O câncer de pulmão permanece a neoplasia mais letal com cerca de 1,59 milhões de mortes por ano em todo o mundo, com eficácia terapêutica limitada e mau prognóstico. Aproximadamente 80 % dos casos são de câncer de pulmão de não pequenas células (CPNPC), desses, cerca de 50 % são os adenocarcinomas (AdC). Atualmente o tratamento padrão-ouro no tratamento de AdC pulmonar é baseado em agentes de platina (cisplatina, carboplatina) normalmente administrados em combinação com outros agentes, entretanto a doença é raramente curável. Vários fatores contribuem para a alta taxa de mortalidade e um dos mais comuns é a quimioresistência. Portanto, existe uma necessidade urgente de terapias mais eficazes em aumentar a sobrevida global dos pacientes com essa patologia. Tendo em vista que o desenvolvimento de novos medicamentos requer muito tempo e investimento financeiro, o uso de drogas existentes como terapia combinada torna-se uma boa abordagem. Estudos anteriores de nosso grupo demonstraram que a agressividade do AdC de pulmão está associada com elevado estresse oxidativo onde o peróxido de hidrogênio (H2O2) tem um papel crucial, uma vez que o tratamento com a enzima antioxidante catalase (CAT) atenuou a agressividade do tumor. Assim, este estudo avaliou a eficácia da terapia combinada com CAT na linhagem celular A549 de AdC humano. Primeiramente, a adição de CAT causou uma inibição dose-dependente da proliferação celular com um efeito máximo na dose de 1000 U/mL. Essa inibição foi relacionada a um efeito citostático, não citotóxico, do consumo de H2O2 intracelular, já que a retirada da CAT restaurou a taxa proliferativa celular a níveis de controle. Após, a avaliação do “status” de ativação do NFB mostrou um aumento de 2,9 vezes no imunoconteúdo citosólico da subunidade p65, sugerindo uma diminuição da ativação do NFB em células tratadas com CAT. A análise do efeito do tratamento de CAT em parâmetros redox mostrou uma redução nos níveis intracelulares de tióis (-SH) e no potencial antioxidante total (TRAP) e um aumento na produção de H2O2 e nos níveis de glutationa (GSH). Entre as drogas testadas (Cisplatina, 5-Fluorouracil, Paclitaxel, Hidroxiuréia e Daunorrubicina), apenas o tratamento com paclitaxel e hidroxiureia mostraram aumento da produção de H2O2 quando comparados com o veículo. No entanto, o co-tratamento com CAT e paclitaxel não alterou a produção de H2O2. Hidroxiuréia e CAT promoveu uma redução na produção de H2O2, quando comparado com a droga sozinha. A cisplatina, por si só não teve efeito na produção de H2O2, mas cisplatina e CAT promoveram um aumento na produção de H2O2. A eficácia da terapia combinada com CAT em potencializar a citotoxicidade dos quimioterápicos, foi analisada pelo ensaio SRB e pelo software CalcuSyn®. Pela análise dos valores de “Combination Index” (CI) observamos que, com a exceção da combinação de CAT e paclitaxel, que gerou antagonismo, as combinações de CAT com cisplatina, 5-fluorouracil e hidroxiuréia exibiram um efeito sinérgico na eliminação de células da linhagem de AdC humano A549. Dessa forma, os dados aqui apresentados sugerem que a terapia combinada de CAT com cisplatina, 5-fluorouracil e hidroxiuréia pode surgir como uma nova estratégia terapêutica para o AdC. / Lung cancer remains the most lethal malignant disease with nearly 1.59 million deaths annually worldwide, limited efficacy of current therapeutics and dismal prognostic. Approximately 80% of the cases are non-small cell lung cancer (NSCLC), of these, roughly 50% are adenocarcinomas (AdC). Currently, the gold standard treatment for AdC is based on platinum agents, usually given in combination with other agents. Despite these therapies, the disease is rarely curable. Several factors contribute to the high mortality rate and one of the most common includes tumor cell chemoresistance to cytotoxic drugs. Therefore, there is an urgent need for more effective therapies that could increase the overall survival of lung AdC patients. With the notion that the development of novel drugs require much time and financial investment, the use of existing drugs as adjuvant treatment becomes a good approach. Previous studies of our group demonstrated that lung AdC aggressiveness is associated with elevated intracellular oxidative stress, in which hydrogen peroxide (H2O2) plays a crucial role, since the exogenous treatment with the antioxidant enzyme catalase (CAT) attenuated tumor aggressiveness. Then, this study evaluated the efficacy of CAT adjuvant treatment in the human AdC cell line A549. Firstly, exogenous addition of CAT caused a dose dependent inhibition on cellular proliferation with a maximal dose effect of 1000 U/mL. Growth inhibition was related to a cytostatic, not cytotoxic, effect of intracellular H2O2 consumption, since CAT washout readily restored cellular proliferative rate similar to control. After that, evaluation of NFB activation status showed a 2.9-fold