Modifying substrate specificity of β-glucuronidase (GUS) would be helpful in various enzyme prodrug systems in delivering drug dose to the site of action in the cancer treatment. Due to the presence of endogenous enzyme in human tissues, GUS-based Antibody-Directed Enzyme Prodrug Therapy (ADEPT) requires a novel substrate to avoid undesirable systemic activation. GUS is a glycosyl hydrolase, highly specific towards the glucuronide derivatives. It catalyzes the glycosidic cleavage of β-D-glucuronides to β-D-glucuronic acid and aglycone moiety. In order to gain insight on the substrate specificity of GUS, C6 carboxyl group of glucuronic acid was modified to C6 carboxamide (amide derivative). We have examined amide derivatized substrates with a variety of different aglycone groups including p-nitrophenyl, phenyl and 4-methylumbelliferone to further probe the activity profile of GUS. In an effort to optimize GUS activity, docking studies have been performed which indicated that amino acid point mutations near C6 carboxyl group of glucuronic acid could improve binding of the derivatized substrates. As a result point mutations to Arg-562 and Lys-568 which make the active site less positively charged either by glutamine or glutamate lead to an enzyme with much lower native substrate activity but abolished activity for the amide-derivatized substrate. This research study showed that there is still a further need of finding appropriate mutations required to make glucuronamide a better substrate for the mutated version of GUS.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-11Bitstream added on 2014-06-13T20:56:20Z : No. of bitstreams: 1 rombola_th_me_jabo.pdf: 290913 bytes, checksum: 08f244b9cd35a229582ac7563c4b4ff9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Espécies de Burkholderia filogenéticamente distantes do complexo B. cepacia, são versáteis solubilizadores de minerais insolúveis, através da produção de ácidos, viabilizando nutrientes para as plantas. Descrevemos a purificação e caracterização da fosfatase ácida ligada à membrana, produzida pela bactéria do gênero Burkholderia, isolada de solo agrícola em Ponta grossa-PR-BRASIL, e identificada através da análise do 16s rDNA. A expressão da enzima se mostrou estritamente dependente ao fósforo (expressão ótima a 5 mM). A enzima ligada à membrana foi purificada por ultracentrifugação a 100.000g por 1 hora a 4°C e testada com atividade enzimática para p-nitrofenilfosfato (PNFF) e Pirofosfato. O pH ótimo da enzima foi 6 e não foi afetado pela concentração de PNFF. Em pH 6 a hidrolise do PNFF seguiu os seguintes parâmetros cinéticos com n=1.5, Vm= 103.8 U.mg-1 e K0,5=0,06 mM em uma faixa de 0,003 e 10 mM de PNFF. Estudos do pH sobre os parâmetros cinéticos demonstraram 26 pontos de variação na faixa de 3,5 a 6 e em seguida diminui até pH 8 enquanto K0,5 não mostrou variação nesta faixa. O H para hidrólise do PNFF foi de 5,74 kcal.mol-1. A atividade PNFFase foi inibida pelo arsenato e Cálcio, mas não por levamisol, EDTA, zinco e cobalto. A hidrolise do pirofosfato na presença de 2 mM de MgCl2 seguiu os parâmetros cinéticos de Michaelis-Menten” com Km= 0.142 mM e Vm= 237 U.mg-1.Os resultados sugerem que a produção da fosfatase ácida ligada à membrana pode ser um mecanismo de solubilização de fosfato mineral por essa bactéria. / Burkholderia species phylogenetically greatly distant from the B. cepacia complex species are versatile organisms that solubilize insoluble minerals through the production of acids, increasing the availability of nutrients to plants. Here we describe the purification and characterization a membrane-bound acid phosphatase produced by bulkholderia isolated from an agricultural soil in Ponta grossa-PR-BRASIL and identified through analysis of the 16S rDNA gene. The expression of this enzyme was demonstrated to be strictly regulated by phosphorus (optimal expression at 5 mM). The membrane bound enzyme was purified by centrifugation at 100.000g for 1 h at 4ºC and the enzyme was assayed for p-Nitrophenyl phosphatase (PNPPase) and pyrophosphatase activities. The optimal pH (6.0) was not affect by p-Nitrophenyl phosphate concentration. At pH 6.0 the hydrolysis of PNPP following a cooperative kinetics with n= 1.5, Vm= 103.8 U.mg-1 and K0.5= 0.60 mM in the range between 0.003 and 10 mM. Studies of pH effects on kinetics parameters revealed a 26-folds variation in the range of pH 3.5 to 6 following decreases until pH 8.0, wile K0,5 and cooperativity did not varied considerably in this range. The H for hydrolysis of PNPP was 5,74 kcal.mol-1. PNPPase activity was inhibited by arsenate, phosphate and calcium, but not for levamisole, EDTA, zinc, magnesium and cobalt. The hydrolysis of pyrophosphate in presence of 2 mM MgCl2 follows Michaelis-Menten” kinetics with Km= 0.142 mM and Vm= 237 U.mg-1. Our results suggesting that the production of membranebound acid phosphatase might be one mechanism of mineral phosphate solubilization by this genus of Bacterium.

