Le développement d’un système de détection sensible, sélectif, rapide, fiable et portable à coût modéré est devenu une nécessité pour prévenir le risque chimique lors d’attaques operationnelles ou terroristes potentielles. Ainsi, ce projet porte sur l’élaboration d’un capteur pour la détection d’agents chimiques de guerre de type organophosphorés (Sarin, Tabun, Soman, VX). L’utilisation de microleviers comme capteur pour augmenter la sensibilité est une méthode prometteuse. La surface faible des microleviers conventionnels limite, la sensibilité de la méthode. Ainsi, pour augmenter la surface de capture, nous avons crée un réseau de nanotubes de TiO2 verticalement alignés. Cette nanostructuration est réalisée par une anodisation électrochimique d’une couche de titane pour obtenir les nanotubes de TiO2. L’influence des paramètres du dépôt de titane et de l’anodisation a été optimisée sur des surfaces modèles puis les conditions ont été transferées sur les microleviers. Afin d’augmenter la sélectivité des capteurs (mais aussi la sensibilité) une fonctionnalisation a été réalisée avec une famille originale de ligands bifonctionnels capables de promouvoir la reconnaissance moléculaire des composés organophosphorés cibles et adaptés à la liaison avec une surface de TiO2. / The development of a sensitive, selective, fast, reliable, and moderate cost portable detection system has become a necessity to prevent chemical risk during operational or terrorist attacks. Thus, this project is focused on the elaboration of sensor for the detection of chemical warfare agents (Sarin, Tabun, Soman, VX). The use of microcantilevers as sensors is a promising method to increase sensitivity of detection. The low surface area of conventional microcantilevers limits the sensitivity of the method. Thus, to increase the surface of capture, we create a nanotubular titanium oxide structures. This nanostructuration is performed by anodization of titanium layer to obtain titania nanotubes. The influence of Ti deposition and anodization parameters was studied and the synthesis was optimized onto model surfaces, then beeing transferred to the microcantilevers. In order to increase the selectivity (but also sensitivity) of the sensors functionalization has been carried out with an original family of bifunctional ligands able to promote the molecular recognition of target organophosphorus compounds and suitable for the binding with a TiO2 surface.

Issue de l'Archaea extrêmophile Sulfolobus solfataricus, SsoPox est une enzyme à fort potentiel biotechnologique du fait de son extrême stabilité. Nous avons caractérisé son activité lactonase naturelle ainsi que son activité de promiscuité phosphotriestérase. Elle est capable d'hydrolyser les organophosphorés (OPs), des agents neurotoxiques utilisés en tant qu'insecticide et arme chimique de guerre dont les méthodes actuelles de décontamination restent insatisfaisantes. Non optimale, l'activité phosphotriestérase de SsoPox a été améliorée en redessiné in silico son site actif de afin qu'il ressemble à celui des PTEs, hydrolysant très efficacement les OPs. L'enzyme ainsi développée offre des perspectives d'utilisation pour la décontamination des pollutions aux OPs. L'activité lactonase de l'enzyme a également été améliorée envers certaines N-Acyl Homosérine Lactones (AHLs), des molécules impliquées dans la communication inter-bactérienne (quorum sensing). Or, le quorum sensing régule notamment la virulence bactérienne (e.g. Pseudomonas aeruginosa). Ainsi, l'utilisation de lactonases telles que SsoPox constituerait une alternative aux antibiotiques par interférence au quorum sensing (i.e. quorum quenching). L'ensemble de ces travaux a conduit au dépôt d'un brevet protégeant les séquences des variants améliorés ainsi que leurs domaines d'applications. Ils permirent également d'étudier des phénomènes clés dans l'évolution des enzymes tels que l'épistasie, la promiscuité