Superoxide Dismutase (SOD) activity in cell-free extracts prepared from healthy mature flowers of Petunia hybrida (variety 'Hurrah') was studied. The SOD activity in the crude extracts was stable for more than one month when stored at -20 oC. It was found that pH 7.8 is optimal for SOD activity. Different flower tissues of petunia (stigma, style and ovary) at various stages of development were extracted and analysed for SOD activity. SOD activity was found to be significantly highest in the ovary tissue of dehiscent petunia flowers. Three SOD isozymes were detected after crude extracts of the different female reproductive tissues of petunia flowers were analysed on a non-denaturing polyacrylamide gel electrophoresis system. Based on a difference in the sensitivity of the SOD isoforms to H2O2 and KCN, it is suggested that Mn-SOD, Fe-SOD and Cu/Zn-SOD were present in the crude extracts of the female reproductive tissues of petunia flowers. The response of the female reproductive parts of petunia flowers was also tested under water deficiency and high temperature (35 oC) stress. The SOD activity seemed to increase more in response to the high temperature than the water deficiency stress. Intense blue staining was observed from developing younger buds, and much lower formazan deposition was detected at the later stage. This indicates the lower O2- produced during later stages mainly due to increasing SOD synthesis. DEAE cellulose chromatography was successfully used to partially purify SOD from the ovaries of petunia flowers. The characteristics of the partially purified enzyme fraction were found to be very similar to those of the crude extracts.

Superoxide Dismutase (SOD) activity in cell-free extracts prepared from healthy mature flowers of Petunia hybrida (variety 'Hurrah') was studied. The SOD activity in the crude extracts was stable for more than one month when stored at -20 oC. It was found that pH 7.8 is optimal for SOD activity. Different flower tissues of petunia (stigma, style and ovary) at various stages of development were extracted and analysed for SOD activity. SOD activity was found to be significantly highest in the ovary tissue of dehiscent petunia flowers. Three SOD isozymes were detected after crude extracts of the different female reproductive tissues of petunia flowers were analysed on a non-denaturing polyacrylamide gel electrophoresis system. Based on a difference in the sensitivity of the SOD isoforms to H2O2 and KCN, it is suggested that Mn-SOD, Fe-SOD and Cu/Zn-SOD were present in the crude extracts of the female reproductive tissues of petunia flowers. The response of the female reproductive parts of petunia flowers was also tested under water deficiency and high temperature (35 oC) stress. The SOD activity seemed to increase more in response to the high temperature than the water deficiency stress. Intense blue staining was observed from developing younger buds, and much lower formazan deposition was detected at the later stage. This indicates the lower O2- produced during later stages mainly due to increasing SOD synthesis. DEAE cellulose chromatography was successfully used to partially purify SOD from the ovaries of petunia flowers. The characteristics of the partially purified enzyme fraction were found to be very similar to those of the crude extracts.

Durante a fase de obturação dos canais radiculares, pode ocorrer o contato direto do material obturador com os tecidos periapicais, por tempo indeterminado, o que pode retardar, dificultar ou impedir a ocorrência do processo de reparo, por isso, para o êxito do tratamento endodôntico, a seleção de um material de obturação do canal radicular adequado é tão essencial como a técnica operatória. Esse contato pode ocorrer por extravasamento na forma de puff ou mesmo sem extravasamento, pois componentes derivados desses materiais podem entrar em contato direto com os tecidos, através de numerosas conexões, como, por exemplo, os túbulos dentinários, canais acessórios, canais laterais e o forame apical, causando irritação e possível desconforto pós-operatório. Outra forma pela qual os materiais obturadores de canais radiculares podem entrar em contato com os tecidos periapicais é através do processo realização de tratamento endodôntico em dentes decíduos, devido ao processo de rizólise ou tratamento endodôntico em dentes imaturos (ápice aberto). Por esses motivos, a biocompatibilidade dos materiais obturadores de canais radiculares é de extrema importância, diante disso objetivo deste trabalho foi avaliar a citotoxicidade, por meio do Ensaio do MTT, de seis materiais endodônticos usados em dentes decíduos e permanentes (AH Plus, GuttaFlow 2, Endomethasone N, Vitapex®, Calen® espessada e MTA ProRoot) recém espatulados, em cultura primária de linfócitos do sangue periférico de humanos, por 24 horas. Os resultados foram submetidos a análise estatística pelo teste one-way ANOVA e pós-teste de Tukey, com nível de significância de 5%. Observou-se que o Endomethasone N foi o mais citotóxico sobre linfócitos, O AH Plus, e a Calen® espessada foram citotóxicas a partir de 25 mg/mL, enquanto o MTA ProRoot, GuttaFlow 2 e o Vitapex® foram os menos citotóxicos sobre linfócitos humanos, podendo-se concluir que o Vitapex® (usados em dentes decíduos) e o GuttaFlow 2 e MTA ProRoot (usados em dentes permanentes) foram os materiais obturadores menos citotóxicos sobre linfócitos humanos. / During the filling phase of root canals, there may be direct contact of the filling material with the periapical tissues, for an (indefinite) unknown period, which may delay, hinder or prevent the occurrence of the repair process. The objective of this study is to evaluate the cytotoxicity by MTT assay of six endodontic materials used in primary and permanent teeth (AH Plus , GuttaFlow 2, Endomethasone N, Vitapex®, Calen® thickened and MTA ProRoot) newly spatulate in primary cultures of peripheral human blood lymphocytes for 24 hours. The results were statistically analyzed by one-way ANOVA and Tukey\'s test at 5% significance level. It was observed that the Endomethasone N was the most cytotoxic material for the lymphocytes, the AH Plus, and Calen® thickened were cytotoxic from 25 mg/mL, while the MTA ProRoot, GuttaFlow 2 and Vitapex® were less cytotoxic for human lymphocytes, allowing to conclude that the Vitapex® (used in deciduous teeth) and GuttaFlow 2 and MTA ProRoot (used in permanent teeth) were less cytotoxic sealers on human lymphocytes.