Orientador: João Martins Pizauro Junior / Banca: Maria Inês Tiraboschi Ferro / Banca: Pietro Ciancaglini / Resumo: Espécies de Burkholderia filogenéticamente distantes do complexo B. cepacia, são versáteis solubilizadores de minerais insolúveis, através da produção de ácidos, viabilizando nutrientes para as plantas. Descrevemos a purificação e caracterização da fosfatase ácida ligada à membrana, produzida pela bactéria do gênero Burkholderia, isolada de solo agrícola em Ponta grossa-PR-BRASIL, e identificada através da análise do 16s rDNA. A expressão da enzima se mostrou estritamente dependente ao fósforo (expressão ótima a 5 mM). A enzima ligada à membrana foi purificada por ultracentrifugação a 100.000g por 1 hora a 4°C e testada com atividade enzimática para p-nitrofenilfosfato (PNFF) e Pirofosfato. O pH ótimo da enzima foi 6 e não foi afetado pela concentração de PNFF. Em pH 6 a hidrolise do PNFF seguiu os seguintes parâmetros cinéticos com n=1.5, Vm= 103.8 U.mg-1 e K0,5=0,06 mM em uma faixa de 0,003 e 10 mM de PNFF. Estudos do pH sobre os parâmetros cinéticos demonstraram 26 pontos de variação na faixa de 3,5 a 6 e em seguida diminui até pH 8 enquanto K0,5 não mostrou variação nesta faixa. O H para hidrólise do PNFF foi de 5,74 kcal.mol-1. A atividade PNFFase foi inibida pelo arsenato e Cálcio, mas não por levamisol, EDTA, zinco e cobalto. A hidrolise do pirofosfato na presença de 2 mM de MgCl2 seguiu os parâmetros cinéticos de "Michaelis-Menten" com Km= 0.142 mM e Vm= 237 U.mg-1.Os resultados sugerem que a produção da fosfatase ácida ligada à membrana pode ser um mecanismo de solubilização de fosfato mineral por essa bactéria. / Abstract: Burkholderia species phylogenetically greatly distant from the B. cepacia complex species are versatile organisms that solubilize insoluble minerals through the production of acids, increasing the availability of nutrients to plants. Here we describe the purification and characterization a membrane-bound acid phosphatase produced by bulkholderia isolated from an agricultural soil in Ponta grossa-PR-BRASIL and identified through analysis of the 16S rDNA gene. The expression of this enzyme was demonstrated to be strictly regulated by phosphorus (optimal expression at 5 mM). The membrane bound enzyme was purified by centrifugation at 100.000g for 1 h at 4ºC and the enzyme was assayed for p-Nitrophenyl phosphatase (PNPPase) and pyrophosphatase activities. The optimal pH (6.0) was not affect by p-Nitrophenyl phosphate concentration. At pH 6.0 the hydrolysis of PNPP following a cooperative kinetics with n= 1.5, Vm= 103.8 U.mg-1 and K0.5= 0.60 mM in the range between 0.003 and 10 mM. Studies of pH effects on kinetics parameters revealed a 26-folds variation in the range of pH 3.5 to 6 following decreases until pH 8.0, wile K0,5 and cooperativity did not varied considerably in this range. The H for hydrolysis of PNPP was 5,74 kcal.mol-1. PNPPase activity was inhibited by arsenate, phosphate and calcium, but not for levamisole, EDTA, zinc, magnesium and cobalt. The hydrolysis of pyrophosphate in presence of 2 mM MgCl2 follows "Michaelis-Menten" kinetics with Km= 0.142 mM and Vm= 237 U.mg-1. Our results suggesting that the production of membranebound acid phosphatase might be one mechanism of mineral phosphate solubilization by this genus of Bacterium. / Mestre

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 μmol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 μmol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.

