The role of cell death in atherosclerosis remains ill-defined, however, a growing body of evidence suggests that cell death stimulates atherogenesis through the induction of inflammation and enlargement of the necrotic core. Although there is solid evidence to suggest that lipid oxidation and toxicity are linked, indications that protein oxidation may play an important role in cytotoxicity are numerous. The abundance of dead cells in atherosclerotic plaques and their co-localization with HOCl-modified proteins provides an opening for the suggestion that the products of protein oxidation may be at the heart of oxLDL-induced cell death. Examination of the modification of LDL and albumin by HOCl, and the cytotoxicity of these oxidised molecules were the focus of this study, along with the elucidation of their cell death mechanisms toward U937 cells. Measurement of lipid peroxidation markers, TBARS and 7-ketocholesterol, showed no significant increase in HOCl-oxLDL compared to native levels although all α-tocopherol had been lost. In contrast there was a large loss of tyrosine, of which a small percentage went to dityrosine, indicating that the protein moiety of LDL was the main target of HOCl attack. Albumin became fragmented and smeared on SDS-PAGE gels with increasing HOCl/BSA molar ratios. In addition there was significant reduction in tyrosine levels and a small increase in dityrosine. Both HOCl-oxLDL and oxidised albumin (oxALB) caused concentration-dependent cell viability loss in U937 cells following a significant drop in intracellular GSH concentration, coinciding with a peak in oxidative stress. Removal of chloramines with methionine significantly reduced the toxicity of oxALB, but at higher concentrations this effect was reduced. This was in contrast to HOCl-oxLDL where the removal of chloramines had no effect on its toxicity. Morphological observations of cell swelling, cell membrane integrity loss and rupture, along with flow cytometry results indicate that U937 cells underwent necrosis, but only after intracellular GSH was lost. Intracellular GSH and cell viability loss were prevented by 200 μM extracellular 7,8-dihydroneopterin (78NP), indicating that 78NP scavenging of ROS generated in response to the oxidised proteins was sufficient to prevent cell death. This study demonstrates the cytotoxicity of HOCl-damaged LDL and albumin is likely due to a common oxidative product or structural motif which may be active within atherosclerotic plaques.

No description available.

Estudos envolvendo cultura de células, animais e humanos tem sido extensivamente aplicados no monitoramento da aterosclerose, visando seu controle ou mesmo reversão. Entre os modelos animais atualmente utilizados, tem-se observado uma resposta adequada à indução de dislipidemia e inflamação, porém elevada resistência à indução de estresse oxidativo. Uma alternativa para superar essa limitação seria a ingestão de dietas contendo ácidos graxos oxidados. Entretanto, os estudos que investigaram o efeito do consumo de peróxidos e produtos secundários da oxidação lipídica na progressão da aterosclerose têm mostrado resultados inconclusivos. Portanto, o objetivo deste estudo foi de alterar um modelo animal de referência para desenvolvimento de aterosclerose, visando elevar a variação de biomarcadores de estresse oxidativo, através do consumo crônico de ácidos graxos poli-insaturados parcialmente oxidados, em concentrações naturalmente presentes na dieta humana. Camundongos \"knockout\" para receptores LDL (C57BL/6), foram divididos em 4 grupos experimentais. Todos os grupos receberam uma dieta hiperlipídica modificada composta por 30% de lipídios durante 90 dias. Três níveis de oxidação foram induzidos no óleo de linhaça, representando neste estudo um nível baixo, moderado e elevado, caracterizados por uma concentração de hidroperóxidos de 2,47 ± 0,02 (LOW), 3,87 ± 0,04 (MED) e 4,69 ± 0,04 meq LOOH/Kg (HIGH), respectivamente. O grupo que recebeu a ração LOW pode ser considerado o grupo controle negativo, pois o óleo utilizado na formulação de sua ração não sofreu o processo de oxidação. O quarto grupo recebeu a dieta LOW, além da indução de diabetes mellitus do tipo 1 através de estreptozotocina no inicio do estudo sendo esse o grupo controle positivo (CONT+). O óleo de linhaça pré-oxidado utilizado