Haemophilus influenzae encounters niches within the human host that are predicted to differ in availability of oxygen and reactive nitrogen species (RNS: nitrite and nitric oxide), which influence the environmental redox state. Previously reported data has indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. To elucidate the role of redox signaling in virulence, we examined regulation by the FNR homolog of H. influenzae, whose counterpart in E. coli has been reported to be a direct oxygen sensor and a regulator of genes responsible for RNS metabolism and resistance. Many members of the FNR regulon are subject to coordinated transcriptional control by NarP, a regulator in E. coli that is activated by cognate sensor NarQ in response to environmental nitrite. To study the regulatory activities of FNR and NarQ-NarP in H. influenzae, I targeted a gene predicted to be FNR-regulated, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration. The fnr, narP and nrfA mutants were assayed for nitrite reduction, which implicated the roles of FNR, NarP and NrfA in RNS metabolism. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrf promoter (Pnrf-HA), I demonstrate that FNR and NarP, but not NarQ, are required for full activation of the nrf promoter. Additionally, Pnrf-HA expression increases as oxygen becomes depleted and decreases when exposed to high concentrations of nitrite, implying that the nrfpromoter is modulated by environmental redox signals. FNR of E. coli has been implicated in regulation of resistance mechanisms to a reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection as a host defense mechanism. A mutant lacking FNR is more sensitive to NO exposure and killing by activated macrophages than wild type H. influenzae after anaerobic pre-growth. Mutants of nrfA and narP have been tested and initial experiments have shown both mutants have a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors that might be involved to this phenotype, we discovered FNR-regulated gene, ytfE, which contributes to defense against nitrosative stress. The fnr and ytfE mutants are more susceptible to killing by activated macrophages indicating that FNR regulation of ytfE might be important for in vivo infection.

Orientador: Adriana Zerlotti Mercadante / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T21:40:04Z (GMT). No. of bitstreams: 1 Chiste_RenanCampos_D.pdf: 8233764 bytes, checksum: 20989b604d256eba3bcdcfda79445208 (MD5) Previous issue date: 2011 / Resumo: Os compostos bioativos presentes no reino vegetal possuem importantes funções e ações biológicas, podendo ser considerados promotores da saúde humana. Já é reconhecida a associação entre a ingestão de frutas e vegetais e a diminuição do risco de desenvolvimento de diversas desordens crônico-degenerativas, tais como câncer, inflamações, doenças cardiovasculares, catarata, degeneração macular e outras, sendo os carotenóides e compostos fenólicos alguns dos grupos de compostos bioativos aos quais são atribuídas tais ações. As sementes e os extratos de urucum (Bixa orellana L.) são utilizados como corantes nas indústrias alimentícias, farmacêutica e de cosméticos, devido à presença majoritária do carotenóide bixina. Até então, não havia sido relatada na literatura a composição de compostos fenólicos das sementes de urucum. Dessa forma, foi desenvolvido e validado um método por cromatografia líquida de alta eficiência acoplada aos detectores de arranjo de diodos e espectrômetro de massas (HPLC-DAD-MS/MS) para separar, identificar e quantificar bixina e os compostos fenólicos em semente de urucum. Adicionalmente, foi otimizado um procedimento de extração simultânea desses compostos através da metodologia de superfície de resposta. Além de bixina, conhecida por ser o principal carotenóide em sementes de urucum, a hipolaetina e um derivado de ácido caféico foram identificados, pela primeira vez, como os principais compostos fenólicos. O procedimento otimizado envolveu 15 extrações com acetona:metanol:água (50:40:10, v/v/v) como solvente, razão sólido-líquido de 1:9 (m/v) e 5 min para cada extração em ultrassom. O método cromatográfico proposto foi validado com sucesso para a análise simultânea de compostos fenólicos e bixina em sementes de urucum. Extratos líquidos de urucum com elevada capacidade antioxidante e potencial de cor foram obtidos a partir da extração de bixina e de compostos fenólicos de sementes de urucum utilizando solventes com diferentes polaridades (água, etanol:água, etanol, etanol:acetato de etila e acetato de etila). Os valores mais elevados de compostos fenólicos totais foram encontrados nos extratos obtidos com água, etanol:água e etanol (0,5 mg equivalentes de ácido gálico/mL), e o valor mais elevado de bixina foi encontrado no extrato obtido com etanol:acetato de etila (5,2 mg/mL), que foi caracterizado como o mais vermelho e o mais vívido (a* = 40,5, h°=46,1, C* = 58,4). O extrato obtido com etanol:acetato de etila também apresentou a maior atividade anti-radical livre (4,7 umol