No description available.

Yes / We have set out an equation for partition of 87 neutral molecules from water to o-nitrophenyl octyl ether, NPOE, an equation for partition of the 87 neutral molecules and 21 ionic species from water to NPOE, and an equation for partition of 87 neutral molecules from the gas phase to NPOE. Comparison with equations for partition into other solvents shows that, as regards partition of neutral (nonelectrolyte) compounds, NPOE would be a good model for 1,2-dichloroethane and for nitrobenzene. In terms of partition of ions and ionic species, NPOE is quite similar to 1,2-dichloroethane and not far away from other aprotic solvents such as nitrobenzene.

Micelas e agregados de cloreto de dimetildialquilamônio (Herquat) foram utilizados como modelos de interfaces para o estudo de reações químicas. As reações de tiólise de acetato de p-nitrofenila (NPA) e octanoato de p-nitrofenila (NPO) por n-heptilmercaptana (HM) foram estudadas na presença de concentrações variáveis de brometo de hexadeciltrimetilamônio(CTAB), dodecilsulfato de sódio (SDS) e 3-(N,N dimetildodecilamônio) propano-l-sulfohato(SDP). O detergente positivo (CTAB) provoca uma alteração máxima da reação de tiólise de NPA de 6.103 vezes e da reação de tiólise de NPO por um fator máximo de 1,6.104 vezes. O efeito da concentração de CTAB na constante de pseudo-primeira-ordem das reações de tiólise (kψ) e no pK aparente (pKap) da HM foi analisado segundo o modelo de Troca Iônica (Quina, F.H. e Chaimovich, H. (1979). Os parâmetros necessários para a análise dos resultados descritos acima foram determinados por métodos independentes dos experimentos cinéticos. Um ajuste excelente das curvas teóricas aos dados experimentais foi obtido utilizando-se uma relação entre as constantes de velocidade de segunda ordem nas fases micelar (km2) e aquosa (ko2) de 1 e 5 para as reações de tiólise de NPA e NP0, respectivamente. Assim, o efeito de CTAB sobre a kψ das reações de tiólise de NPA e NPO pode ser atribuído a uma concentração de substratos na fase micelar, desvio do pKap da HM e concentração do íon hidróxido na interface. A velocidade da reação de tiólise de NPO por HM diminui cerca de 70 vezes em 0,1 M de SDS e, na presença de SDP, aumenta por um fator máximo de 270 vezes, O pKap da HM aumenta com a concentração de ambos os detergentes. O efeito da concentração de SDS e SDP no pKap da HM e na kψ da reação de tiólise de NPO foi analizado segundo o modelo de pseudo-fase (Yatsimirski, A.K., Martinek, K. e Berezin, I.V., (1971). Um ajuste muito bom das curvas teóricas aos dados experimentais foi obtido utilizando-se um valor de 0,085 para a relação km2/ko2 em SDS e 1 para a relação em SDP. Assim, a inibição da tiólise de NPO por SDS é devida a um aumento do pKap do substrato, uma km2 menor que ko2 e uma pequena constante de associação entre o SDS e a forma aniônica da mercaptana. A aceleração da reação de tiólise de NPO por SDP pode ser atribuída unicamente a concentração de reagentes na pseudo-fase. A reação de hidrólise de 2-Feniliminotetrahidrofurano (FI) foi estudada em presença de SDS observando-se que o detergente aumenta o pKap do substrato e a velocidade da reação de hidrólise (a pHs maiores que 4). A análise destes resultados empregando-se equações derivadas a partir do modelo de Troca-Iônica mostrou que a relação km/ko é praticamente 1 a todos os pHs estudados. Desde que a reação é inibida a alta força iônica em fase aquosa, podemos inferir que, no microambiente no qual o FI é solubilizado (camada de Stern do SDS), a reação ocorre com uma constante de velocidade similar à observada em baixa força iônica. Poi feita uma caracterização parcial dos agregados de Herquat (vesículas) por filtração em Sephadex G-25. Demonstrou-se que as vesículas incorporam substratos hidrofóbicos (4-(-1-pireno) butanoato de metila) e hidrofílicos ((14C) glicose) e que o contraíon do anfifílico (cloreto) pode ser deslocado do Herquat pelo ânion do tampão. Estes agregados aceleram a reação de tiólise de NPA por HM por um fator máximo de 5.104 vezes e a reação de tiólise de NPO por HM por um fator de 7.106 vezes. O efeito mais impressionante destes agregados é que a reação de tiólise de NPO por HM em Herquat pode ser estudada a pH 4, portanto, cerca de 6,7 unidades de pH abaixo do pK da mercaptana em água. Após a análise deste sistema de reações utilizando-se o modelo de Troca-Iônica obteve-se uma relação km2/ko2 da ordem de 5 para a reação de HM e NPA e da ordem de 13 para a reação de tiólise de NPO. A aceleração provocada