This research examines the hypothesis that the satiety effect of cholecystokinin octapeptide (CCK-8) is mediated by changes in gastric emptying. A method for collection of gastric emptying data, the double sampling procedure, is developed and validated for use in the rat. The double sampling technique permits repeated measurements of liquid gastric volume and thus describes the time course of emptying within a single experimental session. Further, the method allows determination of the amount of gastric secretion, volume emptied into the intestines, and amount of gastric load remaining in the stomach. Experiments are presented which: i) demonstrate the utility of the technique; ii) validate its accuracy in determining gastric volume; iii) indicate the stability of measurements obtained with this procedure; and iv) provide a procedure for quantitative evaluation of data obtained with this technique. Using the double sampling procedure, the ability of CCK-8 to delay gastric emptying and to influence feeding are then compared under similar experimental conditions. The effect of CCK-8 on gastric emptying is assessed in 6 hr deprived rats receiving 10 ml intragastric test loads of either .15M saline or 15% sucrose. Intraperitoneal (ip) injections of CCK-8 in doses of 1.4-22.4 ug/kg produce a dose-dependent retardation of gastric emptying of both saline and nutrient. Lower doses of CCK-8, 0.01 and 0.1 ug/kg, have no effect on gastric emptying. The effect of CCK-8 on feeding is assessed in rats tested under the same experimental conditions used in the gastric emptying studies. Doses of CCK-8 capable of retarding gastric emptying also suppress eating in a dose-dependent manner. These findings provide necessary correlational support for the hypothesis that satiety produced by CCK-8 is mediated by inhibition of gastric emptying. However, a further quantitative analysis of the correspondence of the gastric emptying and feeding effects of CCK-8 suggest that retardation of emptying may not account for the entire satiety effect of the peptide. The next set of studies provide direct tests of whether changes in gastric emptying mediate CCK-induced satiety. If gastric emptying plays a significant role in the satiety produced by CCK-8 then: i) the effects of CCK-8 on emptying and feeding should share similar kinetics, and ii) peptides that inhibit emptying should also inhibit feeding. I show that CCK-8 (5.6 ug/kg) injected coincident with introduction of an intragastric load or presentation of a test meal produces a rapid inhibition of both emptying and feeding. In contrast, the identical dose of CCK-8 administered 15 min before testing causes no inhibition of emptying, even though the peptide retains its ability to produce satiety. I also test the abilities of the peptides pentagastrin (100 ug/kg), bombesin (8 & 16 ug/kg) and secretin (2.86, 14.3 & 28.6 ug/kg) to reduce food intake and inhibit gastric emptying. Pentagastrin does not affect food intake or gastric emptying. Bombesin causes a small transient delay in emptying but a large and sustained suppression of eating. High dose secretin (14.3 ug/kg) causes no significant reduction of food intake, even though this dose of secretin inhibits emptying to the same degree as 1.4 ug/kg CCK-8, which does reduce intake. Thus, although CCK-8 does influence the rate of gastric emptying, the present results indicate that the inhibition of emptying by CCK is neither necessary nor sufficient to explain its satiety effect. / Thesis / Doctor of Philosophy (PhD)

Os azo corantes atualmente são considerados um assunto preocupante no que se refere à saúde pública e ambiental, pois quando lançados nos efluentes industriais contaminam o meio ambiente. Infelizmente, o método convencional de tratamento de efluentes têxteis, bem como de águas brutas que os recebem não são capazes de remover de maneira eficaz os corantes bem como sua toxicidade. Dentro deste contexto, este trabalho teve como objetivo avaliar a eficiência do tratamento de amostras aquosas por fotoeletrocatálise em comparação com a cloração convencional como método alternativo de degradação de azo corantes, usando os corantes Disperse Orange 1, Disperse Red 1 e Disperse Red 13 como modelo. Adicionalmente, foi avaliada a citotoxicidade dos corantes originais em condrócitos bovinos e células HepG2 em cultura em monocamadas e 3D. Para tanto, soluções desses corantes originais, clorados e fotoeletrocatalisados foram avaliadas utilizando ensaios de genotoxicidade/mutagenicidade, citotoxicidade e ecotoxicidade. Todos os corantes originais e clorados foram genotóxicos para as células HepG2 no ensaio cometa. Para o ensaio com Salmonella, a cloração reduziu a mutagenicidade dos corantes para a linhagem YG1041 e aumentou o efeito para a linhagem TA98, exceto o Disperse Red 13 que teve a mutagenicidade reduzida para as duas linhagens após cloração. A fotoeletrocatálise removeu tanto a genotoxicidade quanto a mutagenicidade. Somente o Disperse Orange 1 induziu apoptose pelo ensaio com anexina V, mas essa citotoxicidade foi removida após os