no preparo das rações foi mantido a 4ºC. A peletização das rações alterou a concentração dos marcadores primários e secundários, porém de forma proporcional, mantendo assim a diferença entre os três níveis. O grupo CONT+ apresentou menor ganho de peso que os demais. Nenhuma diferença foi observada entre os grupos em relação ao perfil lipoproteico. Os grupos CONT+ e HIGH apresentaram menor concentração de ácidos graxos no fígado, em especial de ácidos graxos poli-insaturados. Igualmente, ambos os grupos CONT+ e HIGH apresentaram maior concentração de MDA hepático que os grupos MED e LOW, sugerindo um aumento do estresse oxidativo decorrente da ingestão de ácidos graxos poli-insaturados parcialmente oxidados. O grupo HIGH também apresentou um aumento da espessura da parede vascular e do tamanho do lúmen da aorta. Este perfil de resultados sugere que a ingestão crônica de ácidos graxos poli-insaturados parcialmente oxidados contribui para o aumento do estresse oxidativo em camundongos LDLr (-/-), promovendo um aumento na concentração de MDA hepático similar àquele obtido com modelos que utilizam formas mais agressivas de indução. Considerando-se a tendência de aumento no consumo de ácidos graxos poli-insaturados Omega 3, os resultados deste estudo realizados com o óleo de linhaça apontam para a necessidade controle da estabilidade oxidativa desses óleos durante seu processamento e armazenamento, visto que níveis moderados de oxidação já poderiam apresentar potencial aterogênico. / Studies involving cell culture, animals and humans have been extensively applied in the monitoring of atherosclerosis, aiming its control or even this reversal. Among the animal models, it has been observed an appropriate response to the induction of dyslipidemia and inflammation, but a high resistance to oxidative stress induction. An alternative to overcome this limitation would be by the intake of diets containing oxidized fatty acids. However, studies that investigated the effect of consumption of peroxides and secondary products of lipid oxidation in the progression of atherosclerosis have shown controversial results. Therefore, the aim of this study was to alter an animal model for the development of atherosclerosis, aiming to raise the variation in biomarkers of oxidative stress through chronic consumption of polyunsaturated fatty acids partially oxidized under concentrations naturally present in the human diet. LDL receptor \"knockout\" mice (C57BL / 6) were divided into 4 experimental groups. All groups received a modified fat diet containing 30% fat for 90 days. Three levels of oxidation were induced in flaxseed oil, representing low, moderate and high levels, characterized by a hydroperoxide concentration of 2.47 ± 0.02 (LOW), 3.87 ± 0.04 (MED) and 4.69 ± 0.04 LOOHs meq / kg (HIGH), respectively. The fourth group received the LOW diet, beyond diabetes mellitus type 1 through induction by streptozotocin at baseline (CONT +). The pre-oxidized flaxseed oil used to prepare the diets was maintained at 4 °C. The pelletization changed the concentration of primary and secondary products, but proportionally, maintaining the difference between the three levels. CONT + group showed less weight gain than the others. No differences were observed between groups in relation to the lipoprotein profile. The CONT + and HIGH groups showed lower concentration of fatty acids in the liver, especially polyunsaturated fatty acids. In addition both CONT+ and HIGH groups showed higher concentrations MDA in the liver than MED and LOW groups, suggesting increased oxidative stress resulting from the intake of polyunsaturated fatty acids partially oxidized. The HIGH group also showed an increase in the thickness of the vascular wall and the size of the aorta lumen. This profile suggests that the chronic ingestion of partially oxidized polyunsaturated fatty acids contributes to increase oxidative stress in mice LDLr (-/-) , similar to that obtained with models using more aggressive forms of induction. Considering the trend of increased consumption of polyunsaturated fatty acids Omega 3, the results of this study conducted with flaxseed oil highlight to the need of controlling the oxidative stability of these oils during processing and storage, since moderate oxidation levels already present atherogenic potential.

