the social-economic context of Brazil. It is responsible for annual production of more than 2 million ton of meat, one of the essential sources of animal protein on Brazilian diet. The large productivity should be always accomplished by an efficient sanitary control of the animals. The economic losses caused by internal and external parasites are the main limiting factors of meat production. Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and it is responsible for severe economic losses, mainly by reducing the quality of leather and the production of milk and meat. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. Changes in a specific carboxylesterase (E3) activity have been associated with OP resistance in Lucilia cuprina, one important myiasis-causing fly in sheep in Australia and also a member of the Calliphoridae family. The recent introduction of other insecticides for C. hominivorax control, associated with the development of OP resistance in L. cuprina in less than ten years, shows the importance of resistance investigation in C. hominivorax. Based on foregoing studies, the same mutations responsible for conferring OP resistance in L. cuprina and Musca domestica (Muscidae) were identified in C. hominivorax. Besides characterization of these mutations, a rapid and efficient method for identifying the major mutation related to OP resistance (Gly137/Asp) was developed. Moreover, the entire codifying region of carboxylesterase E3 was amplified and sequenced, allowing its characterization in this species. Using a bioassay, the major mutation (Gly137/Asp) was associated with OP resistance in C. hominivorax. The characterization of this gene in natural populations can be an important tool for surveying resistance to insecticides in different regions of Brazil. This will provide information for the selection and for the implementation of more effective pest management programs / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Au cours des années, l'utilisation de composés nocifs a augmenté de façon exponentielle, et c'est la raison pour laquelle développer des méthodes pour l'identification de composés dangereux, tels que les composés organophosphorés (OP) et les explosifs organiques nitroaromatiques, est indispensable. L'analyse des composés organophosphorés et des explosifs est un enjeu important dans la sécurité intérieure, la médecine légale et les sciences de l'environnement. Il est crucial de développer des méthodes analytiques fiables, sensibles et efficaces pour identifier précisément les composés organophosphorés et les explosifs. Dans ce contexte, le but de ce travail de recherche était de développer des méthodes basées sur la spectrométrie de masse pour l'identification précise et sans ambiguïté de ces composés. Un arbre d'identification a été développé pour l'élucidation structurale de composés organophosphorés. Cette approche a été évaluée à l'aide d'une matrice biologique. D'autre part, les explosifs nitroaromatiques ont été étudiés et plusieurs nouveaux résultats ont été trouvés et reportés. L'application de l'analyse directe en temps réel (DART) couplée à la spectrométrie de masse Orbitrap (DART-FTMS) est décrite ici pour le screening rapide et la caractérisation des échantillons de frottis sur coton obtenus à partir d'armes militaires. / Over the years, the widespread use of harmful compounds has increased exponentially, and this is the main reason to develop methods for the identification of dangerous compounds, such as organophosphorus (OP) compounds and organic nitroaromatic explosives. The analysis of OP compounds and explosives is an important issue in homeland security, forensic and environmental sciences. To this aim, it is crucial to develop reliable, sensitive and efficient analytical methods to accurately identify OP compounds and explosives. In this context, the goal of this research work was to develop accurate mass spectrometric-based methods for the unambiguous identification of these compounds. An identification tree was developed for the structural elucidation of OP compounds. This approach was assessed using a biological matrix. On the other hand, nitroaromatic explosives have been investigated and several new findings were found and reported. Furthermore, the application of Direct Analysis in Real Time (DART) coupled to the Orbitrap mass spectrometer (DART-FTMS) is discussed herein for the fast screening and characterization of cotton swab samples obtained from military weapons.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is responsible to terminate acetylcholine activity in the terminal nervous junctions with its effector organs or post-synaptic sites. The activity of this enzyme could be inhibited by organophosphorus (OP) compounds, and this inactivation leads to an accumulation of acetylcholine in the cholinergic receptors, leading to a cholinergic crisis that may result in death. In this way, the OP compound methamidophos has been related to its broad use in various agriculture cultures in Brazil, with high intoxication rate. Actually, the only compounds able to revert the AChE inhibition by OP, are the oxime, such compounds may reactive the enzyme activity due its high nuclephilic power, attacking the phosphoryl group of the inhibited enzyme and displacing it. However, such compounds show toxic effects, and its use is limited by the high specificity, with each oxime acting only in the reactivation of AChE induced by specific OP coumponds. These limitations raise the need of development of new compounds with AChE reactivator potency, with minor side effects. In this way, have been utilized a series of computational tools (in silico models), with the aim of understand the interaction occurring at a molecular level and, so, rationalize the new compounds development. Thus, the aim of this thesis is to evaluate the activity of three new compounds in reactivate the AChE inhibited by methamidophos, in comparison with two others oximes used in clinical (obidoxime and pralidoxime), both in in silico and in vitro models. Our work demonstrate that the newly synthesized compounds are able to reactivate human erythrocyte AChE, however less efficiently than pralidoxime and obidoxime, and reactivate human plasma butyrylcholinesterase (BChE), where the classical oximes failed. We also show that pralidoxime, which obtained the best reactivation constant among all tested compounds, attack the phosphorus-oxygen moiety (formed between the methamidophos and the AChE catalytic triad residue Ser203) via a region known as oxyanion-hole , composed by the residues Gly120, Gly121 and Ala204. Such found may help in the development of new compounds with better reactivatory activity on AChE inhibited by OP compounds. Furthermore, we show for the first time the individual contribution of each amino acid of AChE, in a radii of 14 Å from the ligand, to the oxime bonding to its active site, using quantum chemistry methods. Here, we demonstrate the important of a quaternary nitrogen to the stabilization of the oximes into the active site; as well as, we obtained evidences that the active form of oximes should be the unprotonated one, instead the protonated, which has been target of debate in the scientific society. Particularly important, we show the critical contribution of amino acids that lies distant from the ligand to the adopted conformation and stabilization of the compounds into the active site of AChE, which has been neglected until far. Finally, our study also evaluates the toxic effects of the compound isatin-3-N4-benzilthiosemicarbazone (IBTC) in mice, which presented low toxicity, with median lethal dose superior at 500 mg/kg. Concluding, this study contributes significantly to the development of new drugs able to restore the AChE activity with minor toxic effects. / A enzima acetilcolinesterase (EC 3.1.1.7, AChE) é responsável por terminar a ação da acetilcolina nas junções das terminações nervosas com seus órgãos efetores ou sítios pós-sinaptícos. A atividade desta enzima pode ser inibida por compostos organofosforados (OP), e sua inativação resulta em um acúmulo de acetilcolina nos receptores colinérgicos, levando a crise colinérgica que pode levar a morte. No Brasil, se destaca o uso do composto OP metamidofós, que é largamente utilizado no controle de pragas em culturas agrícolas e tem sido relacionado com altas taxas de intoxicação. Atualmente, os únicos compostos capazes de reverter a inibição da AChE por OP são as oximas, tais compostos podem reativar a enzima devido a seu alto poder nucleofílico, podendo atacar e retirar o grupamento fosforil da enzima inibida. Porém, tais compostos apresentam efeitos tóxicos, e tem seu uso limitado pela alta especificidade, com cada oxima atuando na AChE inibida por apenas alguns compostos OP. Tais limitações criam a necessidade do desenvolvimento de novos fármacos com potencial reativador da AChE com menores efeitos colaterais. Neste sentido, se tem utilizado uma série de ferramentas computacionais (modelos in silico), com o objetivo de entender as interações que ocorrem em nível molecular e, desta forma, racionalizar o desenvolvimento de novos compostos. Sendo assim, o objetivo desta tese consiste em avaliar a atividade de três novos compostos, em comparação com duas oximas já utilizadas na clínica (obidoxima e pralidoxima), sobre a atividade da AChE inibida por metamidofós, tanto em modelos in silico como in vitro. Como resultados, observamos que os três novos compostos foram capazes de reativar a AChE de eritrócitos humanos inibida por metamidofós, contudo com menor eficiência que as oximas já utilizadas na clínica. Porém, todos os novos compostos foram capazes de reativar a enzima butirilcolinesterase (BChE), uma enzima acessória à AChE no sistema colinérgico, inibida por metamidofós, enquanto nenhumas das oximas clássicas tiveram qualquer atividade reativadora nesta enzima. Nosso trabalho também demonstrou que a pralidoxima, que obteve a melhor constante de reativação entre todos os compostos testados, ataca a ligação fosforo-oxigênio (formada entre o metamidofós e o resíduo Ser203, da tríade catalítica da AChE) via uma região conhecida como oxyanion-hole , que compreende os resíduos Gly120, Gly121 e Ala204. Tal achado pode ajudar no desenvolvimento de novos compostos com melhor atividade reativatória na AChE inibida por OPs. Além disso, mostramos aqui pela primeira vez, a contribuição de cada resíduo de aminoácido da AChE, num raio de 14 Å do ligante, para a ligação das oximas no seu sítio ativo, usando métodos de química quântica. Tais achados mostraram a importância da presença de um nitrogênio quaternário para a estabilização das oximas no sítio ativo; assim como colheu evidências que a forma ativa das oximas seria a sua forma desprotonada, ao invés da forma protonada, o que tem sido alvo de algum debate no meio cientifico. Particularmente importante, foi demonstrado a contribuição fundamental de aminoácidos que se encontram distantes do ligante, para a estabilização e conformação adotada pelos compostos, e que até o momento tem sido negligenciados em estudos in silico. Por fim, nosso estudo também avaliou os efeitos tóxicos do composto isatina-3-N4-benziltiosemicarbazona (IBTC) em camundongos, o qual apresentando baixa toxicidade, com valores de dose letal mediana (LD50) superiores a 500 mg/kg. Desta forma, este estudo contribui para a desenvolvimento de novos fármacos capazes de reativar a AChE e que apresentem menos efeitos tóxicos.