equivalente Trolox/mL) e a maior porcentagem de proteção ao triptofano contra o oxigênio singlete (63,6 %). Por outro lado, acetato de etila e a mistura etanol:água foram os solventes menos eficazes para a extração de compostos fenólicos e bixina, respectivamente. De acordo com a análise estatística multivariada, etanol:acetato de etila e acetato de etila foram os solventes mais promissores para obtenção de extratos de urucum com ambas as propriedades antioxidantes e de cor. A partir da extração com diferentes solventes (água, etanol:água, etanol, etanol:acetato de etila e acetato de etila), foram também obtidos extratos liofilizados de urucum, e a capacidade antioxidante na desativação de diferentes espécies reativas de oxigênio (ROS) e de nitrogênio (RNS) foi avaliada. Além disso, os teores de compostos fenólicos e de bixina dos extratos de urucum foram determinados por HPLC-DAD. Todos os extratos de urucum foram capazes de desativar todas as espécies reativas testadas (peróxido de hidrogênio, ácido hipocloroso, oxigênio singlete, radical óxido nítrico, ânion peroxinitrito e radical peroxila), em baixas concentrações na faixa de ?g/mL, com exceção do radical superóxido. O extratos de sementes de urucum obtidos com etanol:acetato de etila e acetato de etila, que apresentaram os maiores níveis de hipolaetina e bixina, respectivamente, foram os extratos com a maior capacidade antioxidante. Adicionalmente, o padrão de bixina apresentou os menores valores de IC50 na desativação de todas as ROS e RNS testadas. O piquiá (Caryocar villosum (Aubl.) Pers), fruta nativa da região Amazônica, pode ser considerado uma fonte inexplorada de compostos bioativos, uma vez que poucos estudos sobre seus constituintes químicos e fitoquímicos estão disponíveis. Dessa forma, a composição química e fitoquímica da polpa de piquiá foi determinada, incluindo a composição de carotenóides e de compostos fenólicos por HPLC-DAD-MS/MS. De acordo com a composição nutricional, água (52 %) e lipídios (25 %) foram os principais componentes encontrados na polpa e o valor energético total foi 291 kcal/100 g. Sobre os compostos bioativos, a polpa apresentou (base seca) maior teor de compostos fenólicos (236 mg equivalentes de ácido gálico/100 g), flavonóides totais (67 mg equivalentes de catequina/100 g) e taninos totais (60 mg equivalentes de ácido tânico/100 g) em relação ao teor de carotenóides totais (7 mg/100 g) e alfa-tocoferol (1 mg/100 g). Os principais compostos fenólicos identificados por HPLC-DAD-MS/MS, foram ácido gálico (182 ug/g polpa), seguido por ácido elágico ramnosídeo (107 ug/g polpa) e ácido elágico (104 ug/g polpa). Os principais carotenóides identificados foram all-trans-anteraxantina (3 ug/g), all-trans-zeaxantina (3 ug/g), all-trans-neoxantina (2 ug/g), all-trans-violaxantina (1 ug/g) e all-trans-?-caroteno (0,7 ug/g). A capacidade anti-radical livre da polpa (3,7 mmol equivalente Trolox/100 g) indica que a polpa pode ser considerada um eficiente sequestrador do radical peroxila. Foram obtidos também extratos liofilizados de polpa de piquiá a partir da extração com diferentes solventes (água, etanol:água, etanol, etanol:acetato de etila e acetato de etila). Todos os extratos naturais foram caracterizados em relação ao teor de compostos bioativos (compostos fenólicos totais, flavonóides, taninos, carotenóides e tocoferóis). Além disso, a capacidade de desativação do radical peroxila, assim como a porcentagem de proteção contra o oxigênio singlete foi determinada para todos os extratos. Os extratos obtidos com água e a mistura etanol:água apresentaram os maiores teores (base seca) de compostos fenólicos totais (9,2 e 6,3 mg de equivalentes de ácido gálico/g, respectivamente), flavonóides totais (3,8 e 2,5 mg equivalente de catequina/g, respectivamente) e taninos totais (7,6 e 2,4 mg de ácido tânico/g, respectivamente). O extrato obtido com etanol:água também apresentou a maior capacidade de desativação do radical peroxila (ORAC) (0,3 mmol equivalente Trolox/g extrato). Por outro lado, o extrato obtido com etanol, que foi classificado como o de cor mais vívida e amarelo (C*ab = 13,7 e b* = 13,3), apresentou o maior teor de carotenóides totais (0,1 mg/g) e maior percentual de proteção contra o oxigênio singlete (10,6 %). Com base nos resultados deste estudo, etanol:água, água e etanol são os solventes mais promissores para obtenção de extratos de piquiá com alto teor de compostos bioativos, proteção contra o oxigênio singlete e capacidade sequestradora do radical peroxila. Portanto, tais informações são importantes para as indústrias alimentícia, cosmética e farmacêutica, sabendo que o piquiá e o urucum são fontes naturais acessíveis de compostos bioativos para serem usados como potencial matéria-prima para obtenção de extratos contra os danos oxidativos em alimentos ou sistemas biológicos / Abstract: The bioactive compounds found in the plant kingdom have important biological functions and actions and may be considered human health promoters. The association between the intake