increase in the cytosolic immunocontent of the NFB subunit p65, suggesting a decreased NFB activation in CAT-treated cells. Analysis of CAT treatment effect in redox parameters showed decreased intracellular thiol levels (-SH) and non-enzymatic antioxidant potential (TRAP) and increases the H2O2 production and glutathione levels (GSH). Among drugs tested (cisplatin, paclitaxel, 5-fluorouracil, daunorubicin, hydroxurea) (GI50), only paclitaxel and hydroxyurea showed increased production of H2O2 when compared with vehicle. However, co-treatement with CAT and paclitaxel had no alteration in the H2O2 production. Hydroxyurea plus CAT had a decreased in H2O2 production when compared with the drug alone. Cisplatin, alone had no effect in H2O2 production, but cisplatin plus CAT, had an increased in H2O2 production. Regarding the effectiveness of adjuvant CAT treatment in potentiate chemotherapeutic drugs cytotoxicities, we used de SRB assay and Calcusyn Software to access this interaction. From analyzes of combination index (CI) values, generated by CalcuSyn, we observed that with the exception of CAT plus paclitaxel, all combinations exhibited a synergistic effect. Taken together, data presented here suggest that adjuvant CAT treatment can act synergistically with chemotherapeutics and modulate tumorassociated signaling pathways providing a new therapeutic strategy for AdC therapy.

The research presented in this thesis focuses on the molecular design of an oxidation-sensitive nanocarrier and its enzyme conjugate with a view of their application in the field of biomaterials. I have polarised our attention on a specific class of polymers, the polysulfides, for their environmental responsiveness (towards oxidising substances, a condition often associated with inflammatory reactions), interesting physico-chemical properties, ease of the preparation and multiple possibilities for further modifications and bioconjugations, which are perfectly suitable for the development as systems for drug delivery applications. In this work we firstly have focused on the synthesis of amphiphilic poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) block copolymers by employing vinyl sulfone as the functional group to link the blocks and modify the end of the PEG. This study was followed by an investigation of the macromolecular interchange and payload exchange of the formed polymeric micelles to understand the 'co-formulation' events, employing fluorophores (dansyl groups) and quenchers (dabsyl groups) either as terminal groups in macroamphiphiles or as encapsulated hydrophobic payloads. In another part of the work, I have developed a micellar system with which simultaneously to two of the most important ROS: superoxide and hydrogen peroxide, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity towards superoxide with that of hydrophobic thioethers towards hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then 'mopped-up' by the polysulfides. This mode of operation is functionally analogous to the SOD/catalase combination, with the advantage of being based on a single and more stable system.

No description available.

Vitamin C is used in the diets in order to improve growth, resistance to stress and disease, as well as the survival of fish. It can also be effective in the conservation of fish during processing and storage, inhibiting the degradation of lipids by oxidation. This study evaluated the stocking density and the effects of supplementation of vitamin C in the final quality of the fillet tilapia (Oreochromis nilotius, Linneaus, 1875). We used one hundred and twenty fingerlings for each trial, the study of fish stocking density had an initial weight of 4.0 g and evaluation of supplementation with vitamin C, 11.0 g. Both studies had a randomized design, comprising four treatments of different stocking densities (50, 75, 100 and 125 fish/m3) with five replications. Already working with vitamin C (ascorbic acid 2 - sulfate acid - protected form) was carried out in four treatments with six replicates, characterized by four levels of supplementation of vitamin C in the diets (250, 500 and 750 mg/kg diet) and control group (zero mg/kg diet) with two different stocking densities (50 and 100 fish/m3). The ration given to the first study contained 36% crude protein and 3100 kcal/DE/kg feed and the second was 28% crude protein and 3400 kcal/DE/kg. After ninety days, the fish were slaughtered and evaluated the growth performance parameters for the study of stocking density, chemical composition, cholesterol content, fatty acid profile, quality of lipid fraction in both studies. The liver of fish from the second stage of labor was frozen at - 80°C for lipid peroxidation analysis and determination of enzyme activity of oxidative stress (catalase and superoxide dismutase). It follows that for the study of stoking significant difference ρ <0.05 between the final average weight