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Malachite green(MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in aquaculture. Malachite green is reduced to and persists as leucomalachite green(LMG) in the tisssues of fish. Concern over MG and LMG are due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Several hepatotxicants and liver carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC).This study wanted to determine if disruption of cell-cell interactions occurs in rat liver epithelial cells in response to MG and LMG treatment. Rat liver epithelial cells(WB) were treated with LMG(0~6£gg/ml) or MG.(0~5£gg/ml) for one hour and gap junction was analyzed using the scrape- loading/dye transfer assay. The viability and proliferation of rat liver epithelial cells treated with LMG or MG were determined by MTT and colony forming efficiency. In addition, expression and intracellular localization of connexin43, E-cadherin,£]-catenin,£\-tubulin, ZO-1 and occludin were determined by immunoblot and immunostain analysis. A clear decrease in the distance of dye transfer was evident following treatment with MG(0~5£gg/ml) or LMG(0~6£gg/ml). Treatment with LMG and MG at different concentrations resulted in a decrease in cell viability and cell proliferation. Preincubation of cells with protein kinase C(PKC) inhibitor decreased the inhibition of GJIC by 5ug/ml MG and the specific MEK 1 inhibitor decreased substantially the inhibition of GJIC by 5£gg/ml MG and 5£gg/ml LMG. On the other hand, the specific PI3 kinase inhibitor decreased the inhibition of GJIC only by 3£g/mlLMG and we treated WB cells with EDTA to chelate extracellular calcium ion. The decrease of free calcium ion caused the expression of GJIC. At the transcriptional level, 10£gg/ml LMG and 10£gg/ml MG after treatment for one hour caused no change in the level of connexin43 mRNA. At the translational level, the different concentrations of MG or LMG after treatment for one hour or 24 hours caused a decrease in the level of the concentrations of connexin43 protein, E-cadherin protein,£]-catenin protein,£\-tubulin protein, ZO-1 protein and changed the distribution of occludin and ZO-1. Therefore, these data speculated the hypothesis that disruption of cell-cell signaling by interference with GJIC may contribute to LMG and MG toxicity, carcinogenicity and apoptosis.

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

The malachite green aptamer was originally engineered for binding specificity to malachite green (MG). The environment inside the aptamer’s electronegative binding pocket was previously harnessed to catalyze an ester cleavage via the stabilization of positively charged intermediates. In order to further explore and expand the catalytic potential of this molecule we have analyzed the binding and chemical properties of MG derivatives. The catalyzed reaction rate is approximately 2000-fold faster than the non-catalyzed reaction rate. This catalytic activity demonstrates the possible significance of electrostatic forces in RNA enzymes. The ability of RNA to catalyze different reactions depending on the substrate provided would have been beneficial in an early RNA world. We have investigated the interactions between the malachite green aptamer and its ligands. This investigation contributes to a better understanding of RNA aptamer interactions with small molecules which are crucial for drug development and RNA based catalysis. Equilibrium dialysis data suggests that MG binds exclusively within the binding pocket. On the other hand, pyronin Y which lacks the third phenyl ring of MG intercalates non-specifically. This confirms that all three phenyl rings have crucial interactions with RNA bases. Isothermal calorimetry data shows a more negative enthalpy value for the binding of tetramethylrosamine (TMR) to the MG aptamer than binding of MG to the MG aptamer. This agrees with the crystal structure of TMR bound to the MG aptamer that shows more extensive base stacking interactions compared to the MG : MG aptamer complex. TMR differs from MG by the addition of an oxygen between two of its phenyl rings, that gives TMR a more planar structure. MG binding to the MG aptamer shows an entropy value increase compared to TMR binding to the MG aptamer. This agrees with available ab initio calculations which show the development of an asymmetrical charge distribution across MG when bound to the MG aptamer.