of fruits and vegetables and the decreased risk of developing several chronic-degenerative disorders is already recognized, being the carotenoids and phenolic compounds, groups of the bioactive compounds responsible for such actions. The annatto (Bixa orellana L.) seeds and extracts are used as colourant in the food, pharmaceutical and cosmetic industries, due to the presence of bixin. As far as we are concerned, no information about the composition of phenolic compounds present in the annatto seeds and extracts was reported. Thus, a method by high performance liquid chromatography coupled to diode array detector and mass spectrometer (HPLC-DAD-MS/MS) was developed and validated for separation, identification and quantification of phenolic compounds and bixin in annatto seeds. Furthermore, using response surface methodology, an optimized procedure for simultaneous extraction of both compound classes was established. In addition to bixin, known to be the main carotenoid in annatto seeds, hypolaetin and a caffeoyl acid derivative were identified as the main phenolic compounds. The optimized procedure involved 15 extractions using acetone:methanol:water (50:40:10, v/v/v) as solvent, a solid¿liquid ratio of 1:9 (w/v) and an extraction time of 5 min per extraction in a sonicator. Validation data indicated that the developed HPLC method is suitable for the simultaneous analysis of phenolic compounds and bixin in annatto seeds. Liquid annatto extracts with high antioxidant capacity and colour potential were obtained from annatto seeds by extraction of phenolic compounds and bixin using solvents with different polarities (water, ethanol:water, ethanol, ethanol:ethyl acetate and ethyl acetate). The highest levels of total phenolic compounds were found in the water, ethanol:water and ethanol extracts (0.5 mg gallic acid equivalent/mL), and the highest level of bixin was found in the ethanol:ethyl acetate extract (5.2 mg/mL), which was characterised as the reddest and the most vivid one (a* = 40.5, h° = 46.1, C* = 58.4). The ethanol:ethyl acetate extract also showed the highest antioxidant activity (4.7 umol Trolox equivalent/mL) and the highest percentage of tryptophan protection against singlet oxygen (63.6 %). On the other hand, ethyl acetate and ethanol:water were the least effective solvents for the extraction of phenolic compounds and bixin, respectively. According to the multivariate statistical analysis, ethanol:ethyl acetate and ethyl acetate were the most promising solvents to obtain annatto extracts with both antioxidant and colour properties. From the extraction with different solvents (water, ethanol:water, ethanol, ethanol:ethyl acetate and ethyl acetate), freeze-dried annatto extracts were also obtained, and the capacities to scavenge reactive oxygen (ROS) and reactive nitrogen (RNS) species were evaluated. In addition, the levels of phenolic compounds and bixin in the annatto extracts were determined by HPLC-DAD. All annatto extracts were able to scavenge all the tested reactive species (hydrogen peroxide, hypochlorous acid, singlet oxygen, nitric oxide radical, peroxynitrite anion and peroxyl radical) at low concentration in the ug/mL range, with the exception of superoxide radical. The ethanol:ethyl acetate and ethyl acetate extracts of annatto seeds, which presented the highest levels of hypolaetin and bixin, respectively, were the extracts with the highest antioxidant capacity, and the bixin standard presented the lowest IC50 values. Piquia (Caryocar villosum (Aubl.) Pers), a native fruit from Amazon region, can be considered an unexploited source of bioactive compounds, since few studies about chemical and phytochemical constituents are available. Thus, the chemical and phytochemical composition of piquia pulp was determinaed, including the composition of carotenoids and phenolic compounds by HPLC-DAD-MS/MS. According to the nutritional composition, water (52 %) and lipids (25 %) were the major components found in the pulp and the total energetic value was 291 Kcal/100 g. Regarding the bioactive compounds, the pulp presented (dry basis) higher content of phenolic compounds (236 mg gallic acid equivalent/100 g), total flavonoids (67 mg catechin equivalent/100 g) and total tannins (60 mg tannic acid equivalent/100 g) than total carotenoids (7 mg/100 g) and alfa-tochopherol (1 mg/100 g). The major phenolic compounds identified by HPLC-DAD-MS/MS were gallic acid (182 ug/g pulp), followed by ellagic acid rhamnoside (107 ug/g pulp) and ellagic acid (104 ug/g pulp). The main carotenoids identified were all-trans-antheraxanthin (3 ug/g), all-trans-zeaxanthin (3 ug/g), all-trans-neoxanthin (2 ug/g), all-trans-violaxanthin (1 ug/g) and all-trans-beta-carotene (0.7 ug/g). The antioxidant capacity of the pulp (3.7 mmol Trolox/100 g) indicates that the pulp can be considered a good peroxyl radical scavenger. Freeze-dried extracts of piquia using five solvents with different polarities (water, ethanol:water, ethanol, ethanol:ethyl acetate and ethyl acetate) were