pelos agregados de Herquat pode ser atribuída a concentração de reagentes nas vesículas, efeito dos agregados sobre o pKap da HM e uma constante de segunda ordem na pseudo-fase maior que na fase aquosa. / Micelles and dialkyldimethylammonium chloride aggregates (Herquat) were employed as model interfaces for the study of interfacial effects on chemical reactions. The thiolyses of p-nitrophenyl acetate (NPA) and p-nitrophenyloctanoate (NPO) by n-heptylmercaptan (HM) were investigated as a function of concentration of added hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and 3- (N,N-dimethyl-N-dodecyl ammonium) - propane-l-sulfonate (SDP). In the presence of the cationic detergent (CTAB), maximum rate acceleration of 6x103 and 1.6xl04 were observed for the thiolyses of NPA and NPO, respectively. The CTAB concentration dependences of the pseudo first-order rate constants (kψ) for these reactions and of the apparent pK (pKap) of HM were analyzed within the framework of the conceptual Model for Ion Exchange (Quina, F.H. and Chaimovich, H., 1979), the requisite parameters for the analysis being determined independently of the kinetic results. Excellent simulations of the experimental results were obtained using ratios of second order rate constants in the micellar (km2) and aqueous (ko2) phases of 1 and 5 for thiolysis of NPA and NPO, respectively. Thus, in this case, the effect of CTAB on kψ may be attributed to the interplay of concentration of the substrate in the micellar phase, changes in the pKap of HM and concentration of the hydroxide ion at the interfaces. The rate of thiolysis of NPO by HM diminishes by roughly a factor of 70 upon addition of O.1M SDS (anionic detergent) and increases by a factor of 270 at the maximum in the presence of SDP (zwitterionic detergent). The pKap of HM increases in the presence of both detergents. The effect of detergent concentration on the pKap of HM and on kψ for thiolysis of NPO were analyzed in terms of the pseudo-phade model (Yatsimirski, Martinek, Berezin, 1971). Ressonable simulation of the experimental data were obtained using values of 0.085 for the ratio km2/ko2 in SDS and 1 for this some ratio in SDP. Thus, the inhibition of the thiolysis of NPO by SDS may be ascribed to an increase in the pKap of HM, coupled with a small value of the constant for association of the mercaptide ion of HM with the micellar phase and an apparent decrease in km2 relative to ko2. The acceleration of this same reaction by SDP can be attibuted exclusively to extrinsic factors, concentration of the reagents in the micellar phase predominating over the increase in the pKap of HM. The hydrolysis of 2-Phenyliminotetrahydrofuran (FI) was studied in the presence of SDS, it being verified that both the pKap of the substrate and the velocity of hydrolysis (at pH values higher than 4) increase in the presence of the detergente Analysis of these results, using equations derived from the conceptual Model for Exchange, demonstrates that the ratio km/ko is essentially 1 over the entire pH vange investigated. Since this reaction is inhibited at high ionic strength in aqueous solution, one may infer that, in the micro environment in which FI is solubilized (Stern layer of SDS), the reaction occurs with a rate constant similar to that observed in aqueous medium at low ionic strength. A partial characterization of the aggregates (vesicles) formed by Herquat in aqueous dispersion was performed by gel filtration on Sephadex G-25. In this manner, it was demonstrated that the vesicles incorporate hydrophobic(methyl 4-(1-pireno)butanoate) and hydrophilic (14C-glucose) substrates and that the chloride counterion of Herquat can be exehanged with anionic buffer components. These aggregates accelerate the thiolysis of NPA by HM by up to a factor of 5x104 and that of NPO by up to a fator of 7x106. The most impressive aspeet of these Herquat agregates is the fact that the thiolysis of NPO by HM can be studied at pH 4, some 6.7 pH units below the pKa of HM in water. Applying an ion-exchange formalism analogous to that amployed for reactions in CTAB to the analysis of these thiolyses, km2/ko2 ratios of about 5 for the reaction between NPA and HM and of roughly 13 for the reaction between NPO and HM were obtained. The acceleration observed in the presence of these aggregates may thus be attributed to concentration of the reagents in the vesicle phase, effects on the pKap of HM and a somewhat larger second-order rate constant in the vesicle phase relative to the aqueous phase.