and total weight gain between the different densities tested, the highest weight was found in the group density of 75 fish/m3. The body composition analysis, differences were observed ρ <0.05 between the densities on moisture, total lipid content, protein, calories and cholesterol. The ratio polyunsaturated / saturated was higher in density of 11.76 for 100 fish/m3, since the density of 50 fish/m3 presented a sum of 6.85 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the densities studied showed the contents of n-6/n-3, hypocholesterolemic/hypercholesterolemic index of polyunsaturated fatty acids of atherogenicity and index of thrombogenicity as favorable for consumption. Fish reared at a density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. Regarding the second stage of labor was observed difference ρ<0.05 between the levels of vitamin C supplementation levels in total lipid content, crude protein, calories and cholesterol. The ratio polyunsaturated/saturated was higher in the supplementation level of 250 mg of vitamin C (1.26), since the level of 500 mg of vitamin C showed a sum of 6.48 for the levels of EPA + DHA. In assessing the nutritional quality of lipids, the levels of vitamin C study showed rates of n-6/n-3, hypocholesterolemic / hypercholesterolemic index of atherogenicity and index of thrombogenicity for subsequent food consumption. Fish reared with diets supplemented with 750 mg of vitamin C in the density of 50 fish/m3 can be seen as a good food from a nutritional standpoint, they presented a good level of fatty acids, especially omega-3 fatty acid and its value in hypocholesterolemic/hypercholesterolemic. In the study of redox balance results showed that there was an increase in lipid peroxidation, suggesting that there may have been damage to the liver of animals and the antioxidant defenses were not sufficient to prevent oxidative stress. Density of 50 fish/m3, the dietary supplementation with vitamin C can be pro-oxidant. Since the density 100 fish/m3, vitamin C supplementation may be an antioxidant. The catalase activity of the enzyme showed low activity, suggesting an impairment in hepatic redox balance for this enzyme in animals receiving diets containing different levels of vitamin C. Since superoxide dismutase activity of the enzyme showed high activity, suggesting a redox balance in protecting liver for this enzyme in animals receiving diets containing different levels of vitamin C. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A vitamina C é usada nas dietas com a finalidade de melhorar o crescimento, a resistência ao estresse e às doenças, assim como a sobrevivência de peixes. Também pode ser eficiente na conservação do pescado durante o processamento e estocagem, inibindo a degradação dos lipídeos pela oxidação. O presente trabalho avaliou a densidade de estocagem e os efeitos da suplementação de vitamina C na qualidade final do filé de Tilápia-do-Nilo (Oreochromis nilotius, Linneaus, 1875). Foram utilizados cento e vinte alevinos para cada estudo, sendo que os peixes do estudo da densidade de estocagem tinham peso inicial de 4,0 g e os da avaliação com a suplementação de vitamina C de 11,0 g. O delineamento inteiramente casualizado foi utilizado em ambos os estudos perfazendo quatro tratamentos de diferentes densidades de estocagem (50, 75, 100 e 125 peixes/m3) com cinco repetições. Já o trabalho com a vitamina C (ácido 2 - sulfato ascórbico - forma protegida) foi realizado em quatro tratamentos com seis repetições, caracterizado pela suplementação de quatro níveis de vitamina C nas rações (250, 500 e 750 mg/kg de ração) e o grupo controle (zero mg/kg de ração) com duas diferentes densidades de estocagem (50 e 100 peixes/m3). A ração fornecida para o primeiro estudo continha 36% de proteína bruta e 3100 kcal/energia digestiva/kg de ração e a segunda tinha 28% de proteína bruta e 3400 kcal/energia digestiva/kg. Após noventa dias, os peixes foram abatidos e foram avaliados os parâmetros de desempenho zootécnico para o estudo de densidade de estocagem, composição centesimal, teor de colesterol, perfil de ácidos graxos, qualidade da fração lipídica para ambos os estudos. O fígado dos peixes da segunda etapa do trabalho foi congelado a – 80ºC para análises de peroxidação lipídica e determinação da atividade das enzimas do estresse oxidativo (catalase e superóxido dismutase). Conclui-se que para o estudo de densidade de estocagem houve diferença significativa ρ<0,05 entre o peso médio final e no ganho de peso total entre as diferentes densidades populacionais testadas; o maior peso foi encontrado no grupo de densidade de 75 peixes/m3. Quanto à análise da composição corporal observou-se diferença ρ<0,05 entre as densidades nos teores de umidade, lipídeos totais, teor de proteína bruta, valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada na densidade de 100 peixes/m3 de 11,76, já a densidade de 50 peixes/m3 