et la flexibilisation. Ce projet, mêlant Sciences fondamentales et appliquées, pourrait, in fine, mener à la valorisation d'un projet de recherche académique. / Issued from the extremophilic Archaea Sulfolobus solfataricus, SsoPox exhibits an important biotechnological potential because of its extreme stability. Belonging to the phosphotriesterase-like lactonase family, its natural lactonase and promiscuous phosphotriesterase activities were characterized. The enzyme is able to hydrolyze organophosphates (OPs), i.e. nerve agents used as insecticides and chemical warfare agents for which current methods of decontamination are unsatisfactory. The phosphotriesterase activity of SsoPox is suboptimal unlike some mesophilic enzymes (PTEs). To develop a robust bio-decontaminant of OPs, we redesigned, in silico, SsoPox active site in the aim to mimic that of PTEs. Using a mutation database in an in vitro evolution protocol, we screened, selected and characterized efficient enzymes for OPs insecticides hydrolysis, offering prospects in OPs decontamination. SsoPox lactonase activity was also improved for some N-Acyl Homoserine Lactones (AHLs), molecules involved in the inter-bacterial communication (quorum sensing). Quorum sensing regulates several functions such as virulence of some pathogens (e.g. Pseudomonas aeruginosa). The use of lactonases such as SsoPox could constitute an alternative to antibiotics by quorum sensing (and thus virulence) inhibition (i.e. quorum quenching). These works were patented, protecting the sequences of improved variants and their fields of application. They also allowed to study key phenomena in enzyme evolution such as epistasis, promiscuity and flexibility. This project, combining basic and applied Sciences, could lead, in fine, to the promotion of an academic research project.

Quatro linhagens capazes de crescer com acefato foram isoladas a partir de solos com históricos de aplicação de deste composto. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. e Pandoreae sp. foram identificadas através do rDNA 16S e perfil de ácidos graxos de membrana. Rhodococcus sp. foi o isolado mais eficiente na degradação de acefato, removendo 99.24% deste composto em meio de cultura com acefato como única fonte de carbono. Quando avaliado com acefato como fonte combinada de carbono e nitrogênio, este organismo degradou 19% de acefato com formação de metamidofós (17%). Staphylococcus sp. apresentou 21% de degradação de acefato utilizando-o como fonte de carbono e nitrogênio, mas não manteve o crescimento com este composto como fonte de carbono. Stenotrophomonas maltophilia e Pandoreae sp. não mantiveram crescimento com acefato como única fonte de carbono isoladamente. Estas linhagens apresentaram crescimento em acefato como fonte de nitrogênio e enxofre, porém, as análises de GC/MS demonstraram que não houve degradação nestas condições. / Four strains of microorganisms capable of growth on acephate were isolated from soil samples with a history of acephate application. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. and Pandoreae sp. were identified based on 16S rRNA gene sequencing and fatty acid profiling. Rhodococcus sp. was the most efficient acephate degrader of the isolates, it removed 99.24% of acephate from defined growth media when the compound was provided as sole carbon source. When provided as a combined carbon and nitrogen source, the organisms degraded 19% of acephate with formation of methamidophos (17%). Staphylococcus sp. degraded 21% of acephate when provided as sole nitrogen and carbon source but did not grow on the compound as a sole source of carbon. Pandoreae sp. and Stenotrophomonas maltophilia failed to grow on acephate as sole source of carbon in defined medium. These strains grew in media where the pesticide was provided as a combined nitrogen and carbon source, but no acephate biodegradation could be demonstrated in these instances.