Micelas e agregados de cloreto de dimetildialquilamônio (Herquat) foram utilizados como modelos de interfaces para o estudo de reações químicas. As reações de tiólise de acetato de p-nitrofenila (NPA) e octanoato de p-nitrofenila (NPO) por n-heptilmercaptana (HM) foram estudadas na presença de concentrações variáveis de brometo de hexadeciltrimetilamônio(CTAB), dodecilsulfato de sódio (SDS) e 3-(N,N dimetildodecilamônio) propano-l-sulfohato(SDP). O detergente positivo (CTAB) provoca uma alteração máxima da reação de tiólise de NPA de 6.103 vezes e da reação de tiólise de NPO por um fator máximo de 1,6.104 vezes. O efeito da concentração de CTAB na constante de pseudo-primeira-ordem das reações de tiólise (kψ) e no pK aparente (pKap) da HM foi analisado segundo o modelo de Troca Iônica (Quina, F.H. e Chaimovich, H. (1979). Os parâmetros necessários para a análise dos resultados descritos acima foram determinados por métodos independentes dos experimentos cinéticos. Um ajuste excelente das curvas teóricas aos dados experimentais foi obtido utilizando-se uma relação entre as constantes de velocidade de segunda ordem nas fases micelar (km2) e aquosa (ko2) de 1 e 5 para as reações de tiólise de NPA e NP0, respectivamente. Assim, o efeito de CTAB sobre a kψ das reações de tiólise de NPA e NPO pode ser atribuído a uma concentração de substratos na fase micelar, desvio do pKap da HM e concentração do íon hidróxido na interface. A velocidade da reação de tiólise de NPO por HM diminui cerca de 70 vezes em 0,1 M de SDS e, na presença de SDP, aumenta por um fator máximo de 270 vezes, O pKap da HM aumenta com a concentração de ambos os detergentes. O efeito da concentração de SDS e SDP no pKap da HM e na kψ da reação de tiólise de NPO foi analizado segundo o modelo de pseudo-fase (Yatsimirski, A.K., Martinek, K. e Berezin, I.V., (1971). Um ajuste muito bom das curvas teóricas aos dados experimentais foi obtido utilizando-se um valor de 0,085 para a relação km2/ko2 em SDS e 1 para a relação em SDP. Assim, a inibição da tiólise de NPO por SDS é devida a um aumento do pKap do substrato, uma km2 menor que ko2 e uma pequena constante de associação entre o SDS e a forma aniônica da mercaptana. A aceleração da reação de tiólise de NPO por SDP pode ser atribuída unicamente a concentração de reagentes na pseudo-fase. A reação de hidrólise de 2-Feniliminotetrahidrofurano (FI) foi estudada em presença de SDS observando-se que o detergente aumenta o pKap do substrato e a velocidade da reação de hidrólise (a pHs maiores que 4). A análise destes resultados empregando-se equações derivadas a partir do modelo de Troca-Iônica mostrou que a relação km/ko é praticamente 1 a todos os pHs estudados. Desde que a reação é inibida a alta força iônica em fase aquosa, podemos inferir que, no microambiente no qual o FI é solubilizado (camada de Stern do SDS), a reação ocorre com uma constante de velocidade similar à observada em baixa força iônica. Poi feita uma caracterização parcial dos agregados de Herquat (vesículas) por filtração em Sephadex G-25. Demonstrou-se que as vesículas incorporam substratos hidrofóbicos (4-(-1-pireno) butanoato de metila) e hidrofílicos ((14C) glicose) e que o contraíon do anfifílico (cloreto) pode ser deslocado do Herquat pelo ânion do tampão. Estes agregados aceleram a reação de tiólise de NPA por HM por um fator máximo de 5.104 vezes e a reação de tiólise de NPO por HM por um fator de 7.106 vezes. O efeito mais impressionante destes agregados é que a reação de tiólise de NPO por HM em Herquat pode ser estudada a pH 4, portanto, cerca de 6,7 unidades de pH abaixo do pK da mercaptana em água. Após a análise deste sistema de reações utilizando-se o modelo de Troca-Iônica obteve-se uma relação km2/ko2 da ordem de 5 para a reação de HM e NPA e da ordem de 13 para a reação de tiólise de NPO. A aceleração provocada pelos agregados de Herquat pode ser atribuída a concentração de reagentes nas vesículas, efeito dos agregados sobre o pKap da HM e uma constante de segunda ordem na pseudo-fase maior que na fase aquosa. / Micelles and dialkyldimethylammonium chloride aggregates (Herquat) were employed as model interfaces for the study of interfacial effects