apresentou uma somatória de 6,85 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, as densidades estudadas mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de ácidos graxos poliinsaturados de aterogenicidade e índice de trombogenicidade como favoráveis para consumo alimentar. Os peixes criados na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Com relação à segunda etapa do trabalho foi observado diferença ρ<0,05 entre os níveis de suplementação da vitamina C nos teores de lipídeos totais; teor de proteína bruta; valor calórico e de colesterol. A relação poliinsaturados/saturados foi mais elevada no nível de suplementação de 250 mg de vitamina C (1,26), já o nível de 500 mg de vitamina C apresentou um somatório de 6,48 para os teores de ácido eicosapentaenóico + ácido docosahexaenóico. Na avaliação da qualidade nutricional dos lipídeos, os níveis de suplementação de vitamina C estudados mostraram os índices de n-6/n-3, hipocolesterolêmicos/hipercolesterolêmicos, índice de aterogenicidade e índice de trombogenicidade favoráveis quanto ao consumo alimentar. Os peixes criados com ração suplementada com 750 mg de vitamina C na densidade de 50 peixes/m3 podem ser considerados como um bom alimento do ponto de vista nutricional, pois apresentaram um bom teor de ácidos graxos, principalmente de ácido ômega-3 e pelo seu valor em hipocolesterolêmicos/hipercolesterolêmicos. Já no estudo do balanço redox os resultados mostraram que houve aumento na peroxidação lipídica, sugerindo que podem ter existido danos ao fígado dos animais e as defesas antioxidantes não foram suficientes para evitar o estresse oxidativo. Na densidade de 50 peixes/m3, a ração suplementada com vitamina C pode ser pró-oxidante. Já na densidade 100 peixes/m3, a vitamina C suplementada pode ser antioxidante. A atividade da enzima catalase apresentou uma baixa atividade, sugerindo um comprometimento no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C. Já atividade da enzima superóxido dismutase apresentou elevada atividade, sugerindo uma proteção no balanço redox hepático para esta enzima nos animais submetidos à ração com diferentes níveis de suplementação de vitamina C.

Tumourigenic cells utilize aberrant metabolic process that supports the biosynthetic requirements for hyperproliferation, survival and prolonged maintenance characterised by glucose metabolism to lactate dehydrogenase independent of oxygen availability (Warburg effect). In addition, tumourigenic cells exert increased glycolytic- and glutaminolytic activity in order to provide increased quantities of adenosine triphosphate. The aim of this research project was to investigate the influence of glutamine deprivation on proliferation, morphology, oxidative stress, mitochondrial membrane potential, cell cycle progression, antioxidant defences, deoxyribonucleic acid (DNA) damage, energy status, cell survival signaling and cell death induction in tumourigenic- and non-tumourigenic breast cell lines. In this study it was found that glutamine deprivation results in differential antiproliferative activity where the MCF-7 cell line was the most affected with decreased cell growth to 61% after 96 h of glutamine deprivation. Aberrant redox activity was most prominently observed in the MCF-7 cell line accompanied with biphasic mitochondrial membrane potential- and reactive oxygen species production. The MCF-7 cell line showed significant mitochondrial membrane depolarisation after 24 h and 96 h deprivation from glutamine (1.5- and 1.37 fold). Cell cycle progression analysis illustrated an increase in the amount of cells present in the S-phase in the MCF-7 cell line after 72 h of glutamine deprivation. The MDA-MB-231 cell line resulted in a significant increase in cells occupying the G2/M phase after 24 h of glutamine deprivation. Glutamine deprivation in the BT-20 cell line resulted in a significant increase in cells occupying G1 phase after 72 h of glutamine deprivation. The MCF-7 cell line demonstrated the least amount of viable cells when analysing apoptosis induction, when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines after glutamine deprivation suggesting that the MCF-7 cell line is the most affected cell line. Analysis of antioxidant mechanism via superoxide dismutase (SOD) inhibition illustrated increased SOD activity in the MCF-7 cell line (9.1%) after 72 h of glutamine deprivation. Evaluation of catalase protein concentration indicated that the MCF-7 catalase expression increased to 1.28 fold after 24 h of glutamine deprivation when compared to cell propagated in complete growth medium. DNA damage was demonstrated by visualising the presence of fluorescent 8-hydroxydeoxyguanosine and showed that the MCF-7 cell line presented with significant 8-hydroxydeoxyguanosine staining. Survival signaling was also evaluated through visualising extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) signaling which demonstrated increased ERK activation in the non-tumourigenic MCF-10A cell line and decreased PI3K activation. This study provides evidence that there are differential- and time-dependent responses in breast tumourigenic cells versus non-tumourigenic cells, to glutamine deprivation thus unraveling the crosstalk between glutamine deprivation, oxidative stress and cell death and different cell types will enable us to better understand the basics of tumour cell metabolism and thus develop therapeutics that provide promising pre-sensitization potential for chemotherapeutic agents. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