Estudos envolvendo cultura de células, animais e humanos tem sido extensivamente aplicados no monitoramento da aterosclerose, visando seu controle ou mesmo reversão. Entre os modelos animais atualmente utilizados, tem-se observado uma resposta adequada à indução de dislipidemia e inflamação, porém elevada resistência à indução de estresse oxidativo. Uma alternativa para superar essa limitação seria a ingestão de dietas contendo ácidos graxos oxidados. Entretanto, os estudos que investigaram o efeito do consumo de peróxidos e produtos secundários da oxidação lipídica na progressão da aterosclerose têm mostrado resultados inconclusivos. Portanto, o objetivo deste estudo foi de alterar um modelo animal de referência para desenvolvimento de aterosclerose, visando elevar a variação de biomarcadores de estresse oxidativo, através do consumo crônico de ácidos graxos poli-insaturados parcialmente oxidados, em concentrações naturalmente presentes na dieta humana. Camundongos \"knockout\" para receptores LDL (C57BL/6), foram divididos em 4 grupos experimentais. Todos os grupos receberam uma dieta hiperlipídica modificada composta por 30% de lipídios durante 90 dias. Três níveis de oxidação foram induzidos no óleo de linhaça, representando neste estudo um nível baixo, moderado e elevado, caracterizados por uma concentração de hidroperóxidos de 2,47 ± 0,02 (LOW), 3,87 ± 0,04 (MED) e 4,69 ± 0,04 meq LOOH/Kg (HIGH), respectivamente. O grupo que recebeu a ração LOW pode ser considerado o grupo controle negativo, pois o óleo utilizado na formulação de sua ração não sofreu o processo de oxidação. O quarto grupo recebeu a dieta LOW, além da indução de diabetes mellitus do tipo 1 através de estreptozotocina no inicio do estudo sendo esse o grupo controle positivo (CONT+). O óleo de linhaça pré-oxidado utilizado no preparo das rações foi mantido a 4ºC. A peletização das rações alterou a concentração dos marcadores primários e secundários, porém de forma proporcional, mantendo assim a diferença entre os três níveis. O grupo CONT+ apresentou menor ganho de peso que os demais. Nenhuma diferença foi observada entre os grupos em relação ao perfil lipoproteico. Os grupos CONT+ e HIGH apresentaram menor concentração de ácidos graxos no fígado, em especial de ácidos graxos poli-insaturados. Igualmente, ambos os grupos CONT+ e HIGH apresentaram maior concentração de MDA hepático que os grupos MED e LOW, sugerindo um aumento do estresse oxidativo decorrente da ingestão de ácidos graxos poli-insaturados parcialmente oxidados. O grupo HIGH também apresentou um aumento da espessura da parede vascular e do tamanho do lúmen da aorta. Este perfil de resultados sugere que a ingestão crônica de ácidos graxos poli-insaturados parcialmente oxidados contribui para o aumento do estresse oxidativo em camundongos LDLr (-/-), promovendo um aumento na concentração de MDA hepático similar àquele obtido com modelos que utilizam formas mais agressivas de indução. Considerando-se a tendência de aumento no consumo de ácidos graxos poli-insaturados Omega 3, os resultados deste estudo realizados com o óleo de linhaça apontam para a necessidade controle da estabilidade oxidativa desses óleos durante seu processamento e armazenamento, visto que níveis moderados de oxidação já poderiam apresentar potencial aterogênico. / Studies involving cell culture, animals and humans have been extensively applied in the monitoring of atherosclerosis, aiming its control or even this reversal. Among the animal models, it has been observed an appropriate response to the induction of dyslipidemia and inflammation, but a high resistance to oxidative stress induction. An alternative to overcome this limitation would be by the intake of diets containing oxidized fatty acids. However, studies that investigated the effect of consumption of peroxides and secondary products of lipid oxidation in the progression of atherosclerosis have shown controversial results. Therefore, the aim of this study was to alter an animal model for the development of atherosclerosis, aiming to raise the variation in biomarkers of oxidative stress through chronic consumption of polyunsaturated fatty acids partially oxidized under concentrations naturally present in the human diet. LDL receptor \"knockout\" mice (C57BL / 6) were divided into 4 experimental groups. All groups received a modified fat diet containing 30% fat for 90 days. Three levels of oxidation were induced in flaxseed oil, representing low, moderate and high levels, characterized by a hydroperoxide concentration of 2.47 ± 0.02 (LOW), 3.87 ± 0.04 (MED) and 4.69 ± 0.04 LOOHs meq / kg (HIGH), respectively. The fourth group received the LOW diet, beyond diabetes mellitus type 1 through induction by streptozotocin at baseline (CONT +). The pre-oxidized flaxseed oil used to prepare the diets was maintained at 4 °C. The pelletization changed the concentration of primary and secondary products, but proportionally, maintaining the difference between the three levels. CONT + group showed less weight gain than the others. No differences were observed between groups in relation to the lipoprotein profile. The CONT + and HIGH groups showed lower concentration of fatty acids in the liver, especially polyunsaturated fatty acids. In addition both CONT+ and HIGH groups showed higher concentrations MDA in the liver than MED and LOW groups, suggesting increased oxidative stress resulting from the intake of polyunsaturated fatty acids partially oxidized. The HIGH group also showed an increase in the thickness of the vascular wall and the size of the aorta lumen. This profile suggests that the chronic ingestion of partially oxidized polyunsaturated fatty acids contributes to increase oxidative stress in mice LDLr (-/-) , similar to that obtained with models using more aggressive forms of induction. Considering the trend of increased consumption of polyunsaturated fatty acids Omega 3, the results of this study conducted with flaxseed oil highlight to the need of controlling the oxidative stability of these oils during processing and storage, since moderate oxidation levels already present atherogenic potential.

