Os extratos obtidos a partir da própolis vermelha brasileira (PVB) têm sido investigados devido às suas amplas atividades biológicas. Recentemente, em nosso grupo de pesquisa, demonstramos a viabilidade da incorporação de um extrato n-hexânico de PVB em nanoemulsões de uso tópico, bem como sua permeação/retenção em pele de orelha suína. No presente estudo, avaliamos as propriedades físico-químicas e reológicas de hidrogéis contendo essas nanoemulsões visando a obtenção de um produto semissólido adequado para aplicação tópica. Em uma primeira fase, foram preparadas nanoemulsões compostas de núcleo oleoso contendo extrato n–hexânico de PVB, miristato de isopropila, lecitina de ovo (NE) e DOTAP (NE/DT), e fase externa aquosa. O polímero gelificante hidroxietilcelulose foi incorporado às formulações após a sua obtenção por emulsificação espontânea (H-NE e H-NE/DT). As formulações apresentaram-se monodispersas com diâmetro médio na faixa de 200-300 nm, confirmado por microscopia eletrônica de transmissão. H-NE apresentou um potencial zeta negativo (-38mV), enquanto o mesmo parâmetro para H-NE/DT foi positivo (+36mV), devido à presença do lipídeo catiônico DOTAP na formulação. O teor de benzofenonas totais, determinado por cromatografia líquida de alta eficiência (CLAE), foi de cerca de 85 mg/g de extrato. Esses parâmetros mantiveram-se constantes durante 90 dias de armazenamento a 4C. As formulações H/NE e H-NE/DT apresentaram um comportamento não-Newtoniano pseudoplástico. Estudos de permeação/retenção das benzofenonas através da pele de orelha suína foram realizados utilizando células de difusão do tipo Franz. A maior retenção das benzofenonas na pele (18,11 μg/cm² após 8h) foi observada para a formulação H-NE/DT, demonstrando o efeito do lipídeo catiônico DOTAP nesse parâmetro. Em uma última etapa, investigou-se a capacidade das formulações de conferirem proteção à pele de orelha suína frente ao dano oxidativo gerado pela sua exposição à luz UVA/UVB. A proteção da pele de orelha suína foi evidenciada pelas técnicas de TBARS, carbonilação de proteínas e grupamentos tióis totais. Os resultados obtidos sugerem que os Hidrogéis contendo extrato de PVB apresentam propriedades físico-químicas e reológicas adequadas para serem utilizadas topicamente para a prevenção do dano oxidativo causado pela exposição à luz UVA/UVB. / Brazilian red propolis (BRP) extracts have been investigated due to their extensive biological activities. Recently, in our research group, we demonstrated the feasibility of incorporating an n-hexane extract of BRP into topical nanoemulsions, as well as the permeation/retention of these compounds in porcine ear skin. In the present study, we evaluated the physicochemical and rheological properties of hydrogels containing these nanoemulsions in order to obtain a semi-solid product suitable for topical application. In a first step, nanoemulsions composed of an oil nucleus containing BRP n-hexane extract, isopropyl myristate, egg lecithin (NE) and DOTAP (NE/DT), and an aqueous external phase were prepared. The hydroxyethylcellulose gelling polymer was incorporated into the formulations after being obtained by spontaneous emulsification (H-NE and H-NE/DT). The formulations were monodisperse exhibiting a mean diameter in the 200-300 nm range, confirmed by transmission electron microscopy. H-NE presented a negative zeta potential (-38mV), while H-NE/DT showed a positive value (+ 36mV) due to the presence of the cationic lipid DOTAP in the formulation. The total benzophenone content, determined by high performance liquid chromatography (HPLC), was about 85 mg/g extract. These parameters were maintained constant for 90 days of storage at 4°C. The formulations H/NE and H-NE/DT presented a non-Newtonian pseudoplastic behavior. Permeation/retention studies of benzophenones through porcine ear skin were performed using Franz type diffusion cells. The highest retention of benzophenones in the skin (18.11 μg/cm² after 8h) was observed for the H-NE/DT formulation, demonstrating the effect of the DOTAP cationic lipid on this parameter. In a last step, the ability of the formulations to confer protection of porcine ear skin against oxidative damage, generated by its exposure to UVA/UVB light, was investigated. The protection was evidenced by the TBARS, carbonylation of proteins, and total thiol groups techniques. The results obtained suggest that the hydrogels containing BRP have adequate physicochemical and rheological properties to be used topically for the prevention of oxidative damage caused by exposure to UVA/UVB light.