The purpose of this research is that investigate the effects of time and concentration of exposure for the accumulation and depletion of malachite green and leucomalachite green in tilapia by pharmacokinetics. LC-MS/MS was used as the analytical instrument in this research, and the detection limit of malachite green and leucomalachite green is 0.51 ppb and 0.48 ppb. The results show that malachite green is unstable at high temperature. Addition of TMPD into the standard can stabilize malachite green. The malachite green in exposure water is easy to adhere to the fiberglass tub and cause the decreases of concentration of malachite green in water. The concentrations of malachite green and leucomalachite green in tilapia are positive related to the time and concentration of exposure. In experiments of exposure of malachite green, the highest concentration of malachite green occurs in liver, accumulation rate constant is 21.62 h-1. Liver is also the major organ for transforming malachite green into leucomalachite green, the net leucomalachite green accumulation rate constant is 213.67 h-1. In the period of water bath, Gill is the fastest organ for eliminating malachite green and leucomalachite green, the elimination rate constant is 0.7799 h-1 and 0.4658 h-1; the leucomalachite green concentration in fat is still increase until 6h of water bath.

This research investigate the matrix effect, operation time, cost and solvent comsumption for triarylmethanes and corresponding metabolites in different aquaculture products by different extraction methods. Triarylmethane was determined by LC-ESI-MS/MS in positive mode. The analytical method validation were followed the regulation of 2002/657/EC. HPLC separation was conducted with the RP-18 column. The mobile phases consisted of 0.5 mM ammonium acetate buffer (pH 3.8, adjusted with acetic acid) ¡V acetonitrile (contained 0.1% formic acid) solution. The ratios of standard deviation to average residual time for all compounds are less than 0.05. The ion ratio is stable and allowed by the regulation of 2002/657/EC. The regression constant of calibration curve is in the range from 0.998 to 0.999. The CC£\ for MG, LMG, CV, LCV are 0.09 ¡Ó 0.05, 0.19 ¡Ó 0.14, 0.15 ¡Ó 0.13, 0.15 ¡Ó 0.10 £ggkg-1, respectively. The CC£] for MG, LMG, CV, LCV are 0.09 ¡Ó 0.05, 0.19 ¡Ó 0.14, 0.15 ¡Ó 0.13, 0.15 ¡Ó0.10 £ggkg-1, respectively. The results for SPE and liquid-liquid extraction are not significantly different; the liquid-liquid extraction have shorter operation time, cheaper cost and less solvent consumption in comparison with SPE.

Orientador: Felix Guillermo Reyes Reyes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T19:05:58Z (GMT). No. of bitstreams: 1 Hashimoto_JulianaCampos_M.pdf: 16051938 bytes, checksum: 650dab34d83af879bb5279035b7aa9eb (MD5) Previous issue date: 2010 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Mestre em Ciência de Alimentos