also obtained. All natural extracts were characterised in relation to the contents of bioactive compounds (total phenolic compounds, flavonoids, tannins, carotenoids and tocopherols). In addition, the scavenging capacity of all piquia extracts against peroxyl radical, as well as the quenching capacity against singlet oxygen were determined. All the data were used for classification of the piquia extracts applying multivariate statistical analysis. The water and ethanol:water extracts presented the highest levels of total phenolic compounds (9.2 and 6.3 mg gallic acid equivalent/g, respectively), total flavonoids (3.8 and 2.5 mg catechin equivalent/g, respectively) and total tannins (7.6 and 2.4 mg tannic acid/g, respectively). The ethanol:water extract also showed the highest scavenging capacity against peroxyl radical (ORAC) (0.3 mmol Trolox equivalent/g extract). On the other hand, the ethanol extract, which was classified as the most vivid and yellow one (C*ab = 13.7 and b* = 13.3), presented the highest level of total carotenoids (0.1 mg/g) and highest percentage of protection against singlet oxygen (10.6 %). Based on the results of this study, ethanol:water mixture, water and ethanol are the most promising solvents to obtain piquia extracts with high contents of bioactive compounds, protection against singlet oxygen and peroxyl radical scavenging capacity. Therefore, such informations are important for the food, cosmetic and pharmaceutical industries, since piquia and annatto are natural sources of bioactive compounds available for use as a potential raw material to obtain extracts against oxidative damage in foods or biological systems / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

The adenylate cyclase toxin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates CR3-expressing phagocytes and catalyzes the uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by oxidative burst and opsonophagocytic mechanisms. Here we show that CyaA suppresses the production of bactericidal reactive oxygen and nitrogen species in neutrophils and macrophages, respectively. The inhibition of reactive oxygen species (ROS) production is most-likely achieved by the combined PKA-dependent inhibition of PLC and Epac-dependent dysregulation of NADPH oxidase assembly. Activation of PKA or Epac interfered with fMLP-induced ROS production and the inhibition of PKA partially reversed the CyaA-mediated inhibition of ROS production. CyaA/cAMP signaling then inhibited DAG formation, while the PIP3 formation was not influenced. These results suggest that cAMP produced by CyaA influences the composition of target membranes. We further show here that cAMP signaling through the PKA pathway activates the tyrosine phosphatase SHP-1 and suppresses the production of reactive nitrogen species (RNS) in macrophages. Selective activation of PKA interfered with LPS- induced iNOS expression...

Excess nitrogen (N) from terrestrial landscapes poses environmental challenges as it moves via surface runoff and groundwater flows into aquatic ecosystems. Managing and anticipating the environmental challenges associated with these altered N inputs from terrestrial to aquatic ecosystems requires a deep understanding of how N is biogeochemically transformed, retained, and/or transported in streams and rivers. Here, I used long-term data on surface stream and groundwater chemistry as well as discharge to determine the main sources of N and estimate the N mass balance of a 1.4 km boreal stream reach. The goal was to evaluate daily net uptake or production rates of different N forms (ammonium - NH4-N, nitrate NO3-N and dissolved organic N - DON) throughout the seasons, and assess physical and chemical factors that may drive changes in net processing. The mass balances analysis revealed distinct patterns in net uptake among N forms. Notably, there was clear evidence of NH4-N and DON uptake (removal) in the stream, while NO3-N processing patterns showed neither clear uptake nor production. Further, variation in net uptake for NH4-N and DON was positively related to stream DOC, DOC:DIN, and C:N ratios, indicating that carbon rich conditions promoted greater N demand in this ecosystem. By comparison, variations in net NO3-N uptake or production at the reach scale were only weakly correlated with these carbon rich conditions. Finally, I assessed these patterns within the nutrient processing domains (NPDs) framework to characterize the behavior/character of the study reach. Accordingly, during the open water season, the stream reach acted mostly as a consumer for both NH4-N and DON, while on many dates it acted as a weak enhancer for NO3-N. These findings contribute to the broader understanding of N dynamics in boreal stream ecosystems and emphasize the complex interplay among organic and inorganic N forms, carbon dynamics, and nutrient processing in these environments. This knowledge is crucial for effective environmental management and conservation efforts in the region.

No description available.