Grâce à leurs fortes acidités, les milieux superacides permettent d’accéder par polyprotonation à des intermédiaires superélectrophiles polycationiques, capables d’être piégés par des nucléophiles très faibles. Ces espèces très réactives ont permis de développer des méthodologies de synthèse sans équivalent en conditions « classiques ».Dans la première partie de ce travail, le comportement de composés organophosphorés dans le milieu HF/SbF5 a été évalué et les sites de protonation de différentes fonctions phosphorées ont pu être observés par RMN in situ à basse température. La réactivité d’oxydes de phosphines insaturés a également été évaluée et des composés organophosphorés cycliques et/ou fluorés ont été synthétisés avec de bons rendements. Des expériences de RMN in situ à basse température ont permis de mettre en évidence un intermédiaire superélectrophile de type phosphonium-carbénium inédit dont l’implication a été confirmée par des calculs théoriques.La deuxième partie est consacrée à l’exploitation de la contrainte benzylique d’espèces superélectrophiles. A partir d’éphédrines tosylées, des benzosultames cycliques ont été obtenus par cyclisation intramoléculaire diastéréospécifique dans l’acide trifluorométhanesulfonique. L’analyse des intermédiaires réactionnels par RMN a permis de révéler l’implication d’une contrainte benzylique contrôlant la spécificité de la réaction. Les benzosultames chiraux N-F obtenus après fluoration ont été utilisés comme réactifs de fluoration électrophile énantiosélective et la synthèse de dioxydes de méthanodibenzothiazocines chiraux inédits a également été envisagée. / Thanks to their exceptional acidity, superacid allow access to polycationic superelectrophiles by polyprotonation. These highly reactive species are capable of being trapped by very weak nucleophiles and can be used to develop new synthetic methodologies without equivalents under “classical” conditions. In the first part of this work, the behavior of organophosphorus compounds in HF/SbF5 was evaluated and the protonation sites of different phosphorus functions have been observed by low temperature NMR spectroscopy. The reactivity of unsaturated phosphine oxides has also been evaluated and cyclic and/or fluorinated organophosphorus compounds have been synthetized in good yields. In situ low-temperature NMR experiments revealed a phosphonium-carbenium superelectrophilic intermediate whose implication was confirmed by theoretical calculations. The second part deals with the study of the benzylic strain applied to superelectrophilic species. From tosylated ephedrines, cyclic benzosultams were obtained by diastereospecific intramolecular process in trifluoromethanesulfonic acid. Analysis of the reaction intermediates by NMR revealed the implication of a benzylic strain controlling the specificity of the reaction. After fluorination, the obtained N-F chiral benzosultams were used as enantioselective electrophilic fluorination reagents and the synthesis of new chiral methanodibenzothiazocine dioxides was also considered.

Os dispositivos biossensores foram primeiramente pesquisados por Clark e Lyons, em 1962, visando medir o nível de glicemia. Atualmente, há uma popularização dos medidores de glicemia e diversas tecnologias foram desenvolvidas, principalmente, para aplicações em diagnósticos médicos e monitoramento ambiental. No entanto, há uma ampla quantidade de aplicações para biossensores ainda não disponível no mercado. A imobilização de espécies bioativas que interagem diretamente com o analito, chamada de superfície bioativa, é frequentemente considerada a etapa mais difícil no desenvolvimento de biossensores para novas aplicações. O objetivo deste trabalho é demonstrar a caracterização da camada bioativa desenvolvida pela técnica de construção de camadas auto-organizadas usando o princípio da adsorção eletrostática. Para isto, foram utilizadas três diferentes concentrações de soluções compostas por ligações covalentes entre nanotubos de carbono de paredes múltiplas e três diferentes biomateriais, Polietilenoimina, Ácido Desoxirribonucléico e enzimas do tipo Hidrolase de Organofosforados. As diferentes soluções polieletrolíticas foram caracterizadas pela técnica de ressonância de plasmons de superfície e simultaneamente formaram camadas auto-organizadas na superfície de um eletrodo de carbono vítreo. A atividade das enzimas foi analisada por voltametria cíclica para as diferentes concentrações das soluções utilizadas. Os resultados mostraram que a caracterização por ressonância de plasmons de superfície promove o controle da adsorção eletrostática. A variação do ângulo de ressonância de plasmons de superfície corresponde à intensidade e à saturação das interações eletrostáticas entre as camadas. Biossensores desenvolvidos com soluções de maior concentração, consequentemente, ampliaram os limites de detecção e elevaram a resolução do sinal elétrico. Em aplicações para medir baixas concentrações do analito, o adequado controle das concentrações das soluções poli eletrolíticas reduz os custos no desenvolvimento de biossensores especialmente, com relação ao custo de enzimas isoladas. / The biosensor devices were first researched by Clark and Lyons in 1962, aiming to measure the blood glucose level. Currently, there is a popularization of blood glucose meters and several technologies have been developed mainly for applications in medical diagnostics and environmental monitoring. However, there is a wide range of applications for biosensors not yet available. The immobilization of bioactive species that interact directly with the analyte, called bioactive surface, is often considered the most difficult step in the