Au cours de cette thèse, de nouveaux dérivés organométalliques basés sur une structure triphénylméthylium ont été synthétisés. Ces composés, notamment grâce à leur structure multiplolaire, sont pressentis pour avoir des propriétés remarquables en optiques non linéaire (ONL) qui pourraient donner lieu à des développements appliqués dans plusieurs domaines. Ces caractéristiques, et plus précisément leur absorption biphotonique (A2P), en font des composés particulièrement intéressants pour l'industrie de pointe (microfabrication, imagerie, dispositifs optiques, etc.). Cependant, ces propriétés d'ONL et d'absorption biphotoniques n'ont été jusqu’à lors que peu étudiées, malgré leur potentiel prometteur. Dans un premier temps, une famille de nouveaux composés organiques présentant cette structure particulière, a donc été synthétisée afin de confirmer le potentiel d'absorption à deux photons de ces chromophores, notamment par des mesures Z-scan. Dans un second temps, la synthèse de d’analogues organométalliques ayant des groupes donneurs tels que «Fe(η5-C5Me5)(κ2-dppe)C≡C− , « Ru(Cl)(κ2-dppe)2C≡C− » ou ethynylferrocene a été réalisée et ces dérivés ont été extensivement caractérisés. Ces dérivés organométalliques devraient présenter des sections efficaces d’A2P (ainsi que des réponses ONL de troisième ordre) dans le proche-IR bien supérieures à celles de leurs analogues organique. De plus les groupements métalliques redox-actifs devraient permettre d’obtenir des chromophores électro-commutables. Dans cet objectif, les études déjà réalisées sur ces composés joints à l’étude à venir de leur propriétés ONL de troisième ordre et permettra de mieux comprendre les phénomènes electroniques sous-jacents à ces propriétés et le rôle joué par les parties organique et organométalliques de leur structure. / During this PhD, new organic and organometallic triphenylmethylium dyes were synthesized. Due to their multipolar structure, these compounds have been anticipated to possess strong nonlinear optical (NLO) properties which could be of interest in many fields. These characteristics, and more precisely their two-photon absorption (TPA) properties, make them attractive for the design of high technology materials (microfabrication, imaging, optical devices, etc…). However, their multiphoton absorption properties and other related nonlinear optical (NLO) effects have not been thoroughly investigated to date, in spite of their promising potential. In this respect, a family of organic derivatives has been synthesized to confirm their potential as two-photon absorbers, which was established thanks to Z-scan measurements. Organometallic compounds bearing electron-releasing groups such as «Fe(η5-C5Me5)(κ2-dppe)C≡C−» , « Ru(Cl)(κ2-dppe)2C≡C− » or ethynylferrocene were then synthesized and fully characterized. These organometallic dyes should possess larger TPA cross-sections (and also larger third-order NLO properties) in the NIR range than their organic counterparts. Furthermore, these redox-active metal centers permit access to electro-switchable molecules. In this respect, along with the data already gathered on these derivatives, the study of their cubic NLO properties in their different redox states will now contribute to a better understanding of the underlying electronic phenomena and the role played by the organic and organometallic moieties in such architectures.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-19T11:13:21Z No. of bitstreams: 2 Tese - Denise Ramos Silveira Alves - 2016.pdf: 1503752 bytes, checksum: 84aa2ffe80e8b6013fb3b3cbdf580374 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-19T11:13:44Z (GMT) No. of bitstreams: 2 Tese - Denise Ramos Silveira Alves - 2016.pdf: 1503752 bytes, checksum: 84aa2ffe80e8b6013fb3b3cbdf580374 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-19T11:13:44Z (GMT). No. of bitstreams: 2 Tese - Denise Ramos Silveira Alves - 2016.pdf: 1503752 bytes, checksum: 84aa2ffe80e8b6013fb3b3cbdf580374 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-05-05 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Objective: Evaluate the effect of photodynamic therapy (PDT) on Enterococcus faecalis biofilm in infected root canals and on dentin structure. Methods: Twenty-one root canals of a sample of 24 extracted single-rooted human teeth were infected by E. faecalis for 60 days to form biofilm. The antimicrobial strategies tested were (n=3 in each group): root canal preparation using NiTi rotary instruments, 2.5% NaOCl and 17% EDTA irrigation, and PDT with 0.01% methylene blue (group I) or 0.01% malachite green (group II); root canal preparation using NiTi rotary instruments, 2.5% NaOCl and 17% EDTA irrigation, and PDT with 0.01% methylene blue (group III); PDT with 0.01% methylene blue without root canal preparation (group IV); root canal preparation using NiTi rotary instruments, 22.5% NaOCl and 17% EDTA irrigation, and no PDT (group V); 2.5% NaOCl irrigation with no root canal preparation, and 17% EDTA irrigation (group VI); positive control (group VII). Three roots were not infected and were used as negative controls (group VIII). Samples for microbiological tests were collected using three sterile paper points, later stored in BHI and incubated at 37o C for 48 hours at three time points: before (S1) and after (S2) root canal preparation, and after PDT application (S3). Bacterial growth was analyzed according to turbidity of culture medium, presence of bacteria, and