on chemical reactions. The thiolyses of p-nitrophenyl acetate (NPA) and p-nitrophenyloctanoate (NPO) by n-heptylmercaptan (HM) were investigated as a function of concentration of added hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and 3- (N,N-dimethyl-N-dodecyl ammonium) - propane-l-sulfonate (SDP). In the presence of the cationic detergent (CTAB), maximum rate acceleration of 6x103 and 1.6xl04 were observed for the thiolyses of NPA and NPO, respectively. The CTAB concentration dependences of the pseudo first-order rate constants (kψ) for these reactions and of the apparent pK (pKap) of HM were analyzed within the framework of the conceptual Model for Ion Exchange (Quina, F.H. and Chaimovich, H., 1979), the requisite parameters for the analysis being determined independently of the kinetic results. Excellent simulations of the experimental results were obtained using ratios of second order rate constants in the micellar (km2) and aqueous (ko2) phases of 1 and 5 for thiolysis of NPA and NPO, respectively. Thus, in this case, the effect of CTAB on kψ may be attributed to the interplay of concentration of the substrate in the micellar phase, changes in the pKap of HM and concentration of the hydroxide ion at the interfaces. The rate of thiolysis of NPO by HM diminishes by roughly a factor of 70 upon addition of O.1M SDS (anionic detergent) and increases by a factor of 270 at the maximum in the presence of SDP (zwitterionic detergent). The pKap of HM increases in the presence of both detergents. The effect of detergent concentration on the pKap of HM and on kψ for thiolysis of NPO were analyzed in terms of the pseudo-phade model (Yatsimirski, Martinek, Berezin, 1971). Ressonable simulation of the experimental data were obtained using values of 0.085 for the ratio km2/ko2 in SDS and 1 for this some ratio in SDP. Thus, the inhibition of the thiolysis of NPO by SDS may be ascribed to an increase in the pKap of HM, coupled with a small value of the constant for association of the mercaptide ion of HM with the micellar phase and an apparent decrease in km2 relative to ko2. The acceleration of this same reaction by SDP can be attibuted exclusively to extrinsic factors, concentration of the reagents in the micellar phase predominating over the increase in the pKap of HM. The hydrolysis of 2-Phenyliminotetrahydrofuran (FI) was studied in the presence of SDS, it being verified that both the pKap of the substrate and the velocity of hydrolysis (at pH values higher than 4) increase in the presence of the detergente Analysis of these results, using equations derived from the conceptual Model for Exchange, demonstrates that the ratio km/ko is essentially 1 over the entire pH vange investigated. Since this reaction is inhibited at high ionic strength in aqueous solution, one may infer that, in the micro environment in which FI is solubilized (Stern layer of SDS), the reaction occurs with a rate constant similar to that observed in aqueous medium at low ionic strength. A partial characterization of the aggregates (vesicles) formed by Herquat in aqueous dispersion was performed by gel filtration on Sephadex G-25. In this manner, it was demonstrated that the vesicles incorporate hydrophobic(methyl 4-(1-pireno)butanoate) and hydrophilic (14C-glucose) substrates and that the chloride counterion of Herquat can be exehanged with anionic buffer components. These aggregates accelerate the thiolysis of NPA by HM by up to a factor of 5x104 and that of NPO by up to a fator of 7x106. The most impressive aspeet of these Herquat agregates is the fact that the thiolysis of NPO by HM can be studied at pH 4, some 6.7 pH units below the pKa of HM in water. Applying an ion-exchange formalism analogous to that amployed for reactions in CTAB to the analysis of these thiolyses, km2/ko2 ratios of about 5 for the reaction between NPA and HM and of roughly 13 for the reaction between NPO and HM were obtained. The acceleration observed in the presence of these aggregates may thus be attributed to concentration of the reagents in the vesicle phase, effects on the pKap of HM and a somewhat larger second-order rate constant in the vesicle phase relative to the aqueous phase.