MSc (Eng) dissertation - Faculty of Engineering and the Built Environment / Fertilizers are defined in the broadest sense as products that improve the levels of available plant nutrients or chemical and physical components that directly or indirectly enhance plant growth, yield and quality. The aim of this study was to produce slow controlled release fertilizers from oxidised coal. Two types of coals namely, Waterberg and Twistdraai (products, middlings) were utilised for the production of humic acids through slurry phase oxidation. The highest yields of humic acids were obtained in Waterberg and Twistdraai products samples. Subsequent to that, a nitrogen element was successfully inserted into the humic acid substrate. Humic acids are potential feedstock for modern manufacturing of organic fertilizers. The chemical substances regarded as hazardous to human consumption or those elements that negatively impact on the soil were significantly less detectable.

Introdução: A lipoproteína de baixa densidade (LDL) e suas formas oxidadas (LDLox) possuem múltiplas propriedades aterogênicas, atuando na deposição de colesterol, indução e manutenção da inflamação, disfunção endotelial, surgimento de células espumosas na parede arterial e conseqüente formação da placa de ateroma. Adicionalmente, LDLox induz apoptose de células endoteliais humanas (HMEC). A lipoproteína de alta densidade (HDL) possui inúmeras atividades antiaterogênicas, incluindo ações antioxidante, anti-inflamatória e anti-trombótica. A HDL é capaz de proteger as HMEC contra apoptose. As subfrações de HDL (sHDL) são heterogêneas em sua composição físico-química e atividades biológicas. A atividade antioxidante das sHDL aumenta com a densidade (HDL2b<HDL2a<HDL3a<HDL3b <HDL3c) e está deficiente em pacientes com SMet. Contudo, a heterogeneidade da atividade anti-apoptótica das sHDL é ainda desconhecida. Objetivos: (i) avaliar a heterogeneidade da atividade protetora das sHDL de indivíduos normolipidêmicos (n=7) e de pacientes com SMet (n=16) contra apoptose de HMEC induzida por LDLox; (ii) definir os mecanismos moleculares envolvidos nesta atividade. Métodos: Através de ultracentrifugação por gradiente de densidade, isolamos cinco diferentes sHDL. HMEC foram incubadas com LDLox (200 ?g apoB/ml) na presença ou não das sHDL (5-100 ?g proteína/ml). Os marcadores de toxicidade (MTT) e de apoptose celular (microscopia de fluorescência, marcagem com anexina V, cit c, AIF, degradação Bid, atividade caspase-3 e fragmentação do ADN) foram analisados. Resultados: Todas sHDL protegeram as HMEC contra a toxicidade e apoptose induzidas pela LDLox. Com mesma concentração de proteína, as subfrações HDL3c (60% proteção - MTT - e >100% - anexina V) e 3b (43% e 67%, respectivamente) de indivíduos normolipidêmicos apresentaram atividade anti-apoptótica mais potente do que as subfrações HDL2a (29% e 28%; p<0,01 vs. HDL3c, respectivamente) e 2b (25% e 62%; p<0,001 vs. HDL3c, respectivamente). Todas sHDL reduziram geração de espécies reativas de oxigênio (ROS) induzida pela LDLox, sendo a HDL3c (54%) mais potente do que HDL2b (21%; p<0.05 vs. HDL3c). Houve correlação positiva entre as atividades anti-apoptótica e antioxidante intracelular com conteúdo de apoA-I e esfingosina 1-fosfato (E1F) das sHDL, senda HDL3b e 3c ricas em E1F. A atividade anti-apoptótica da E1F e das sHDL parece depender da interação com as células endoteliais via apoA-I e seu receptor SR-BI. Finalmente, as