biosensors development for new applications. The objective of this work is to demonstrate the characterization of the bioactive surface developed by self-assembled monolayers technique using the electrostatic adsorption principle. For this, we used three different concentrations of solutions composed of covalent bonds between multi-walled carbon nanotubes and three different biomaterials, Polyethyleneimine, Deoxyribonucleic Acid and Organophosphorous Hydrolase enzymes. These enzymes catalyze the hydrolysis of phosphorus compounds such as organophosphorous neurotoxins. The different polyelectrolyte solutions were characterized by the technique of surface plasmon resonance, and simultaneously, self-assembled monolayers were formed on the surface of a glassy carbon electrode. The enzymes activity was analyzed by cyclic voltammetry for the different concentrations of the solutions used. The results showed that the surface plasmon resonance characterization promote the control of electrostatic adsorption. The variation of the surface plasmon resonance angle corresponds the intensity and saturation of electrostatic interactions between the layers. Biosensors developed with solutions of higher concentration, consequently, widened the detection limits, and increased the resolution of the electrical signal. In applications to measure low concentrations of analyte, adequate control of the concentrations of polyelectrolytes solutions reduces costs in the development of biosensors especially, the cost of isolated enzymes.

Os dispositivos biossensores foram primeiramente pesquisados por Clark e Lyons, em 1962, visando medir o nível de glicemia. Atualmente, há uma popularização dos medidores de glicemia e diversas tecnologias foram desenvolvidas, principalmente, para aplicações em diagnósticos médicos e monitoramento ambiental. No entanto, há uma ampla quantidade de aplicações para biossensores ainda não disponível no mercado. A imobilização de espécies bioativas que interagem diretamente com o analito, chamada de superfície bioativa, é frequentemente considerada a etapa mais difícil no desenvolvimento de biossensores para novas aplicações. O objetivo deste trabalho é demonstrar a caracterização da camada bioativa desenvolvida pela técnica de construção de camadas auto-organizadas usando o princípio da adsorção eletrostática. Para isto, foram utilizadas três diferentes concentrações de soluções compostas por ligações covalentes entre nanotubos de carbono de paredes múltiplas e três diferentes biomateriais, Polietilenoimina, Ácido Desoxirribonucléico e enzimas do tipo Hidrolase de Organofosforados. As diferentes soluções polieletrolíticas foram caracterizadas pela técnica de ressonância de plasmons de superfície e simultaneamente formaram camadas auto-organizadas na superfície de um eletrodo de carbono vítreo. A atividade das enzimas foi analisada por voltametria cíclica para as diferentes concentrações das soluções utilizadas. Os resultados mostraram que a caracterização por ressonância de plasmons de superfície promove o controle da adsorção eletrostática. A variação do ângulo de ressonância de plasmons de superfície corresponde à intensidade e à saturação das interações eletrostáticas entre as camadas. Biossensores desenvolvidos com soluções de maior concentração, consequentemente, ampliaram os limites de detecção e elevaram a resolução do sinal elétrico. Em aplicações para medir baixas concentrações do analito, o adequado controle das concentrações das soluções poli eletrolíticas reduz os custos no desenvolvimento de biossensores especialmente, com relação ao custo de enzimas isoladas. / The biosensor devices were first researched by Clark and Lyons in 1962, aiming to measure the blood glucose level. Currently, there is a popularization of blood glucose meters and several technologies have been developed mainly for applications in medical diagnostics and environmental monitoring. However, there is a wide range of applications for biosensors not yet available. The immobilization of bioactive species that interact directly with the analyte, called bioactive surface, is often considered the most difficult step in the biosensors development for new applications. The objective of this work is to demonstrate the characterization of the bioactive surface developed by self-assembled monolayers technique using the electrostatic adsorption principle. For this, we used three different concentrations of solutions composed of covalent bonds between multi-walled carbon nanotubes and three different biomaterials, Polyethyleneimine, Deoxyribonucleic Acid and Organophosphorous Hydrolase enzymes. These enzymes catalyze the hydrolysis of phosphorus compounds such as organophosphorous neurotoxins. The different polyelectrolyte solutions were characterized by the technique of surface plasmon resonance, and simultaneously, self-assembled monolayers were formed on the surface of a glassy carbon electrode. The enzymes activity was analyzed by cyclic voltammetry for the different concentrations of the solutions used. The results showed that the surface plasmon resonance characterization promote the control of electrostatic adsorption. The variation of the surface plasmon resonance angle corresponds the intensity and saturation of electrostatic interactions between the layers. Biosensors developed with solutions of higher concentration, consequently, widened the detection limits, and increased the resolution of the electrical signal. In applications to measure low concentrations of analyte, adequate control of the concentrations of polyelectrolytes solutions reduces costs in the development of biosensors especially, the cost of isolated enzymes.