tratamentos. Os corantes Disperse Red 1 e Disperse Red 13 foram tóxicos para D. similis enquanto somente o Disperse Red 1 foi tóxico para V. fischeri, sendo que os tratamentos por cloração e fotoeletrocatálise diminuíram a toxicidade apresentada. Os corantes Disperse Orange 1 e Disperse Red 13 passaram a ser tóxicos para V. fischeri após cloração, sendo que a fotoeletrocatálise do Disperse Red 13 também gerou produtos tóxicos para esse organismo. Assim, embora seja um método de tratamento promissor, atenção deve ser dada na avaliação e aplicação da fotoeletrocatálise como um método alternativo à cloração. Os corantes originais Disperse Orange 1 e Disperse Red 13 diminuíram a atividade mitocondrial dos condrócitos, sendo que o Disperse Red 13 também diminuiu a produção de lactato. Todos os corantes reduziram a atividade mitocondrial das células HepG2 em monocamadas, ao passo que o Disperse Orange 1 deixou de exercer esse efeito no cultivo em 3D. Somente o Disperse Red 13 diminuiu a atividade de desidrogenases das células HepG2 e tal efeito foi observado tanto no cultivo em monocamadas quanto em 3D. / The azo dyes are currently considered as a concern regarding the environmental and public health, since when released in industrial effluents they pollute the environment. Unfortunately, the conventional method of treatment of textile effluents is not able to effectively remove both dyes and their toxicity. Within this context, this study aimed to evaluate the effectiveness of the treatment of aqueous samples by photoelectrocatalysis compared to conventional chlorination as an alternative method of degradation of azo dyes, using the dyes Disperse Orange 1, Disperse Red 1 and Disperse Red 13 as a model. Additionally, we evaluated the cytotoxicity of the original dyes using HepG2 cells and chondrocytes cultured in monolayer and in 3D. To this end, solutions of these original dyes, chlorinated and photoelectrocatalysed were evaluated using tests of genotoxicity / mutagenicity, cytotoxicity and ecotoxicity. All the dyes, original and chlorinated, were genotoxic to HepG2 cells in the comet assay. For the test with Salmonella, chlorination reduced the mutagenicity of the dyes for the YG1041 strain and increased the effect for the TA98 strain, except Disperse Red 13, which had the mutagenic effect reduced for both strains after chlorination. The photoelectrocatalysis removed both genotoxicity and mutagenicity. Only Disperse Orange 1 induced apoptosis by annexin V assay, but this cytotoxicity was removed after treatment. The dye Disperse Red 1 and Disperse Red 13 were toxic to D. similis while only the Disperse Red 1 was toxic to V. fischeri, and the treatment by chlorination and photoelectrocatalysis decreased the toxicity showed. The dyes Disperse Orange 1 and Disperse Red 13 began toxic to V. fischeri after chlorination, and the photoelectrocatalysis of the Disperse Red 13 generated toxic products for this organism. So, while it is a promising treatment method, attention should be given in the evaluation and application of photoelectrocatalysis as an alternative to chlorination. The dyes Disperse Orange 1 and Disperse Red 13 decreased the mitochondrial activity of chondrocytes, and the dye Disperse Red 13 also decreased the production of lactate. All the dyes reduced the mitochondrial activity of the HepG2 cells cultured in monolayer, while the Disperse Orange 1 did no show this effect in 3D. Only Disperse Red 13 decreased the activity of dehydrogenases of HepG2 cells and this effect was observed both in monolayer and in 3D.

Os azo corantes atualmente são considerados um assunto preocupante no que se refere à saúde pública e ambiental, pois quando lançados nos efluentes industriais contaminam o meio ambiente. Infelizmente, o método convencional de tratamento de efluentes têxteis, bem como de águas brutas que os recebem não são capazes de remover de maneira eficaz os corantes bem como sua toxicidade. Dentro deste contexto, este trabalho teve como objetivo avaliar a eficiência do tratamento de amostras aquosas por fotoeletrocatálise em comparação com a cloração convencional como método alternativo de degradação de azo corantes, usando os corantes Disperse Orange 1, Disperse Red 1 e Disperse Red 13 como modelo. Adicionalmente, foi avaliada a citotoxicidade dos corantes originais em condrócitos bovinos e células HepG2 em cultura em monocamadas e 3D. Para tanto, soluções desses corantes originais, clorados e fotoeletrocatalisados foram avaliadas utilizando ensaios de genotoxicidade/mutagenicidade, citotoxicidade e ecotoxicidade. Todos os corantes originais e clorados foram genotóxicos para as células HepG2 no ensaio cometa. Para o ensaio com Salmonella, a cloração reduziu a mutagenicidade dos corantes para a linhagem YG1041 e aumentou o efeito para a linhagem TA98, exceto o Disperse Red 13 que teve a mutagenicidade reduzida para as duas linhagens após cloração. A fotoeletrocatálise removeu tanto a genotoxicidade quanto a mutagenicidade. Somente o Disperse Orange 1 induziu apoptose pelo ensaio com anexina V, mas essa