HDL3c (n=5) isoladas de pacientes com SMet possuem conteúdo significativamente menor de apoA-I e reduzida atividade anti-apoptótica (60%, p<0,01), quando comparada aos controles normolipidêmicos (n=5). Houve tendência à diminuição da proteção contra a geração de ROS (SMet, n=10). Conclusão: As subfrações HDL3c protegem de forma potente as células endoteliais humanas contra toxicidade e apoptose induzidas pela LDLox, assim como contra geração de ROS. Esta atividade antiapoptótica está reduzida na SMet. / Background: Low density lipoprotein (LDL) and its oxidized forms (oxLDL) have several atherogenic properties, including cholesterol deposition, inflammation, endothelial dysfunction and foam cell formation on the arterial wall, leading to atherosclerotic plaque development. In addition, oxLDL induces human endothelial cell apoptosis (HMEC). High-density lipoprotein (HDL) has number of antiatherogenic activities, as antioxidative, anti-inflammatory and anti-thrombotic actions. HDL displays anti-apoptotic activity and is able to protect endothelial cells against oxLDL-induced apoptosis. HDL subfractions (sHDL) are highly heterogeneous in their physical and chemical composition and biological functions. Antioxidative activity of HDL subfractions increases with increment in density, HDL2b<HDL2a<HDL3a<HDL3b <HDL3c. Important, HDL subfractions from subjects with metabolic syndrome (MetS), display a significantly lower antioxidative activity as compared to healthy controls. However, the heterogeneity of their anti-apoptotic activity was not demonstrated. Objectives: (i) to evaluate the heterogeneity of antiapoptotic activity of sHDL from normolipidemic controls (n=7) and MetS patients (n=16) towards oxLDL-induced apoptosis of HMEC; (ii) to define molecular mechanisms involved in this anti-apoptotic action. Methods: Five major sHDL were fractionated by density gradient ultracentrifugation. HMEC were incubated with mildly oxLDL (200 ?g apoB/ml) in the presence or absence of each sHDL (5-100 ?g protein/ml). Markers of cellular toxicity (MTT) and apoptosis (fluorescent nucleic acid staining, annexin V binding, cytochrome c, AIF and Bid, caspase-3 activity and DNA fragmentation) were observed. Results: All HDL subfractions isolated from normolipidemic subjects protected HMEC against oxLDL-induced toxicity and apoptosis. At equal protein concentrations, HDL3c (60% protection in the MTT test; >100% in annexin V biding) and 3b subfractions (43% and 67%, respectively) were more potent against oxLDL-induced toxicity and apoptosis as compared to HDL2a (29% and 28%; p<0.01 vs. HDL3c, respectively) and 2b subfractions (25% and 62%; p<0.001 vs. HDL3c, respectively). All HDL subfractions attenuated of reactive oxygen species (ROS) generation in HMEC induced by oxLDL. Again, HDL3c (54% inhibition) were more potent as compared to HDL2b (21%; p<0.05 vs. HDL3c). The anti-apoptotic and intracellular antioxidative activities of HDL3 were positively correlated with apoA-I and sphingosine 1-phosphate (S1P) content of sHDL and, possibly, depend on their cellular interaction through apoA-I and its SR-BI receptor. The sHDL3c isolated from MetS patients (n=5) possess reduced content of apoA-I and less potent anti-apoptotic activity (-60%, p<0.01) than controls (n=5). Conclusion: Normolipidemic small dense HDL3 provide potent protection of human endothelial cells from oxLDL-induced apoptosis; this anti-apoptotic activity is reduced in the MetS.