Em razão dos problemas causados pelos insetos nos armazéns, no Brasil tem-se usado principalmente substâncias químicas para controlar essas pragas, porém, com relativo sucesso na solução do problema. Em fase disso, alguns aspectos sobre a proteção de grãos armazenados foram estudados, entre eles: 1) o efeito do volume de pulverização no teor de umidade dos grãos, 2) a estabilidade de inseticidas durante o processamento de amostras, 3) a influência da formulação concentrado emulsionável nas propriedades físicas do líquido, características do spray e depósito de inseticidas, 4) os resíduos de inseticidas nos grãos e em alguns de seus produtos processados e 5) a eficácia de inseticidas no controle de Sitophilus oryzae (L.), Sitophilus zeamais Motsch. e Rhyzopertha dominica (Fabr.). Para o tratamento dos grãos foi utilizado o bico hidráulico de jato duplo, modelo, TJ-60 8002EVS. O sistema de aplicação foi calibrado de modo a se obter tratamento dos grãos na concentração teórica de 10 e/ou 0,5 mg.kg-1 de fenitrotion e esfenvalerato, respectivamente. Os experimentos de eficácia foram instalados aos 15 dias, 30 dias e, a partir desta em intervalos mensais até 12 meses após o tratamento dos grãos, ou até que não houvesse mais mortalidade nas parcelas. Nos experimentos de resíduo, amostras de grãos foram coletadas ao zero, 15, 30, 60, 90, 120, 180, 240 e 360 dias após o tratamento e as amostras dos produtos processados foram obtidos na mesma data. A umidade dos grãos após a pulverização dos volumes de água estudados teve pequeno acréscimo quando comparado com o teor inicial. Os grãos processados junto com gelo seco proporcionaram a maior porcentagem de recuperação dos inseticidas. Houve grande influência da formulação concentrado emulsinável nas propriedades físicas do líquido, características do spray e depósito dos inseticidas nos grãos. Os resíduos de fenitrotion e esfenvalerato em grãos de trigo concentraram-se preferencialmente no farelo e em menores quantidades na farinha. A taxa de degradação do esfenvalerato foi independente do tipo de grão. O resíduo de fenitrotion no período de carência de 120 dias foi superior ao Limite Máximo de Resíduo (LMR) permitido pela legislação brasileira, e esta se mostra inadequada na regulamentação de ambos os inseticidas. O esfenvalerato foi ineficiente no controle das espécies estudadas; já o fenitrotion foi eficiente no controle de S. oryzae e S. zeamais. A linhagem SR de R. dominica foi mais resistente do que a linhagem CG aos inseticidas testados. As CL50s estimadas concordaram parcialmente com os resultados obtidos nos testes de eficácia. De forma geral, a mortalidade de S. oryzae decresceu mais rapidamente do que a mortalidade de S. zeamais. Houve maior mortalidade das duas espécies de Sitophilus em grãos de trigo do que em grãos de milho. Foi constatado efeito sinergista da mistura fenitrotion + esfenvalerato no controle das duas linhagens de R. dominica. Na ausência de inseticidas S. oryzae apresentou progênie mais numerosa no trigo em relação àquela no milho; já S. zeamais apresentou número similar de progênie nos dois tipos de grãos. Na testemunha, a linhagem CG de R. dominica apresentou progênies mais elevadas do que a linhagem SR, sendo que ocorreu o inverso