Ce projet à caractère dual civil et militaire vise à développer de nouvelles contre-mesures médicales d’urgences hautement efficaces en cas d’intoxications accidentelles ou volontaires aux organophosphorés toxiques (gaz de combat et pesticides). Ces poisons agissent comme des inhibiteurs irréversibles de l’acétylcholinestérase, enzyme clé principalement localisé dans notre système nerveux, par phosphorylation du site actif enzymatique. Cependant, un nucléophile puissant capable de pénétrer jusqu’au site actif de la protéine peut également l’hydrolyser. Notre recherche réside sur l’élaboration de molécules hybrides possédant deux systèmes clés : une partie réactivatrice neutre capable de restaurer l’activité enzymatique et un ligand du site périphérique capable de vectoriser la structure hybride à l’entrée du site actif. Une librairie de nouveaux hybrides bifonctionnels a ainsi été synthétisée, évaluée et décrite dans ces travaux de thèse. L’ensemble de ces hybrides démontre une activité égale ou supérieure à celle des réactivateurs de référence. Nous avons même découvert le premier hybride universel surpassant l’activité de réactivation des antidotes actuellement commercialisés pour des intoxications aux gaz de combat et aux pesticides. / Remediation of both acute and chronic intoxications by organophosphorus nerve agents, both chemical warfare agents and pesticides, continues to be a challenge of paramount importance. These manmade poisons act as covalent and irreversible inhibitors of acetylcholinesterase, a key enzyme mostly located in the nervous system, through phosphylation of its active site. The phosphylated active site residues do not undergo spontaneous hydrolysis. However, hydrolysis can be achieved through the use of strong nucleophiles able to enter the buried active site of the protein. Our research is based on the rational design of hybrid structures containing two key elements: a neutral reactivator to restore the enzyme activity, and a peripheral site ligand giving selectivity by targeted binding to a site at the entrance of the enzyme active site gorge. A library of novel reactivators based on acridine, quinoline and original oxoassoanine analogues was synthesised, evaluated and is herein described. Delightfully, most of these hybrids proved to be equally or more potent than the drugs currently in use. Outstandingly, we have discovered the first broad-spectrum reactivator that outperformed all known reactivators for both chemical warfare agent and pesticide intoxications.