Les maladies cardiovasculaires représentent la première cause de mortalité en France avec une prévalence encore plus importante à La Réunion. La plupart des décès sont attribuables aux cardiopathies ischémiques due à la rupture d'une plaque d'athérosclérose et à la formation d'un caillot entrainant une ischémie cardiaque. L'oxydation des lipoprotéines de basse densité (LDL) et la dysfonction de l'endothélium représentent des étapes importantes dans la déstabilisation de la plaque, d'où l’intérêt de rechercher un traitement capable de diminuer l'oxydation des LDL ou de protéger les cellules endothéliales de leurs effets cytotoxiques. Les polyphénols sont des molécules antioxydantes très présentes dans le règne végétal. Des études préliminaires du laboratoire sur des adipocytes humain on montré que des extraits riches en polyphénols d'Antirhea borbonica et de Doratoxylon apetalum, deux espèces respectivement endémique et indigène de l'Île de La Réunion, possédaient des propriétés antioxydantes et anti-inflammatoires (Marimoutou, M et al. 2015). Le But de cette thèse était, dans un premier temps, de tester les capacités antioxydantes et cytoprotectrices d'extraits riches en polyphénols de ces deux espèces sur des cellules endothéliales. Puis dans un second temps nous avons testé l'extrait de Doratoxylon apetalum sur un modèle murin d'athérosclérose (souris ApoE KO) pour évaluer son effet anti-inflammatoire sur les plaques d'athérome. / Cardiovascular diseases are the leading cause of death in France with an even higher prevalence in Reunion. Most deaths are due to ischemic heart disease because of atherosclerotic plaque rupture and thrombus formation leading to cardiac ischemia. The oxidation of low density lipoproteins (LDL) and endothelial dysfunction represent important steps in the destabilization of plaque, hence the interest of a treatment capable of reducing the oxidation of LDL or of protecting endothelial cells from their cytotoxic effects. Polyphenols are antioxidant molecules very present in the plant kingdom. Preliminary studies on human adipocytes have shown that polyphenol-rich extracts of Antirhea borbonica and Doratoxylon apetalum, respectively endemic and indigenous species of Reunion Island, had antioxidant and anti-inflammatory properties (Marimoutou , M et al., 2015). The aim of this thesis was, first, to test the antioxidant and cytoprotective capacities of polyphenol rich extracts of these two species on endothelial cells. Then in a second time we tested the extract of Doratoxylon apetalum on a murine model of atherosclerosis (ApoE KO mice) to evaluate its anti-inflammatory effect on atheroma plaques.

Introdução: A lipoproteína de baixa densidade (LDL) e suas formas oxidadas (LDLox) possuem múltiplas propriedades aterogênicas, atuando na deposição de colesterol, indução e manutenção da inflamação, disfunção endotelial, surgimento de células espumosas na parede arterial e conseqüente formação da placa de ateroma. Adicionalmente, LDLox induz apoptose de células endoteliais humanas (HMEC). A lipoproteína de alta densidade (HDL) possui inúmeras atividades antiaterogênicas, incluindo ações antioxidante, anti-inflamatória e anti-trombótica. A HDL é capaz de proteger as HMEC contra apoptose. As subfrações de HDL (sHDL) são heterogêneas em sua composição físico-química e atividades biológicas. A atividade antioxidante das sHDL aumenta com a densidade (HDL2b<HDL2a<HDL3a<HDL3b <HDL3c) e está deficiente em pacientes com SMet. Contudo, a heterogeneidade da atividade anti-apoptótica das sHDL é ainda desconhecida. Objetivos: (i) avaliar a heterogeneidade da atividade protetora das sHDL de indivíduos normolipidêmicos (n=7) e de pacientes com SMet (n=16) contra apoptose de HMEC induzida por LDLox; (ii) definir os mecanismos moleculares envolvidos nesta atividade. Métodos: Através de ultracentrifugação por gradiente de densidade, isolamos cinco diferentes sHDL. HMEC foram incubadas com LDLox (200 ?g apoB/ml) na presença ou não das sHDL (5-100 ?g proteína/ml). Os marcadores de toxicidade (MTT) e de apoptose celular (microscopia de fluorescência, marcagem com anexina V, cit c, AIF, degradação Bid, atividade caspase-3 e fragmentação do ADN) foram analisados. Resultados: Todas sHDL protegeram as HMEC contra a toxicidade e apoptose induzidas