quando da aplicação dos inseticidas. / Because of the problems caused by insects in storehouses, chemicals have been chiefly used in Brazil to control these pests. However, success in solving problems with pests has been limited. In view of this, some aspects about stored grain protection were studied, among which are the following: 1) spray volume effect on grain moisture content, 2) stability of insecticides during sample processing, 3) influence of the emulsifiable concentrate formulation on the physical properties of the liquid, spray characteristics, and deposition of insecticides, 4) insecticide residues in grain and some of their processed products, and 5) effectiveness of insecticides in the control of Sitophilus oryzae (L.), Sitophilus zeamaiszeamais Motsch., and Rhyzopertha dominica (Fabr.). Grain treatment was accomplished by using a twin jet, model TJ-60 8002EVS hydraulic nozzle. The application system was calibrated to provide grain treatment at theoretical concentrations of 10 and/or 0.5 mg kg-1 fenitrothion and esfenvalerate, respectively. The effectiveness experiments were installed at 15 days, 30 days and at monthly intervals thereafter for 12 months after treating the grains, or until mortality ceased in the plots. In the residue experiments, grain samples were collected at zero, 15, 30, 60, 90, 120, 180, 240, and 360 days after treatment, and processed product samples were obtained on the same dates. After spraying the water volumes planned in the study, only a small increase in grain moisture was observed when compared with the initial content. Grains processed together with dry ice provided the greatest insecticide recovery percentages. The emulsifiable concentrate formulation had a great influence on the physical properties of the liquid, spray characteristics, and deposition of insecticides on the grain. The fenitrothion and esfenvalerate residues in wheat grain were concentrated preferentially in the meal and at smaller amounts in the flour. The rate of esfenvalerate degradation did not depend on grain type. During a withholding period of 120 days, the fenitrothion residue was higher than the Maximum Residue Limit (MRL) allowed by Brazilian law, which has been demonstrated to be unsuitable for the regulation of both insecticides. Esfenvalerate was ineffective in controlling the species studied; fenitrothion, however, was effective for the control of S. oryzae and S. zeamais. The SR line of R. dominica was more resistant to the insecticides tested than the CG line. The estimated LC50 values agreed in part with the results obtained in the effectiveness tests. In general, S. oryzae mortality decreased more rapidly than S. zeamais mortality. There was greater mortality of both Sitophilus species on wheat grain than on corn grain. A synergistic effect was verified for the fenitrothion + esfenvalerate mixture in controlling both R. dominica lines. In the absence of insecticides, S. oryzae showed a more numerous offspring on wheat in relation to corn; S. zeamais. however, showed a similar number of offspring on both types of grain. In the control, the R. dominica CG line showed higher numbers of offspring than the SR line, and the contrary was verified when insecticides were applied.