Previous studies involving copper (II) complexed with a dissymmetric Schiff base and imidazole derivatives had identified catalase activity of these complexes towards H2O2. Reactions such as this are of great interest due to the important role of copper-based complexes in biological systems. Our research has been conducted to add to the base of knowledge regarding the efforts of other researchers to investigate copper complexes that exhibit similar reactivity as copper-based proteins towards dioxygen. The copper complex chosen for this study contained a tri-dentate Schiff base adduct which, when complexed with an imidazole derivative, limited the manner in which peroxo adducts could bind while providing distinct spectral peaks which were used to conduct kinetic studies. Our results indicate a reaction mechanism by which the role of the complexed copper (II) ion is to activate the peroxo adduct for decomposition through reactions with other peroxide molecules, dioxygen, and water.

The application of Cerium oxide nanoparticles (CNPs) for therapeutic purposes requires a stable dispersion of nanoparticles in biological environment. The objective of this study is to tailor the properties of polyelectrolyte coated CNPs as a function of molecular weight to achieve a stable and catalytic active dispersion. This was achieved by coating CNPs with polyacrylic acid (PAA) which increased the dispersion stability of CNPs and enhanced the catalytic ability. The stability of PAA coating was analysed using the change in the Gibbs free energy computed by Langmuir adsorption model. The adsorption isotherms were determined using soft particle electrokinetics which overcomes the challenges presented by other techniques. The Gibbs free energy was highest for PAA coated CNPs by 250 kg/mole indicating the most stable coating. The free energy for PAA 100 kg/mole coated CNPs is 85% lower than the PAA250 coated CNPs. This significant difference is caused by the strong adsorption of PAA100 on CNPs. Catalytic activity of PAA-CNPs is accessed by the catalase enzymatic activity of nanoparticles. The catalase activity was higher for PAA coated CNPs as compared to bare CNPs which indicated preferential adsorption of hydrogen peroxide induced by coating. Apart from PAA coating the catalase activity is also affected by the structure of the coating layer.

Abiotic stresses like salinity, drought, and extreme temperature are constantly on the rise, posing a very high risk to global agricultural productivity and food security. Hence, understanding stress signaling pathways can help engineer plants that can better withstand stress in unfavorable conditions. The salicylic acid (SA) signaling pathway has been widely studied for its important role in mediating abiotic stress in plants. In tobacco plants, Salicylic Acid Binding Protein 2 (SABP2), a methyl esterase enzyme, catalyzes the conversion of methyl salicylate (MeSA) to SA, which triggers the defense response via the SA-mediated signaling pathway. SIP-428 (SABP2 Interacting Protein-428) is an NAD+ dependent SIR2-like (Silent Information Regulator) deacetylase enzyme that likely interacts with SABP2 during SA biosynthesis. In previous studies, SIP-428 has been shown to be a negative regulator of plant growth under abiotic stress (NaCl and mannitol in vivo). Reactive Oxygen Species (ROS) are oxidizing oxygen products that accumulate under stress conditions, and at high levels can be very harmful to plants. Antioxidant enzymes such as catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD) are actively involved in lowering the ROS levels in the cell by combating the oxidative stress. The objective of this study was to analyze the regulatory functions of SIP-428 in ROS signaling of tobacco plants through the biochemical quantification of POD and CAT activities. We investigated the SIP-428 RNAi-silenced tobacco plants for the POD and CAT enzyme activities in Osmotic (Mannitol) and Salinity (NaCl) stressed plants. Our results showed that SIP-428 plays a significant role in modulating antioxidant enzymes in stressed plants. This study has improved our understanding of some regulatory roles of SIP428, and its application can be used to enhance stress tolerance via the use of synthetic biology.

No description available.