Title: Comlpexes of aminoalkylphosphineoxides Author: Bence Mészáros Department: Department of Inorganic Chemistry, Faculty of Science UK Supervisor: doc. RNDr. Vojt ch Kubí ek, Ph.D. Supervisor's e-mail address: kubicek@natur.cuni.cz Three selected aminoalkylphosphine oxides were synthesized: Aminomethyl(diphenyl)phosphine oxide, N-piperidylmethyl(diphenyl)phosphine oxide, N,N-diethylaminomethylbis(hydroxomethyl)phosphine oxide. All prepared substances were characterized by NMR spectroscopy and mass spectrometry. The aminomethyl(diphenyl)phosphine oxide and N-piperidylmethyl(diphenyl)phosphine oxide were obtained in the solid state as single crystals and their structure was determined by X-ray analysis. Acid-base and complexing properties of the studied compounds were studied by potentiometric titration and NMR titration. Protonation constants of all three compounds were obtained, stability constants were not determined because of the low solubility of the complexes in water or low stability of the complexes. The structure of the aminomethyl(diphenyl)phosphine oxide complex with Cu2+ was determined by X-ray analysis. Keywords: complexes, organophosphorus synthesis, phosphine oxides, acid-base and complexation properties

Reactions leading to cyclization on methylene bridges in PCPCP structure motif were investigated. It was found, that these reactions result in number of various isomers. Some of these isomers were characterized by NMR and MS spectroscopy, but none was isolated. Further, acid-base properties of bis(methylenephosphonato)phosphineic acid were investigated. Formation of complexes was studied with lithium(I), calcium(II), copper(II) and zinc(II) cations. Furthermore, interaction between bis(methylenephosphonato)phosphinic acid and ethylenediamine was investigated by NMR and potentiometric titration. The results show formation of H-bond-based adducts.