Quatro linhagens capazes de crescer com acefato foram isoladas a partir de solos com históricos de aplicação de deste composto. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. e Pandoreae sp. foram identificadas através do rDNA 16S e perfil de ácidos graxos de membrana. Rhodococcus sp. foi o isolado mais eficiente na degradação de acefato, removendo 99.24% deste composto em meio de cultura com acefato como única fonte de carbono. Quando avaliado com acefato como fonte combinada de carbono e nitrogênio, este organismo degradou 19% de acefato com formação de metamidofós (17%). Staphylococcus sp. apresentou 21% de degradação de acefato utilizando-o como fonte de carbono e nitrogênio, mas não manteve o crescimento com este composto como fonte de carbono. Stenotrophomonas maltophilia e Pandoreae sp. não mantiveram crescimento com acefato como única fonte de carbono isoladamente. Estas linhagens apresentaram crescimento em acefato como fonte de nitrogênio e enxofre, porém, as análises de GC/MS demonstraram que não houve degradação nestas condições. / Four strains of microorganisms capable of growth on acephate were isolated from soil samples with a history of acephate application. Stenotrophomonas maltophilia, Rhodococcus sp., Staphylococcus sp. and Pandoreae sp. were identified based on 16S rRNA gene sequencing and fatty acid profiling. Rhodococcus sp. was the most efficient acephate degrader of the isolates, it removed 99.24% of acephate from defined growth media when the compound was provided as sole carbon source. When provided as a combined carbon and nitrogen source, the organisms degraded 19% of acephate with formation of methamidophos (17%). Staphylococcus sp. degraded 21% of acephate when provided as sole nitrogen and carbon source but did not grow on the compound as a sole source of carbon. Pandoreae sp. and Stenotrophomonas maltophilia failed to grow on acephate as sole source of carbon in defined medium. These strains grew in media where the pesticide was provided as a combined nitrogen and carbon source, but no acephate biodegradation could be demonstrated in these instances.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-05-12T14:19:07Z No. of bitstreams: 1 2010 - Henriqueta Talita Guimar?es Barboza.pdf: 10302674 bytes, checksum: 6daf4f88308c3c733615f0772d6db594 (MD5) / Made available in DSpace on 2017-05-12T14:19:07Z (GMT). No. of bitstreams: 1 2010 - Henriqueta Talita Guimar?es Barboza.pdf: 10302674 bytes, checksum: 6daf4f88308c3c733615f0772d6db594 (MD5) Previous issue date: 2010-03-04 / Twelve heterocyclic dialkylphosphorylhydrazones compounds were synthesized in this work. Dialkylphosphorylhydrazones were obtained through 3 steps of reaction. The first step consists on the synthesis of different dialkylphosphonates obtained by reaction of triethyl phosphate (PCl3) with 3 mols of the corresponding alcohol. The second step occurs by the reaction with dialkylphosphonates synthesized and hydrazine, producing dialkylphosphorylhydrazines. The third and last step is the condensation reaction of these dialkylphosphorylhydrazines with different heterocycle substituted aromatic aldehydes. The analysis of the 1H NMR spectra allowed to observe the stereoisomers E and Z, with the formation of E diastereisomer preferencially. All the compounds were characterized by 1H, 13C and 31P NMR, IR and mass spectroscopy. Tests in vitro with, Alternaria sp., Colletotrichum sp., Fusarium sp. e Fusarium solani, known as patogenic in papaya (Carica papaya L.) were performed in order to confirm if the compounds showed fungicide activity. The substance 6 B was the one with the best performance in the inhibition of all fungal growths. Finally, tests with acetylcholinesterase enzyme indicated that all substances did not inhibit this enzyme. Figure 2 - General structures of dialkylphosphoryhidrazones synthesized. R ? Ethyl; propyl; isopropyl e isobuthyl. X = O, Y, Z e W = CH; X = S, Y, Z e W = CH; X = NH, Y, Z e W = CH; X = NH, Y= CH, Z=CH e W = N; X = CH, Y= N, Z=CH e W = N; / Neste trabalho foram sintetizadas doze (12) novas dialquilfosforilidrazonas heteroc?clicas. Esses compostos foram obtidos utilizando-se tr?s etapas de rea??o. A primeira etapa consiste na s?ntese de diferentes fosfonatos de dialquila, obtidos por meio da rea??o do tricloreto de f?sforo (PCl3) com tr?s mols do ?lcool correspondente. A segunda etapa ocorre por meio da rea??o dos fosfonatos de dialquila sintetizados com a hidrazina, formando as dialquilfosforilidrazinas. A terceira e ?ltima etapa ? a condensa??o destas dialquilfosforilidrazinas com diferentes alde?dos heteroc?clicos. A an?lise dos espectros de RMN-1H indica a forma??o de diastereois?meros, E e Z, com predomin?ncia do diastereis?meros E. Todos os compostos obtidos foram caracterizados por RMN de H1 e C13 e P31, IV, an?lises de massas. Em seguida foram realizados testes in vitro para verificar se os mesmos possuem atividade fungicida sobre os fungos Alternaria sp., Colletotrichum sp., Fusarium sp. e Fusarium solani, conhecidos por serem pat?genos da cultura de mam?o (Carica papaya L.). O composto 6 B foi o que apresentou o melhor efeito fungicida sobre o crescimento de todos os fungos. Finalmente foram realizados testes com a enzima acetilcolinesterase a fim de observar a toxicidade dos compostos sintetizados frente a esta enzima. Figura 1- Estrututa gen?rica das dialquilfosforilidrazonas heteroc?clicas sintetizadas. R ? Etil; propil; isopropil e isobutil. X = O, Y, Z e W = CH; X = S, Y, Z e W = CH; X = NH, Y, Z e W = CH; X = NH, Y= CH, Z=CH e W = N; X = CH, Y= N, Z=CH e W = N.