pela LDLox. Com mesma concentração de proteína, as subfrações HDL3c (60% proteção - MTT - e >100% - anexina V) e 3b (43% e 67%, respectivamente) de indivíduos normolipidêmicos apresentaram atividade anti-apoptótica mais potente do que as subfrações HDL2a (29% e 28%; p<0,01 vs. HDL3c, respectivamente) e 2b (25% e 62%; p<0,001 vs. HDL3c, respectivamente). Todas sHDL reduziram geração de espécies reativas de oxigênio (ROS) induzida pela LDLox, sendo a HDL3c (54%) mais potente do que HDL2b (21%; p<0.05 vs. HDL3c). Houve correlação positiva entre as atividades anti-apoptótica e antioxidante intracelular com conteúdo de apoA-I e esfingosina 1-fosfato (E1F) das sHDL, senda HDL3b e 3c ricas em E1F. A atividade anti-apoptótica da E1F e das sHDL parece depender da interação com as células endoteliais via apoA-I e seu receptor SR-BI. Finalmente, as HDL3c (n=5) isoladas de pacientes com SMet possuem conteúdo significativamente menor de apoA-I e reduzida atividade anti-apoptótica (60%, p<0,01), quando comparada aos controles normolipidêmicos (n=5). Houve tendência à diminuição da proteção contra a geração de ROS (SMet, n=10). Conclusão: As subfrações HDL3c protegem de forma potente as células endoteliais humanas contra toxicidade e apoptose induzidas pela LDLox, assim como contra geração de ROS. Esta atividade antiapoptótica está reduzida na SMet. / Background: Low density lipoprotein (LDL) and its oxidized forms (oxLDL) have several atherogenic properties, including cholesterol deposition, inflammation, endothelial dysfunction and foam cell formation on the arterial wall, leading to atherosclerotic plaque development. In addition, oxLDL induces human endothelial cell apoptosis (HMEC). High-density lipoprotein (HDL) has number of antiatherogenic activities, as antioxidative, anti-inflammatory and anti-thrombotic actions. HDL displays anti-apoptotic activity and is able to protect endothelial cells against oxLDL-induced apoptosis. HDL subfractions (sHDL) are highly heterogeneous in their physical and chemical composition and biological functions. Antioxidative activity of HDL subfractions increases with increment in density, HDL2b<HDL2a<HDL3a<HDL3b <HDL3c. Important, HDL subfractions from subjects with metabolic syndrome (MetS), display a significantly lower antioxidative activity as compared to healthy controls. However, the heterogeneity of their anti-apoptotic activity was not demonstrated. Objectives: (i) to evaluate the heterogeneity of antiapoptotic activity of sHDL from normolipidemic controls (n=7) and MetS patients (n=16) towards oxLDL-induced apoptosis of HMEC; (ii) to define molecular mechanisms involved in this anti-apoptotic action. Methods: Five major sHDL were fractionated by density gradient ultracentrifugation. HMEC were incubated with mildly oxLDL (200 ?g apoB/ml) in the presence or absence of each sHDL (5-100 ?g protein/ml). Markers of cellular toxicity (MTT) and apoptosis (fluorescent nucleic acid staining, annexin V binding, cytochrome c, AIF and Bid, caspase-3 activity and DNA fragmentation) were observed. Results: All HDL subfractions isolated from normolipidemic subjects protected HMEC against oxLDL-induced toxicity and apoptosis. At equal protein concentrations, HDL3c (60% protection in the MTT test; >100% in annexin V biding) and 3b subfractions (43% and 67%, respectively) were more potent against oxLDL-induced toxicity and apoptosis as compared to HDL2a (29% and 28%; p<0.01 vs. HDL3c, respectively) and 2b subfractions (25% and 62%; p<0.001 vs. HDL3c, respectively). All HDL subfractions attenuated of reactive oxygen species (ROS) generation in HMEC induced by oxLDL. Again, HDL3c (54% inhibition) were more potent as compared to HDL2b (21%; p<0.05 vs. HDL3c). The anti-apoptotic and intracellular antioxidative activities of HDL3 were positively correlated with apoA-I and sphingosine 1-phosphate (S1P) content of sHDL and, possibly, depend on their cellular interaction through apoA-I and its SR-BI receptor. The sHDL3c isolated from MetS patients (n=5) possess reduced content of apoA-I and less potent anti-apoptotic activity (-60%, p<0.01) than controls (n=5). Conclusion: Normolipidemic small dense HDL3 provide potent protection of human endothelial cells from oxLDL-induced apoptosis; this anti-apoptotic activity is reduced in the MetS.