A new monomer / 1-(4-Nitrophenyl)-2,5-di-2-thienyl-1H-pyrrole SNSNO2 was synthesized through the Knorr-Paal condensation reaction of 1,4-di-2-thienyl-1,4-butanedione and p-nitroaniline. The chemical structure of monomer and polymer were characterized via Nuclear Magnetic Resonance Spectroscopy (NMR) and Fourier Transform Infrared Spectroscopy (FTIR). Chemical polymerization produced a polymer which was completely soluble in organic solvents. Electrochemical behaviors of SNSNO2 and SNSNO2 in the presence of EDOT were studied by cyclic voltammetry (CV). The synthesis of homopolymer and copolymer were achieved via constant potential electrolysis. Both homopolymer P(SNSNO2) and copolymer P(SNSNO2-co-EDOT) were characterized by various techniques including cyclic voltammetry, FTIR, Scanning Electron Microscopy (SEM) and UV-VIS Spectrophotometer. Conductivities of samples were measured by four probe technique. The electrochromic properties of the polymers were investigated via spectroelectrochemistry, colorimetry and switching studies. In addition, dual type electrochromic devices (ECDs) composed of P(SNSNO2), P(SNSNO2-co-EDOT) and poly(3,4-ethylenedioxythiophene) (PEDOT) were constructed and evaluated. Spectroelectrochemistry, switching ability and stability of the devices were investigated by UV-Vis Spectrophotometer and Cyclic Voltammetry. They have shown to possess good switching times, reasonable contrasts and high stabilities.

Philosophiae Doctor - PhD / A highly dispersive gold nanoparticle-dotted 4-nitrophenylazo functionalised graphene nanocomposite (AuNp/G/PhNO2) was successfully synthesised and applied in enhancing sensing platform signals. Three label-free electrochemical immunosensors for the detection of deoxynivalenol mycotoxin (DON) based on the systematic modification of glassy carbon electrodes (GCE) with AuNp/G/PhNO2 was effectively achieved. General electrochemical impedance method was employed for the sensitive and selective detection of DON in standard solutions and reference material samples. A significant increase in charge transfer resistance (Rct) of the sensing interface was observed due to the formation of insulating immune-complexes by the binding of deoxynivalenol antibody (DONab) and deoxynivalenol antigen (DONag). Further attachments of DONab and DONag resulted in increases in the obtained Rct values, and the increases were linearly proportional to the concentration of DONag. The three immunosensors denoted as GCE/PDMA/AuNp/G/PhNH2/DONab, GCE/Nafion/[Ru(bpy)3]2+/AuNp/G/PhNH2/DONab and GCE/Nafion/[Ru(bpy)3]2+/G/PhNH2/DONab have detection range of 6 – 30 ng/mL for DONag in standard samples. Their sensitivity and detection limits were 43.45 ΩL/ng and 1.1 pg/L; 32.14ΩL/ng and 0.3 pg/L; 9.412 ΩL/ng and 1.1 pg/L respectively. This result was better than those reported in the literature and compares reasonably with Enzyme Linked Immunosorbent Assay (ELISA) results. The present sensing methodology represents an attractive alternative to the existing methods for the detection of deoxynivalenol mycotoxin and other big biomolecules of interest due to its simplicity, stability, sensitivity, reproducibility, selectivity, and inexpensive instrumentation. And they could be used to develop high-performance, ultra-sensitive electrochemiluminescence, voltammetric or amperometric sensors as well.