No description available.

Background: Biomonitoring of pesticides is an objective measure of short-term pesticide exposure as it measures possible exposure in the human body. Current evidence on the relationship between demographic, socio-economic and pesticide exposure risk factors and urinary levels of organophosphate (OP) pesticide metabolites among children is generally incomplete and conflicting in some cases. There is therefore a need for further research. Objectives: This study investigated the relationship between socio-economic, demographic and reported pesticide exposure related activities and characteristics in relation to urinary levels of three dialkyl phosphate (DAP) metabolites (diethyl phosphate (DEP), dimethyl phosphate (DMP) and dimethyl triphosphate (DMTP)) among boys living in the rural areas of the Western Cape, South Africa. Methods: This was an analysis of data collected during a cross- sectional study of 183 boys from three agricultural intense areas in the Western Cape of South Africa between April 2007 and March 2008. Measurements included a questionnaire on demographic, socio-economic and pesticide exposure risk factors and analysis of spot urine samples for DAP metabolites. Results: Most of the boys (70%) lived on farms with a median age of 12 years (range: 5.0 - 19.5 years). The median concentrations of DAP, DEP, DMP and DMTP were 68.3 ng/ml (IQR= 27.9; 129.5), 5.5 ng/ml, 32.6 ng/ml and 16.7 ng/ml, respectively. The sum of the three DAP levels wasinversely associated with age. Children older than 14 years had less DAP levels (β = -68.1; 95% CI: -136.8,0.6) than children 9 years and younger. DAP levels also varied significantly with residential area, with the levels highest in Grabouw (apple farming), followed by Hex River Valley (grape farming) (β= -52.1; 95% CI: -97.9, -6.3) then Piketberg (wheat farming) (β= -54.2; 95% CI:-98.8, -9.7). Other weaker and non-significant associations with increased DAP levels were found with increased household income, member of household work with pesticides, living on a farm, drinking water from an open water source and eating crops from the vineyard and or garden. Conclusion: The study found younger age and living in and around an apple and grape farms, to be associated with increased urinary DAP concentrations among the school children provide evidence that younger age and residential area can be associated with increased urinary DAP concentrations among boys. Additionally, there are other household and behavioural characteristics that are associated with elevated urinary DAP levels. Further studies with larger sample sizes and longitudinal designs to improve the statistical power and the associations found are recommended. The study provided more insight to incomplete and inconclusive evidence of previous studies.

No description available.

The syntheses and characterisation of di(cyclohexylmethyl), di(2-cyclohexylethyl), di(2-methylcyclohexyl) and di(4-methylcyclohexyl) phosphoric acids; cyclohexylmethyl phosphonic acid monocyclohexylmethyl ester, di(cyclohexylmethyl), di(4-methylcyclohexyl), di(4-tert- butylcyclohexyl), di(3-methylcyclohexyl), di(3,5-dimethylcyclohexy) and di(2-methylcyclohexyl) phosphinic acids are reported. Problems encountered and how they were resolved during the preparation of the above organophosphorus acids are reported and discussed in detail. These acids are then evaluated as potential hydrometallurgical extractants, for the separation of cobalt from nickel in acid leach liquors, and compared with two commercially available extractants, namely di(2-ethylhexyl)phosphoric acid (D2EHPA) and di(2,4-4- trimethylpentyl)phosphinic acid (Cyanex 272). The effects of variablest such as metal feed solution concentration, extractant concentration, diluent, modifier and temperature are examined experimentally in order to determine which factors are important for optimisation of an extraction system. The extraction characteristics for each acid as a function of pH are presented graphically and the pHO 5 values, distribution coefficients and separation factors are calculated. The dialkylphosphinic acids are found to exhibit much greater selectivity, for cobalt over nickel, than the dialkylphosphoric acids. It is postulated, that steric crowding of the phosphorus atom, by the hydrocarbon groups attached to the phosphorus, increases the selectivity of an extractant. This effect is particularly apparent in the dialkylphosphinic acids with di(2-methylcyclohexyl)phosphinic acid giving the best selectivity; much better than the commercially available Cyanex 272. Several of the dialkylphosphinic acids, evaluated as extractants in this thesis, are protected by a British Patent Application. / Science and Engineering Research Council