Extraintestinal pathogenic Escherichia coli (ExPEC) reside in the enteric tract as a commensal reservoir, but can transition to a pathogenic state by invading normally sterile niches, establishing infection and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. Here, we report that FimX, an ExPEC-associated DNA invertase that regulates the major virulence factor type 1 pili (T1P), is also an epigenetic regulator of a LuxR-like response regulator HyxR. FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the type 1 pili promoter and independent of integration host factor (IHF). In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNIs), primarily through de-repression of hmpA, encoding a nitric oxide-detoxifying flavohaemoglobin. However, in the macrophage, HyxR produced large effects on intracellular survival in the presence and absence of RNI and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. / Dissertation

Sepse, a resposta do organismo a uma infecção, está associada a altas taxas de mortalidade. A razão pela qual um mecanismo protetor resulta num quadro clínico fatal permanece inexplicada. Em trabalho prévio nosso grupo demonstrou que exossomos de origem plaquetária são os mais freqüentes em plasma de pacientes com choque séptico e que estes podem induzir apoptose em células musculares lisas vasculares e células endoteliais em cultura. Demonstramos ainda que tais exossomos possuíam uma fonte enzimática de ROS, uma NADPH oxidase cuja atividade poderia estar associada à indução da apoptose (Janiszewski et al., 2004). No presente trabalho, nós buscamos criar um modelo de geração ex vivo de exossomos similares aos encontrados em pacientes sépticos e identificar possíveis vias responsáveis pela liberação destes e seus efeitos. Choque séptico é uma condição relacionada com exposição a lipopolissacarídeo (LPS) e geração de alta quantidade de trombina, TNF e espécies reativas de nitrogênio. Através de citometria de fluxo revelamos que plaquetas humanas expostas ao doador de NO dietilamina-NONOato e ao LPS geraram exossomos similares àqueles encontrados em pacientes com choque séptico, expondo alta quantidade de tetraspaninas CD9, CD63 e CD81 mas pouca fosfatidilserina. Por outro lado, plaquetas expostas à trombina ou TNF liberaram partículas com características claramente distintas, com alta exposição de fosfatidilserina e baixa de tetraspaninas. Assim como os exossomos sépticos, os exossomos obtidos pela exposição de NO e LPS geraram radical superóxido e NO, como demonstrado pela quimioluminescência da lucigenina (5M) e celenterazinina (5M) e pela fluorescência da 4,5-diaminofluoresceína (10mM) e 2,7-diclorofluoresceína (10mM). A análise por Western Blot nos permitiu identificar as subunidades Nox1, Nox2 e p22phox da NADPH oxidase e a isoforma induzível da enzima NO sintase (NOS) nesses exossomos. Como esperado, inibidores da NOS e da NADPH oxidase reduziram significamente os sinais fluorescentes e quimioluminescentes. Em adição, as células endoteliais em cultura expostas aos exossomos gerados por dietilamina-NONOato e LPS sofreram significativo aumento da taxa de apoptose quando comparadas àquelas expostas a exossomos controle. A inibição da NADPH oxidase assim como da NOS reduziu expressivamente tal efeito. Adição de urato (1mM), mostrou efeito aditivo sobre a inibição do sinal fluorescente, assim como redução adicional da taxa apoptótica, sugerindo papel importante do radical peroxinitrito. Nós propomos, assim, que exossomos derivados de plaquetas podem representar papel adicional no já complexo cenário da sinalização vascular redox. Nesse sentido, uma abordagem baseada em exossomos pode fornecer novas ferramentas para o entendimento e até tratamento da disfunção vascular na sepse / Sepsis, the bodys response to infection, is associated with high mortality rates. Why a protective mechanism turns into a deadly clinical picture is a matter of debate, and goes largely unexplained. In previous work we demonstrated that plateled derived exosomes are found in the plasma of septic patients with septic shock and can induce endothelial and vascular smooth muscle cell apoptosis in culture through an enzymatic superoxide source (Janiszewski et al., 2004). In this work we sought to create a model for ex vivo generation of exosomes, and to identify the pathways responsible for ROS release by exosomes and their effects. Septic shock is a condition related to exposure of lipopolysaccharide (LPS), generation of high amounts of thrombin, TNF and nitrogen reactive species. Through flow cytometry we demonstrated that human platelets exposed to the NO-donor diethylamine-NONOate, and to LPS, generated exosomes similar to those found in the blood of septic shock patients, with high exposure of the tetraspanin CD9, CD63, and CD81, but little phosphatidylserine. On the other hand, platelets exposed to thrombin or TNF released particles with clearly distinct characteristics, such as high phosphatidylserine and low tetraspanin. Like the septic exosomes, the exosomes obtained by NO and LPS exposure generated superoxide radical and NO, as disclosed by lucigenin and coelenterazine chemiluminescence and by 4,5-diaminofluorescein and 2,7-dichlorofluorescein fluorescence. Western Blot analysis revealed the presence of Nox1, Nox2 and p22phox NADPH oxidase subunits and the inducible isoform of NO synthase (NOS) in these exosomes. As expected, NOS inhibitors or NADPH oxidase inhibitors significantly reduced the fluorescence and chemiluminescente signals. In addition, endothelial cells exposed to NO or LPS generated exosomes underwent apoptotic death, while control exosomes had no effects on apoptosis. NADPH oxidase as well as NOS inhibition significantly reduced apoptosis rates. Concomitant generation of NO and superoxide suggests biological effects of the highly reactive radical peroxynitrite. In fact, the peroxynitrite scavenger urate (1 mM) showed an additive effect on fluorescent signal inhibition, as well as on endothelial apoptosis rate reduction. We thus propose that platelet-derived exosomes may be another class of actors in the complex play known as vascular redox signaling. In this sense, an exosome-based approach can provide novel tools for further understanding and even treating vascular dysfunction related to sepsis