Uma parcela significativa das lesões na molécula do DNA é causada por espécies reativas de oxigênio e a sua produção excessiva e/ou o funcionamento deficiente dos sistemas celulares antioxidantes, que neutralizam a sua ação, é conhecido como estresse oxidativo. Os danos em células normais são prontamente detectados por um sistema de defesa e, em conseqüência, uma rede intrínseca de sinalizações é ativada, sendo que uma das vias resulta na ativação dos mecanismos de reparo do DNA. O reparo por excisão de bases (BER) parece ser a via preferencial de reparo de bases oxidadas, mas existem outras vias de reparo implicadas na reversão do dano oxidativo. A doença de Alzheimer (DA), uma patologia causada particularmente por danos oxidativos, acomete atualmente cerca de 25 milhões de pessoas no mundo, sendo o risco aumentado a partir dos 65 anos de idade. Com isso, a necessidade da identificação de fatores de risco, além de fatores protetores relacionados à DA, tornou-se de grande importância. Por outro lado, há também a necessidade de estudos em nível molecular, que possam fornecer informações sobre os mecanismos que levam ao desenvolvimento da doença. Nesse sentido, foi realizado no presente trabalho, um estudo de expressão gênica transcricional pelo método de microarranjos de DNA, bem como uma análise por PCR em tempo real para uma série de genes envolvidos na resposta ao dano oxidativo no DNA (percepção de danos e reparo do dano), além de outros genes relacionados à doença. Adicionalmente, foram também avaliadas as quebras na fita dupla de DNA causadas por bases oxidadas, em linfócitos de pacientes de Alzheimer (grau moderado) e indivíduos sadios, usando-se métodos de detecção de bases oxidadas (8-oxoGuanina). Entre os vinte genes analisados pelo método de PCR quantitativa em tempo real, apenas a APOE mostrou-se induzida, enquanto 19 genes (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) foram reprimidos transcricionalmente. Essa repressão verificada para a maior parte dos genes estudados indica que várias vias de sinalização celular ligadas a respostas ao estresse oxidativo, incluindo-se as várias vias de reparo do DNA, podem estar envolvidas na condição DA. Adicionalmente, a análise de expressão gênica por microarranjos de cDNA indicou uma série de 41 genes significativamente modulados (q < 0,06) (dentre eles, NOTCH1, MARK3, PAK, SMC1L1) mas para a maioria destes não há relatos na literatura sobre uma possível relação com DA. Por essa razão, o método de microarranjos de cDNA aponta novas vias que possam estar alteradas em DA, o que constitui uma informação importante. Em conjunto, os dados obtidos no presente estudo fornecem uma contribuição relevante, que futuramente poderão contribuir em termos de intervenção terapêutica. / A great amount of DNA molecule lesions is caused by reactive oxygen species and its synthesis in excess and/or misfunctioning of antioxidant cell systems, which neutralize its effects, is known as oxidative stress. Damage in normal cells is readily detected by a defence system and as consequence, a complex signaling pathway is activated, among them DNA repair mechanisms. The base excision repair (BER) seems to be the primary repair pathway in base oxidative damages, however there are other pathways that are involved in their repair. The Alzheimers disease (AD), a pathology caused particularly by oxidative damages, hits 25 million people worldwide, and its prevalence increases every 5 years beyond age 65. Therefore, there is an emerging need of finding risk factors, as well as protective factors related do AD. By the other hand, it is also necessary molecular studies, which could provide precious information about the mechanisms which lead to the disease development. In the present work, it was made a study about transcriptional gene expression by cDNA microarray, as well as Real Time PCR analysis in a series of genes involved in oxidative DNA damage response (sensing and damage repair), and others associated with the disease. In addition, it were also evaluated DNA strand breaks induced by oxidized bases in lymphocytes from Alzheimers patients (moderate level) and healthy individuals, by oxidized bases (8- oxoguanine) detection methods. Among the twenty genes tested by the quantitative Real Time PCR assay, only APOE was induced, as the remaining 19 (ADAM17, APEX1, APP, BACE1, OGG1 ATM, ATR, TREX1, FEN1, FANCG, RAD17, DUSP, ERCC1, ERCC3, ERCC6, HUS1, RAD9, RAD1, PRKDC) were found repressed. This observed inhibition in most of genes studied shows that many cell signaling pathways associated to oxidative stress response, including DNA repair pathways, may be also involved in the AD pathology. Additionally, the gene expression analysis by cDNA microarrays showed transcriptional alterations in 41 genes (q < 0.06) (among them, NOTCH1, MARK3, PAK and SMC1L1), but for most of them, there are no reports in the literature about their possible relationship with AD, what brought us new important information. Together, all the data obtained in the preset study provide a relevant contribution, which, in the future, may help on new therapeutic designs.