Thesis (M.Sc. (Chemistry)) -- University of Limpopo, 2015 / The study has focused on the effects of different acids treatments on the nanostructure of MWCNTs; doping metal oxides (copper oxide (CuO), Iron (III) oxide (Fe2O3), nickel oxide (NiO) and cobalt oxide (Co3O4)) on MWCNTs and investigates their electrochemical hydrogen and energy storage capabilities. Fourier transform infrared (FTIR) confirmed the formation of functional groups on the surface of the acid treated MWCNTs. X-ray diffraction (XRD) showed that the graphitic structure of the MWCNTs was retained after treatment with mild acids (nitric acid (HNO3), hydrogen peroxide (H2O2), a mixture of the acids, hydrogen peroxide: nitric acid (H2O2:HNO3) and hydrogen peroxide: sulfuric acid (H2O2:H2SO4)). Transmission electron microscopy (TEM) confirms the removal of bamboo carbon structures inside the inner tubes of the MWCNTs after treatment with mild acids. Brunauer-Emmet- Teller (BET) showed an increase in the surface area of mild acids treated MWCNTs. Thermogravimetric analysis (TGA) results demonstrated that the thermal stability of MWCNTs increases after treatment with mixtures of the acids. Different metal oxides treated at different temperatures were incorporated into MWCNTs (treated by a mixture of H2O2:HNO3). X-ray diffraction (XRD), scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) confirmed the presence of different metal oxides inside/on the surface of the acid treated MWCNTs. The MWCNTs treated by H2O2:HNO3 gave both the highest discharge capacity (72.63 mAh/g) and capacitance (8.61 F/g), as compared to the other electrode materials. The improved hydrogen storage capacity and specific capacitance can be attributed to high surface area, wider pore size distribution and the amount of functional groups on the surface of H2O2:HNO3-treated MWCNTs; with the functional groups acting as electron transmitters. The 5wt.% CuO@300oC-MWCNTs composite showed the highest hydrogen storage capacity of 159 mAh/g. This capacity was further improved by addition of manganese oxide resulting in the highest discharge capacity of 172 mAh/g (which is equivalent to 0.64 wt.% of hydrogen stored). The highest specific capacitance of 9.70 F/g was obtained on 5wt% Fe2O3@400oCMWCNTs composite.

α-Tocopherol has a well known antioxidant action but is also considered likely to exert significant non-antioxidant effects in cell membranes. Due to its lipophilic nature α-tocopherol inserts into biological membranes where it influences the organisation of the component lipids and may therefore influence biophysical parameters including the membrane dipole potential. The dipole potential has been demonstrated to modulate the function of several membrane associated proteins and perturbation of this physical parameter by α-tocopherol may prove to be a significant non-antioxidant mechanism underlying several of its cellular effects. This study investigates the influence of α-tocopherol, and the non-antioxidant structural analogue α-tocopherol succinate, on the membrane dipole potential employing fluorescence spectroscopy techniques with the dipole potential sensitive probe Di-8-ANEPPS. Similar techniques are utilised with the surface potential sensitive probe FPE to investigate the interaction of the charged α-tocopherol succinate molecule with membranes. α-Tocopherol and α-tocopherol succinate are shown to decrease the dipole potential of egg-phosphatidylcholine vesicles and Jurkat T-lymphocyte cell membranes. This effect is placed in the context of the significant influence of membrane cholesterol oxidation on the dipole potential. 7-ketocholesterol, an oxidised form of cholesterol, significantly influences several cellular processes and is thought to mediate these effects, in part, through its physical effects on the cell membrane. These include altering the composition, and therefore biophysical properties, of rafts; structures which are considered to support the function of a host of membrane proteins. This study attempts to correlate the effect of 7-ketocholesterol on the dipole potential of microdomains with the influence of the oxysterol on the function of two microdomains associated receptors: P-glycoprotein and the insulin receptor, assessed by determining the extent of ligand binding using flow fluorocytometry. α-Tocopherol has been suggested to inhibit the raft-mediated effects of 7-ketocholesterol and the influence of this molecule on the effect of 7-ketocholesterol on the dipole potential are investigated as a potential mechanism for this inhibition. It is hypothesized that α-tocopherols may protect against the deleterious effects of cholesterol oxidation in cell membranes by excluding 7-ketocholesterol from specific microdomains, of which rafts are a subset, acting to preserve their dipole potential and maintain the function of the proteins they support. However, where significant cholesterol oxidation has previously occured the concurrent changes in the microdomain landscape of the membrane is suggested to prevent α-tocopherol succinate from eliciting this protective effect.