citotoxicidade foi removida após os tratamentos. Os corantes Disperse Red 1 e Disperse Red 13 foram tóxicos para D. similis enquanto somente o Disperse Red 1 foi tóxico para V. fischeri, sendo que os tratamentos por cloração e fotoeletrocatálise diminuíram a toxicidade apresentada. Os corantes Disperse Orange 1 e Disperse Red 13 passaram a ser tóxicos para V. fischeri após cloração, sendo que a fotoeletrocatálise do Disperse Red 13 também gerou produtos tóxicos para esse organismo. Assim, embora seja um método de tratamento promissor, atenção deve ser dada na avaliação e aplicação da fotoeletrocatálise como um método alternativo à cloração. Os corantes originais Disperse Orange 1 e Disperse Red 13 diminuíram a atividade mitocondrial dos condrócitos, sendo que o Disperse Red 13 também diminuiu a produção de lactato. Todos os corantes reduziram a atividade mitocondrial das células HepG2 em monocamadas, ao passo que o Disperse Orange 1 deixou de exercer esse efeito no cultivo em 3D. Somente o Disperse Red 13 diminuiu a atividade de desidrogenases das células HepG2 e tal efeito foi observado tanto no cultivo em monocamadas quanto em 3D. / The azo dyes are currently considered as a concern regarding the environmental and public health, since when released in industrial effluents they pollute the environment. Unfortunately, the conventional method of treatment of textile effluents is not able to effectively remove both dyes and their toxicity. Within this context, this study aimed to evaluate the effectiveness of the treatment of aqueous samples by photoelectrocatalysis compared to conventional chlorination as an alternative method of degradation of azo dyes, using the dyes Disperse Orange 1, Disperse Red 1 and Disperse Red 13 as a model. Additionally, we evaluated the cytotoxicity of the original dyes using HepG2 cells and chondrocytes cultured in monolayer and in 3D. To this end, solutions of these original dyes, chlorinated and photoelectrocatalysed were evaluated using tests of genotoxicity / mutagenicity, cytotoxicity and ecotoxicity. All the dyes, original and chlorinated, were genotoxic to HepG2 cells in the comet assay. For the test with Salmonella, chlorination reduced the mutagenicity of the dyes for the YG1041 strain and increased the effect for the TA98 strain, except Disperse Red 13, which had the mutagenic effect reduced for both strains after chlorination. The photoelectrocatalysis removed both genotoxicity and mutagenicity. Only Disperse Orange 1 induced apoptosis by annexin V assay, but this cytotoxicity was removed after treatment. The dye Disperse Red 1 and Disperse Red 13 were toxic to D. similis while only the Disperse Red 1 was toxic to V. fischeri, and the treatment by chlorination and photoelectrocatalysis decreased the toxicity showed. The dyes Disperse Orange 1 and Disperse Red 13 began toxic to V. fischeri after chlorination, and the photoelectrocatalysis of the Disperse Red 13 generated toxic products for this organism. So, while it is a promising treatment method, attention should be given in the evaluation and application of photoelectrocatalysis as an alternative to chlorination. The dyes Disperse Orange 1 and Disperse Red 13 decreased the mitochondrial activity of chondrocytes, and the dye Disperse Red 13 also decreased the production of lactate. All the dyes reduced the mitochondrial activity of the HepG2 cells cultured in monolayer, while the Disperse Orange 1 did no show this effect in 3D. Only Disperse Red 13 decreased the activity of dehydrogenases of HepG2 cells and this effect was observed both in monolayer and in 3D.

Introduction: Malignant Melanoma (MM), caused by UV radiation-induced DNA damage, is the most invasive form of skin cancer and has an increasing incidence worldwide. The hallmarks of MM include the presence of reactive oxygen species (ROS) and excessive proliferation of tumour cells. Many treatments are available or under investigation as anticancer therapeutics such as cell therapy, immunotherapy, gene therapy and nanotechnology-based strategies but they all have severe complications and side effects that limit their wider use. Methods: The present in vitro study has evaluated the genotoxic and cytotoxic effects of Se and CBSC-derived exosomes, individually and in combination, on lymphocytes from MM patients and healthy controls, and on the CHL-1 melanoma cell line. The comet assay and cell counting kit-8 (CCK-8) assay were used to measure genotoxicity and cytotoxicity, respectively, in all cell types. Molecular mechanisms underlying the observed effects were explored using transcriptional and protein expression profiling of key cell cycle and apoptosis genes, by employing the RT qPCR and Western blotting techniques. Conclusion: Selenium displays antioxidant and genoprotective effects in human lymphocytes, especially in MM patients. Both Se (10 μM) and CBSC-derived exosomes (120 μL) are well tolerated in lymphocytes, but show significant genotoxicity and cytotoxicity towards the CHL-1 cell line, with combined administration exhibiting a synergistic effect.