spectrophotometric optical density (nm). Specimens were sectioned and prepared for SEM analysis of dentin structure. Results: Bacteria were found at S1, S2 and S3 in all experimental groups. Optical density of culture media at S2 and S3 in groups I, II and III were lower than at S1, but not statistically different. Optical density of culture media at S2 was 28.70% and 24.67% lower than at S1 in groups I and II; after PDT, optical density was 90.00% (group I) and 37.30% (group II) lower. In group III, it was 97.70% lower at S2 and an additional 92.00% lower after PDT. In group IV, optical density increase 3.2%. Dentin analysis after PDT revealed areas of melting and recrystallization, peritubular dentin projections, intertubular dentin erosion and fusion of dentinal tubule openings, which made dentin surface irregular. Some dentinal tubules were obliterated, and there were changes in the shape of their openings. Conclusion: PDT applied after root canal preparation using manual or rotary files was not effective in eliminating E. faecalis completely. PDT changed dentin structure and resulted in dentin melting and recrystallization, as well as in dentinal tubule erosion and obliteration. / Objetivo: Avaliar o efeito da terapia fotodinâmica sobre biofilme de Enterococcus faecalis em canais radiculares infectados e sobre a estrutura dentinária. Metodologia: O estudo foi desenvolvido em vinte e quatro dentes humanos unirradiculares extraídos, dos quais vinte e um canais radiculares foram infectados com E. faecalis por 60 dias para formação de biofilme. As estratégias antimicrobianas testadas foram (n=3): preparo do canal radicular com instrumentos rotatórios de NiTi/ NaOCl 2,5%/ irrigação final EDTA 17%, e TFD com azul de metileno 0,01% (Grupo I) ou verde malaquita 0,01% (Grupo II); preparo do canal radicular com instrumentos manuais de aço inox/ NaOCl 2,5%/ irrigação final EDTA 17% e TFD com azul de metileno 0,01% (Grupo III); TFD com azul de metileno 0,01% sem preparo prévio do canal radicular (Grupo IV); preparo do canal radicular com instrumentos rotatórios de NiTi/ NaOCl 2,5%/ irrigação final EDTA 17% sem emprego da TFD (Grupo V); irrigação com hipoclorito de sódio 2,5% sem preparo do canal radicular/ irrigação final EDTA 17% (Grupo VI); controle positivo (Grupo VII). Três espécimes não foram contaminados, sendo utilizados como controle negativo (Grupo VIII). Coletas microbiológicas foram realizadas, antes (CM1) e após (CM2) o preparo do canal radicular, e depois da aplicação da TFD (CM3), utilizando três pontas de papel absorventes esterilizadas, posteriormente armazenadas em BHI e a seguir , incubadas a 37o C por 48 horas. O crescimento bacteriano foi analisado pela turbidez do meio de cultura, sendo determinada a presença ou ausência de bactérias, e pela densidade óptica do meio de cultura, interpretada por espectrofotometria (nm). A seguir, os espécimes foram seccionados e preparados para análise da estrutura dentinária por meio de imagens de MEV. Resultados: A presença de bactérias foi verificada na CM1 , CM2 e CM3 de todos os grupos experimentais. As medidas da densidade óptica dos meios de cultura das CM2 e CM3 nos grupos experimentais I, II e III apresentaram redução quando comparada a CM1, porém não significativa estatisticamente. Nos Grupos I e II a densidade óptica do meio de cultura foi reduzida em 28.70% e 24,67% em CM2, respectivamente. Após a TFD, a redução da densidade óptica foi 90,00% (Grupo I) e 37,70% (Grupo II). No Grupo III, a redução da densidade óptica do meio de cultura foi de 97,70% na CM2, com redução adicional de 92,00% após TFD. No Grupo IV foi verificado aumento da densidade óptica do meio de cultura em 3,2%. A análise da dentina evidenciou, nos grupos submetidos à TFD, áreas de derretimento e recristalização, projeção da dentina peritubular, e regiões com erosão da dentina intertubular e união das entradas dos túbulos dentinários, tornando a superfície dentinária irregular. Obliteração de túbulos dentinários com alteração do contorno de suas entradas também foi verificada. Conclusão: A TFD, após preparo do canal radicular com sistema rotatório ou manual, não foi efetiva na eliminação completa de E. faecalis, e alterou a estrutura dentinária, determinando derretimento e recristalização de dentina, erosão e obliteração de túbulos dentinários.

The theoretical part of this master’s thesis is focused on the characteristics and forms of phosphorus, occurrence in natural waters and the problems associated with higher phosphorus concentrations, especially in the development of cyanobacteria and algae. There are also described the possibilities of determining the degree of pollution of natural waters by nutrients and the effects of eutrophication. There are also described methods for the determination of phosphates, especially spectrophotometric determination in ternary systems with dyes. Like other methods are mentioned AAS, ion chromatography, electrophoresis and izotachophoresis, electrochemical methods or enzymatic methods. In the experimental part of this work deals with the selection and optimalization of suitable ternary system with organic dyes for spectrophotometric determination of orthophosphates. The system with phosphovanadomolybdic acid is studied too. For analytical procedure with malachite green oxalate, which was found to be most useful, were studied interfering ions, commonly occurring in natural waters . The chosen procedure was finally tested on real water samples.