Sepse, a resposta do organismo a uma infecção, está associada a altas taxas de mortalidade. A razão pela qual um mecanismo protetor resulta num quadro clínico fatal permanece inexplicada. Em trabalho prévio nosso grupo demonstrou que exossomos de origem plaquetária são os mais freqüentes em plasma de pacientes com choque séptico e que estes podem induzir apoptose em células musculares lisas vasculares e células endoteliais em cultura. Demonstramos ainda que tais exossomos possuíam uma fonte enzimática de ROS, uma NADPH oxidase cuja atividade poderia estar associada à indução da apoptose (Janiszewski et al., 2004). No presente trabalho, nós buscamos criar um modelo de geração ex vivo de exossomos similares aos encontrados em pacientes sépticos e identificar possíveis vias responsáveis pela liberação destes e seus efeitos. Choque séptico é uma condição relacionada com exposição a lipopolissacarídeo (LPS) e geração de alta quantidade de trombina, TNF e espécies reativas de nitrogênio. Através de citometria de fluxo revelamos que plaquetas humanas expostas ao doador de NO dietilamina-NONOato e ao LPS geraram exossomos similares àqueles encontrados em pacientes com choque séptico, expondo alta quantidade de tetraspaninas CD9, CD63 e CD81 mas pouca fosfatidilserina. Por outro lado, plaquetas expostas à trombina ou TNF liberaram partículas com características claramente distintas, com alta exposição de fosfatidilserina e baixa de tetraspaninas. Assim como os exossomos sépticos, os exossomos obtidos pela exposição de NO e LPS geraram radical superóxido e NO, como demonstrado pela quimioluminescência da lucigenina (5M) e celenterazinina (5M) e pela fluorescência da 4,5-diaminofluoresceína (10mM) e 2,7-diclorofluoresceína (10mM). A análise por Western Blot nos permitiu identificar as subunidades Nox1, Nox2 e p22phox da NADPH oxidase e a isoforma induzível da enzima NO sintase (NOS) nesses exossomos. Como esperado, inibidores da NOS e da NADPH oxidase reduziram significamente os sinais fluorescentes e quimioluminescentes. Em adição, as células endoteliais em cultura expostas aos exossomos gerados por dietilamina-NONOato e LPS sofreram significativo aumento da taxa de apoptose quando comparadas àquelas expostas a exossomos controle. A inibição da NADPH oxidase assim como da NOS reduziu expressivamente tal efeito. Adição de urato (1mM), mostrou efeito aditivo sobre a inibição do sinal fluorescente, assim como redução adicional da taxa apoptótica, sugerindo papel importante do radical peroxinitrito. Nós propomos, assim, que exossomos derivados de plaquetas podem representar papel adicional no já complexo cenário da sinalização vascular redox. Nesse sentido, uma abordagem baseada em exossomos pode fornecer novas ferramentas para o entendimento e até tratamento da disfunção vascular na sepse / Sepsis, the bodys response to infection, is associated with high mortality rates. Why a protective mechanism turns into a deadly clinical picture is a matter of debate, and goes largely unexplained. In previous work we demonstrated that plateled derived exosomes are found in the plasma of septic patients with septic shock and can induce endothelial and vascular smooth muscle cell apoptosis in culture through an enzymatic superoxide source (Janiszewski et al., 2004). In this work we sought to create a model for ex vivo generation of exosomes, and to identify the pathways responsible for ROS release by exosomes and their effects. Septic shock is a condition related to exposure of lipopolysaccharide (LPS), generation of high amounts of thrombin, TNF and nitrogen reactive species. Through flow cytometry we demonstrated that human platelets exposed to the NO-donor diethylamine-NONOate, and to LPS, generated exosomes similar to those found in the blood of septic shock patients, with high exposure of the tetraspanin CD9, CD63, and CD81, but little phosphatidylserine. On the other hand, platelets exposed to thrombin or TNF released particles with clearly distinct characteristics, such as high phosphatidylserine and low tetraspanin. Like the septic exosomes, the exosomes obtained by NO and LPS exposure generated superoxide radical and NO, as disclosed by lucigenin and coelenterazine chemiluminescence and by 4,5-diaminofluorescein and 2,7-dichlorofluorescein fluorescence. Western Blot analysis revealed the presence of Nox1, Nox2 and p22phox NADPH oxidase subunits and the inducible isoform of NO synthase (NOS) in these exosomes. As expected, NOS inhibitors or NADPH oxidase inhibitors significantly reduced the fluorescence and chemiluminescente signals. In addition, endothelial cells exposed to NO or LPS generated exosomes underwent apoptotic death, while control exosomes had no effects on apoptosis. NADPH oxidase as well as NOS inhibition significantly reduced apoptosis rates. Concomitant generation of NO and superoxide suggests biological effects of the highly reactive radical peroxynitrite. In fact, the peroxynitrite scavenger urate (1 mM) showed an additive effect on fluorescent signal inhibition, as well as on endothelial apoptosis rate reduction. We thus propose that platelet-derived exosomes may be another class of actors in the complex play known as vascular redox signaling. In this sense, an exosome-based approach can provide novel tools for further understanding and even treating vascular dysfunction related to sepsis