Stroke is serious neurological disease and is a major cause of death as well as disability throughout the globe. Stroke has a complex pathophysiology that involves inflammatory pathways, excitotoxicity mechanisms, oxidative damage, apoptosis, ionic imbalances, angiogenesis and neuroprotection. 85% of strokes are ischemic and occurs when a cerebral vessel, or any vessel supplying the brain, narrows or loses pressure resulting in subsequent brain ischemia and infarction downstream to the site of obstruction depriving tissues of vital oxygen and nutrients. This may be caused by either atherosclerotic thrombi or distant emboli defined as a mass of clotted blood or other material. It is estimated that over a billion people currently smoke cigarettes or use other tobacco products, seeing as smoking is a major risk factor for stroke this is of major concern. Platelets are hematopoietic cells produced by bone marrow megakaryocytes. Platelets play a role in the development of ischemic stroke primarily by means of their participation in the formation of thromboemboli, the presence of abnormal platelet function may predispose patients to a pro-thrombotic, pro-inflammatory state. The reorganization of the cytoskeleton in platelets is an important factor in the complex mechanisms found in thrombosis and haemostasis. The platelet membrane contains a large number of receptors which specifically bind agonists that stimulate the physiological platelet response. Oxidative stress is one of the mechanisms involved in the neuronal damage of stroke. Oxidative stress is a state of imbalance between free radical production, in particular, reactive oxygen species (ROS), and the ability of the organism to neutralize them, leading to progressive oxidative damage. Smoking is known to result in the generation of various free radicals. Flow cytometric analysis of the platelets of thrombo-embolic ischemic stroke patients and smokers revealed that the membranes of the two groups were altered in some form as well as an increased activation in both groups when compared to healthy individuals. Superoxide levels in the platelets were higher in smokers when compared to stroke patients, while hydrogen peroxide levels were elevated in the platelets of both groups. Superoxide was elevated in the whole blood samples of both groups. The production and subsequent reactions of reactive oxygen species appear to be influential in stroke and smoking and may likely be a crucial factor in the development of a pro-thrombotic, pro-inflammatory state which may prove to be a hallmark in the pathophysiology of stroke and smoking. Confocal microscopy and Scanning electron microscopy showed that platelets of stroke patients and smokers appear to be more activated and more prone to form tight clots. Furthermore an increased amount of superoxide is present in the platelets of stroke patients and smokers, specifically in the centre of clots. This may be an indication that once platelets have aggregated and started to fuse together, the mitochondria are expelled from the platelets and “trapped” within the clot. Atomic force microscopy also indicated both the stroke patients and smoker‟s platelets appear to be in a more activated state than the control group. Here it is apparent that some form of cytoskeletal rearrangement takes place to a more severe extent in the stroke group than in the smokers. Necrosis may be present in the platelets of stroke patients while neither apoptosis nor necrosis can be identified in the platelets of smokers however some form of membrane alteration is likely present. All the techniques used showed an increase in platelet activation in stroke patients and smokers, necrotic platelets may be present in the stroke patients while the platelet membrane of smokers seems to be altered. ROS is present and alters the platelet function of smokers and stroke patients in some way. It appears as if thrombo-embolic ischemic stroke patients and smokers‟ platelets have similar trends in activation but the processes involved to achieve this differ as there are structural differences present. These differences may prove a useful tool to further understand the pathophysiology behind thrombo-embolic ischemic stroke as well as to discover new therapeutic pathways. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Physiology / Unrestricted

Background: Oxidative damage is one of the most frequent types of cell components damage leading to oxidation of lipids, proteins and the molecule of DNA. As a consequence, there is a higher occurrence of several pathologies such as atherosclerosis, neurodegenerative diseases, cancer; or diabetes. In our study, influence of whole body anesthesia during minor surgery on the level of DNA damage was examined using comet assay technique. Methods: The basic principle of this method is fixing the cells (lymphocytes) in agarose, their lysis for the removal of membranes, incubation with the specific enzymes and electrophoresis of the released cell nuclei. During the electrophoresis, free low-molecular weight and negatively charged fragments of DNA move towards anode which causes the formation of the typical comet cell shape. Finally, the gels are stained by ethidium bromide (DNA intercalating dye) and visualized. Results: We have observed single strand breakages (SSBs) and, with the use of modified assay using specific enzymes for detection of specific lesions, also oxidized purines and pyrimidines. The extent of DNA damage as determined by the intensity of the tail of the comet was quantified using LUCIA Comet Assay (Laboratory Imaging, Czech Republic) software for image analysis. The results were used...

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO4•− were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.

The carbonate radical anion CO3•- is a potent reactive oxygen species (ROS) produced in vivo through enzymatic one-electron oxidation of bicarbonate or, mostly, via the reaction of CO2 with peroxynitrite. Due to the vitally essential role of the carbon dioxide/bicarbonate buffer system in regulation of physiological pH, CO3•- is arguably one of the most important ROS in biological systems. So far, the studies of reactions of CO3•- with DNA have been focused on the pathways initiated by oxidation of guanines in DNA. In this study, low-molecular products of attack of CO3•- on the sugar-phosphate backbone in vitro were analyzed by reversed phase HPLC. The selectivity of damage in double-stranded DNA (dsDNA) was found to follow the same pattern C4′ > C1′ > C5′ for both CO3•- and the hydroxyl radical, though the relative contribution of the C1′ damage induced by CO3•- is substantially higher. In single-stranded DNA (ssDNA) oxidation at C1′ by CO3•- prevails over all other sugar damages. An approximately 2000-fold preference for 8-oxoguanine (8oxoG) formation over sugar damage found in our study identifies CO3•- primarily as a one-electron oxidant with fairly low reactivity toward the sugar-phosphate backbone.

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