La diffusion Raman exaltée de surface (SERS) est utilisée pour la mise au point d’un biocapteur capable de détecter des pesticides dans l’eau, en se basant sur le suivi de l’activité enzymatique de l’Acétylcholinestérase (ACHE). Les nanoparticules d’or sont utilisées comme substrats SERS actifs. Le signal Raman exalté de l’analyte est optimisé en testant plusieurs types de nanoparticules.Le Raman SERS a permis la détection directe du Paraoxon (PO) et du carbaryl (CA) et la possibilité de suivi de l’activité de l’ACHE. En absence d'inhibiteurs, la molécule d’acétylcholine (ATC) est transformée en acide acétique et en choline par l’enzyme ACHE. La mesure de l’activité de l’ACHE repose sur le suivi des concentrations en ATC car sa transformation est inhibée en présence de pesticides. Il a été ainsi possible d’établir une relation linéaire entre la concentration de pesticides et l’exaltation du signal Raman de l’ATC non transformé. La méthode a permis la détection du PO et du CA, avec une limite de détection beaucoup plus faible que la détection directe. Ce biocapteur basé sur l’activité de l’ACHE a ensuite été utilisé pour l'évaluation d’autres polluants (inhibiteurs d’ACHE) comme les additifs contenus dans les plastiques notamment. Enfin, nous avons développé une seconde approche qui consistait à mesurer l’activité de l’ACHE en utilisant la diffusion dynamique de la lumière. En effet, nous avons montré que les paramètres physicochimiques (agrégation) des AuNPs en contact avec certaines molécules, sont fortement influencés par l’activité enzymatique de l’ACHE. C’est ce phénomène d’instabilité qui nous a permis de distinguer entre les deux cas : absence et présence de PO. / Surface-enhanced Raman scattering (SERS) was used to develop a biosensor for the detection of pesticides through the monitoring of the enzymatic activity of acetylcholinesterase (ACHE). Gold nanoparticles (AuNPs) were used as an active SERS substrate. The enhanced Raman signal of the analyte is optimized by testing several types of nanoparticles. Raman SERS allowed the direct detection of Paraoxon (PO) and carbaryl (CA) pesticides and the possibility of follow-up of the activity of the ACHE. In the absence of inhibitors, the acetylcholine (ATC) is transformed into acetic acid and choline by the enzyme ACHE. The measurement of ACHE activity is performed through the monitoring of ATC concentrations because its transformation is inhibited in the presence of pesticides. Results showed a linear correlation between the concentration of pesticides and the SERS signal of the untransformed ATC. The method was optimized for the quantification of paraoxon and carbaryl with a limit of quantification much lower than the one obtained with a direct detection. Their identification was also possible using chemometrics. This biosensors, based on the ACHE activities, was applied to the evaluation of emergent pollutants: additives of commercial polymers. Our results suggested that most of the tested polymers contained molecules that act as inhibitors of the ACHE. Finally, we propose another very simple approach to measure the ACHE activity using dynamic light scattering measurements. We found that the physicochemical parameters (aggregation) of AuNPs were strongly influenced by the enzymatic activity of ACHE when in contact with specified molecules, allowing to detect the presence of PO.