Durante a fase de obturação dos canais radiculares, pode ocorrer o contato direto do material obturador com os tecidos periapicais, por tempo indeterminado, o que pode retardar, dificultar ou impedir a ocorrência do processo de reparo, por isso, para o êxito do tratamento endodôntico, a seleção de um material de obturação do canal radicular adequado é tão essencial como a técnica operatória. Esse contato pode ocorrer por extravasamento na forma de puff ou mesmo sem extravasamento, pois componentes derivados desses materiais podem entrar em contato direto com os tecidos, através de numerosas conexões, como, por exemplo, os túbulos dentinários, canais acessórios, canais laterais e o forame apical, causando irritação e possível desconforto pós-operatório. Outra forma pela qual os materiais obturadores de canais radiculares podem entrar em contato com os tecidos periapicais é através do processo realização de tratamento endodôntico em dentes decíduos, devido ao processo de rizólise ou tratamento endodôntico em dentes imaturos (ápice aberto). Por esses motivos, a biocompatibilidade dos materiais obturadores de canais radiculares é de extrema importância, diante disso objetivo deste trabalho foi avaliar a citotoxicidade, por meio do Ensaio do MTT, de seis materiais endodônticos usados em dentes decíduos e permanentes (AH Plus, GuttaFlow 2, Endomethasone N, Vitapex®, Calen® espessada e MTA ProRoot) recém espatulados, em cultura primária de linfócitos do sangue periférico de humanos, por 24 horas. Os resultados foram submetidos a análise estatística pelo teste one-way ANOVA e pós-teste de Tukey, com nível de significância de 5%. Observou-se que o Endomethasone N foi o mais citotóxico sobre linfócitos, O AH Plus, e a Calen® espessada foram citotóxicas a partir de 25 mg/mL, enquanto o MTA ProRoot, GuttaFlow 2 e o Vitapex® foram os menos citotóxicos sobre linfócitos humanos, podendo-se concluir que o Vitapex® (usados em dentes decíduos) e o GuttaFlow 2 e MTA ProRoot (usados em dentes permanentes) foram os materiais obturadores menos citotóxicos sobre linfócitos humanos. / During the filling phase of root canals, there may be direct contact of the filling material with the periapical tissues, for an (indefinite) unknown period, which may delay, hinder or prevent the occurrence of the repair process. The objective of this study is to evaluate the cytotoxicity by MTT assay of six endodontic materials used in primary and permanent teeth (AH Plus , GuttaFlow 2, Endomethasone N, Vitapex®, Calen® thickened and MTA ProRoot) newly spatulate in primary cultures of peripheral human blood lymphocytes for 24 hours. The results were statistically analyzed by one-way ANOVA and Tukey\'s test at 5% significance level. It was observed that the Endomethasone N was the most cytotoxic material for the lymphocytes, the AH Plus, and Calen® thickened were cytotoxic from 25 mg/mL, while the MTA ProRoot, GuttaFlow 2 and Vitapex® were less cytotoxic for human lymphocytes, allowing to conclude that the Vitapex® (used in deciduous teeth) and GuttaFlow 2 and MTA ProRoot (used in permanent teeth) were less cytotoxic sealers on human lymphocytes.

The work in this thesis is divided into two parts; the first is the synthesis of a ‘switch-on’ fluorophore to measure cell viability, and the second is the development of a fluorescent detection method for protein−peptide affinity assays applied in the identification of protein-protein inhibitors. Tetrazolium salts are often used in cytotoxicity assays as indicators of cell viability as they are reduced to deeply coloured formazans exclusively in healthy cells. However, measuring the absorbance of the formazan is prone to bias from other coloured species in the cell media, requires solubilisation and can be difficult to quantify. A preferable method of detection is direct fluorescence as it is easily quantified, more sensitive and would ideally remove the need to solubilise the insoluble dye. The aim of this project was to synthesise a tetrazolium salt that could be reduced to a soluble fluorescent formazan in healthy cells as an indicator of cell viability. A number of fluorescent formazans were synthesised by incorporation of a fluorophore. The corresponding tetrazolium salts were non-fluorescent and could be reduced to the formazan in vitro. Several formazans were synthesised to attempt to increase the emission wavelength and intensity to overcome cellular autofluorescence. Protein-protein interactions have been implicated in the pathogenesis of many human diseases but until recently were considered undruggable. However, peptides have emerged as ideal compounds for targeting the large and relatively featureless protein interfaces. Work focussed on the discovery of peptide inhibitors for the E3 ubiquitin ligase stationary-phase kinase associated protein (Skp2). Potential peptide inhibitors were identified using CelluSpot synthesis and array technology to screen peptide libraries. Qualitative analysis of the protein affinity assay results by enhanced chemiluminescent detection was found to be misleading, and so a quantifiable and more sensitive fluorescent detection method was developed.