In biological systems, several metabolic and environmental factors are responsible for the production of reactive oxygen (ROS) and nitrogen (RNS) species. The exacerbated production of these reactive species or the significant decrease in the effectiveness of the defenses against them causes the redox imbalance, with consequent damage to biological macromolecules, which is associated to the emergence and progression of several diseases. Among the exogenous antioxidants, phenolic compounds of plants have been highlighted by their ability to scavenge various reactive species. Among the most studied plants, Curcuma longa has many beneficial properties for health, which are mainly associated with the phenolic compounds present in the rhizome, known as curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin), but the benefits are more particularly attributed to curcumin. Thus, the objective of this study was to investigate the antioxidant capacity of C. longa extracts and to determine their contents in curcuminoids, investigating the antioxidant action of the extract and of each isolated curcuminoid against ROS and RNS. The identification and quantification of curcuminoids, the total phenols content analysis and the antioxidant capacity in terms of scavenging of the 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH•) and the FRAP method (ferric reducing antioxidant power) were carried out for methanol (CM), defatted methanol (CHM), ethanol (EC) and hexane (CH) extracts, besides commercial extract (ExtFarC) of C. longa. The elimination capacity of ROS and RNS was performed for CE and isolated curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin). The CM, CHM and CE had shown the three curcuminoids in their compositions, while, for the ExtFarC, curcumin was the predominant compound. The CE and ExtFarC had shown the higher total phenols content and antioxidant activity by the FRAP method, besides the lower IC50 values for to the DPPH• radical. The use of high performance liquid chromatography (HPLC) with spectrophotometric detection (DPPH•-HPLC-UV) indicated that curcumin and demethoxycurcumin had the greater potential for capture of DPPH•, as observed by the reduction of their peaks in HPLC, at equivalent contact time. In the experiments related to hypochlorous acid (HOCl), nitric oxide (•NO) and peroxynitrite (ONOO‾), the curcuminoids had shown a direct efficiency toward their elimination, similarly to the positive control (quercetin). For CE, despite showing higher IC50 values against these species, in comparison to the isolated compounds, it presented lower IC50 values, when compared to other plants’ extracts, studied by the same methods. Although curcumin is the target of many therapeutic studies for a number of diseases, the present data show that the three curcuminoids can play essential roles against ROS and RNS, and thus, may be considered promising in the prevention and treatment of diseases related to oxidative stress. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em sistemas biológicos, diversos fatores metabólicos e ambientais são responsáveis pela produção de espécies reativas de oxigênio (EROs) e de nitrogênio (ERNs). Quando a produção é exacerbada ou quando há uma diminuição significativa na eficácia das defesas contra essas espécies, ocorre o desequilíbrio redox, causando danos a macromoléculas biológicas, o que está associado ao surgimento e progressão de várias doenças. Dentre os antioxidantes exógenos, os compostos fenólicos de plantas têm se destacado pela capacidade de sequestrar diversas espécies reativas. Das plantas mais estudadas, a Curcuma longa apresenta inúmeras propriedades benéficas para a saúde, que são principalmente associadas aos compostos fenólicos presentes no rizoma, conhecidos como curcuminoides (curcumina, desmetoxicurcumina e bisdesmetoxicurcumina), porém os benefícios são mais particularmente atribuídos à curcumina. Dessa forma, o objetivo do estudo foi avaliar a capacidade antioxidante de extratos de C. longa e determinar seus curcuminoides constituintes, investigando a ação antioxidante do extrato e de curcuminoides isolados contra as EROs e ERNs. A identificação e quantificação de curcuminoides, o conteúdo total de fenóis e a capacidade antioxidante, em termos de sequestro do radical 2,2-difenil-1-picrilhidrazila (DPPH•) e pelo método de FRAP (do inglês, ferric reducing antioxidant power), foram realizados para os extratos metanólico (CM), metanólico desengordurado (CHM), etanólico (CE), hexânico (CH) e para o extrato comercial (ExtFarC) de C. longa. A capacidade de eliminação das EROs e ERNs foi realizada para o CE e para os curcuminoides (curcumina, desmetoxicurcumina e bisdesmetoxicurcumina). Os extratos CM, CHM e CE apresentaram, em suas composições, os três curcuminoides, enquanto o ExtFarC apresentou a curcumina como componente majoritário. O extrato etanólico (CE) e o ExtFarC apresentaram maior conteúdo total de fenóis e atividade antioxidante pelo método de FRAP e menores valores de IC50 frente ao radical DPPH•. Cromatografia líquida de alta eficiência (CLAE), com detecção espectrofotométrica (DPPH•-CLAE-UV) indicou que a curcumina e a desmetoxicurcumina apresentaram maior potencial de captura de DPPH•, observado pela redução de seus picos em CLAE, em tempos de contato equivalentes. Nos experimentos de capacidade de eliminação das EROs e ERNs, os curcuminoides apresentaram atividade semelhante ao controle positivo (quercetina) frente ao ácido hipocloroso (HOCl), óxido nítrico (•NO) e peroxinitrito (ONOO‾), mostrando-se eficientes de forma direta contra essas espécies. O extrato CE, apesar de exibir maiores valores de IC50 para essas espécies, quando comparado aos compostos isolados, apresentou valores de IC50 inferiores, em comparação com extratos de outras plantas estudados pelos mesmos métodos. Apesar de a curcumina ser o alvo de estudos com finalidade terapêutica para inúmeras doenças, os dados evidenciam que os três curcuminoides têm papel potencial contra as EROs e ERNs, desta forma são promissores na prevenção e tratamento de doenças relacionadas ao estresse oxidativo.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Oxidative stress has been linked to some neurodegenerative disorders. Current therapies are limited to attenuate the symptoms, however some studies have shown that antioxidant compounds may be able to prevent or delay neuronal oxidative damage, including those present in plant extracts. For these reasons, this study investigated the possible antioxidant activity of the ethanolic extract of Luehea divaricata leaves in brain of rats in vitro and the possible antioxidant mechanisms involved. The extract was evaluated against basal and sodium nitroprusside (SNP) 5 μM induced lipid peroxidation in brain of rats through measurements of thiobarbituric acid reactive substances (TBARS) production. Slices of brain areas were treated with SNP 100 M and extract to determine cellular viability by MTT reduction assay. Scavenger activity was evaluated against NO, DPPH and OH through Griess reagent, DPPH and deoxyribose oxidation assays, respectively. The chelating and reducing capacity for iron were determined by the orto-phenantroline method. The extract was screened by HPLC for the presence of gallic, chlorogenic, and caffeic acids, quercetin, rutin, and kaempferol. Only rutin was detected and then was used, in the same concentrations of the extract, as standard in all assays. L. divaricata extract (1-10 μg/ml) protected against induced lipid peroxidation, decreased basal levels of TBARS (about 50%) and maintained the cells viable. The extract was not able to protect deoxyribose against OH and to chelate iron, however it inhibited NO and DPPH in 33.14% at 20 μg/ml and 53.93% at 50 μg/ml respectively, and showed a reducing power in a concentration and time dependent manner. Therefore, L. divaricata ethanolic leaf extract showed antioxidant properties in vitro at low concentrations. The antioxidant mechanisms were related to scavenger activity on reactive oxygen and nitrogen species and metal reducing property. These effects were similar or more powerful than rutin in same concentrations in all assays, except in NO scavenger activity, and, thus, other unidentified compounds present in the extract appear to be associated with the effects observed in this study. More studies are needed regarding the identification of these compounds and the neuroprotective activity of the extract. / O estresse oxidativo tem sido relacionado a algumas desordens neurodegenerativas. As terapias atuais para essas doenças estão limitadas a atenuar os sintomas apresentados, entretanto alguns estudos têm mostrado que compostos antioxidantes podem ser capazes de prevenir ou retardar o dano oxidativo neuronal, incluindo aqueles presentes em extratos vegetais. Por esses motivos, neste trabalho foi investigada a possível atividade antioxidante do extrato etanólico das folhas de Luehea divaricata em cérebro de ratos in vitro e os possíveis mecanismos antioxidantes envolvidos. O extrato foi avaliado contra a peroxidação lipídica basal e induzida por nitroprussiato de sódio (NPS) 5 μM em cérebro de ratos através da quantificação da produção de espécies reativas ao ácido tiobarbitúrico (TBARS). Fatias de áreas do cérebro foram tratadas com NPS 100 M e extrato para determinar a viabilidade celular através do ensaio da redução do MTT. A atividade scavenger do extrato foi testada contra ON, DPPH e OH através do reagente de Griess e dos ensaios de DPPH e de oxidação da desoxirribose, respectivamente. A capacidade quelante e redutora frente ao ferro foram determinadas através do método da orto-fenantrolina. O extrato foi analisado por HPLC quanto à presença dos ácidos gálico, clorogênico e cafeico, quercetina, rutina e kaempferol. Somente a rutina foi detectada e então usada como padrão, nas mesmas concentrações do extrato, em todos os testes. O extrato de L. divaricata (1-10 μg/ml) protegeu o cérebro contra peroxidação lipídica induzida, diminuiu os níveis basais de TBARS (± 50%) e manteve as células viáveis. O extrato não foi capaz de proteger a desoxirribose contra OH e de quelar o ferro, entretanto inibiu ON e DPPH em 33,14% a 20 μg/ml e 53,93% a 50 μg/ml respectivamente, e mostrou poder redutor de maneira concentração e tempo dependentes. Assim, o extrato etanólico das folhas de L. divaricata demonstrou propriedade antioxidante in vitro em baixas concentrações. Os mecanismos antioxidantes foram relacionados ao efeito scavenger de espécies reativas de oxigênio e de nitrogênio e à atividade redutora de metais. Esses efeitos foram similares ou superiores em comparação às mesmas concentrações de rutina, exceto na capacidade scavenger de ON e, portanto, outros compostos ainda não determinados no extrato parecem estar associados às atividades observadas neste trabalho. Mais estudos devem ser realizados em relação à identificação desses compostos e à atividade neuroprotetora do extrato.