Orientador: Mário Roberto Maróstica Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T19:41:58Z (GMT). No. of bitstreams: 1 Batista_AngelaGiovana_M.pdf: 1042643 bytes, checksum: 8a7d597d0c0012f15fd49f04953138c7 (MD5) Previous issue date: 2013 / Resumo: O sobrepeso e a obesidade têm sido apontados como problemas de saúde pública e estão associados com o surgimento de doenças crônicas. O estresse oxidativo desencadeado pela ingestão de dietas hiperlipídicas estimula o desenvolvimento da síndrome metabólica, a qual é acompanhada de doenças cardiovasculares, dislipidemias, diabetes, resistência à insulina e outros. A casca de jabuticaba (Myrciaria jaboticaba (Vell.) Berg.) é rica em antocianinas, compostos fenólicos antioxidantes extensivamente referenciados pela literatura científica devido ao seu potencial de combater o estresse oxidativo in vivo. A proposta deste trabalho foi verificar a presença de biomarcadores do estresse oxidativo e perfil lipídico de ratos Sprague-Dawley alimentados com dieta hiperlipídica adicionada de casca de jabuticaba liofilizada (CJL). Trinta ratos foram distribuídos em 5 grupos de 6 animais: controle normal (N) ¿ dieta normal AIN-93G; controle hiperlipídico (C) ¿ dieta com 35% de lipídios (31% banha suína e 4% óleo de soja); jabuticaba 1 (J1), jabuticaba 2 (J2) e jabuticaba 4 (J4) ¿ dieta hiperlipídica (C) adicionada de 1, 2 e 4% de CJL, respectivamente. Os animais dos grupos J1, J2 e J4 foram alimentados com dieta C nos primeiros 30 dias e depois receberam as dietas com CJL, completando 70 dias de experimento. Os animais foram mortos por decapitação, e exsanguinados para obtenção do soro, plasma e órgãos para liofilização. As análises da capacidade antioxidante total do plasma e órgãos, enzimas envolvidas no estresse oxidativo em órgãos, além de lipídeos séricos, hepáticos e fecais, foram analisados para verificação do efeito da adição de CJL às dietas. No segundo período do experimento (últimos 40 dias), a ingestão dietética dos animais J4 foi maior em relação ao grupo C. O grupo J2 mostrou os maiores níveis de lipídios hepáticos e fecais, e o grupo J4 a maior média nos níveis de colesterol hepático e fecal. Não houve diferenças significativas para triglicerídeos hepáticos, e os grupos J1 e J4 excretaram mais triglicerídeos via fezes em relação a C. Os parâmetros de avaliação antioxidante no plasma foram aumentados nos grupos alimentados com J2 e J4. Todas as dietas contendo CJL impediram a peroxidação lipídica hepática induzida pela dieta hiperlipídica, bem como aumentaram sua capacidade antioxidante. No cérebro foi percebida uma resposta dependente da dose de CJL: os valores de TBARS diminuíram com o aumento do teor de antocianinas na dieta. Os rins dos animais alimentados com J2 e J4 mostraram uma melhora do seu status antioxidante de acordo com análises de ORAC e GSH. Os grupos J1 e J4 apresentaram os maiores valores de TEAC no baço. Assim, a inclusão na dieta CJL poderia reforçar as defesas antioxidantes sistêmicas,além de promover maior eficiência na excreção de triglicerídeos. Portanto, o produto da casca de jabuticaba pode ser uma alternativa natural para minimizar os danos do estresse oxidativo induzido pela obesidade / Abstract: Overweight and obesity have been identified as public health problems and are associated with chronic diseases. High-fat diet-induced oxidative stress stimulates the development of metabolic syndrome, which is accompanied by cardiovascular diseases,dyslipidemia, diabetes, insulin resistance and others. The peel of jaboticaba (Myrciaria jaboticaba (Vell.) Berg.) is rich in anthocyanins, phenolic antioxidants, which are referenced in the scientific literature because of its in vivo antioxidant potential. The aim of this study was to determine the presence of biomarkers of oxidative stress and to evaluate the lipid profile of rats fed high-fat diet with freeze-dried jaboticaba peel (FJP) added. Thirty rats were divided into 5 groups of 6 animals each: normal control (N) - normal diet AIN-93G; high-fat control (C) - a diet with 35% fat (31% lard and 4% soybean oil); jaboticaba 1 (J1), jaboticaba 2 (J2), and jaboticaba 4 (J4) - fat diet (C) with 1, 2 and 4% CJL added, respectively. The animals of J1, J2 and J4 groups received C diet during the first 30 days and after, they received the FJP diets, completing 70 days. The animals were killed by decapitation and exsanguinated to obtain the serum, plasma and organs for lyophilization. The total antioxidant capacity, oxidative stress-related enzyme activities of the tissues, and serum, liver and feces lipids were analyzed as indicators of FJP diets in vivo effects. In the second period of the experiment (last 40 days), food intake of J4 animals was higher than in C group. The J2 group showed higher levels of hepatic and fecal lipids and the animals from J4 group showed the highest hepatic and fecal cholesterol levels. There were no significant differences in hepatic triglycerides, but J1 and J4 groups excreted more triglycerides relative to C group. The antioxidant parameters evaluated in plasma were increased in the groups fed the J2 and J4 diets. All FJP diets prevented hepatic oxidative stress. Brain exhibited a dependent dose response as lipid peroxidation decreased with increasing of FJP content in the diet. The kidneys of J2 and J4-fed animals showed an improvement in their antioxidant status according to ORAC and GSH assays. The J1 and J4 groups showed the highest TEAC values in the spleen. Thus, the inclusion of FJP in the diet could enhance systemic antioxidant defenses, and promote greater efficiency in the excretion of triglycerides. This product could be a natural alternative to minimize the damage of obesity-induced oxidative stress / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestra em Alimentos e Nutrição

Orientador: Heloísa Helena de Araújo Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T01:09:24Z (GMT). No. of bitstreams: 1 Campos_Daiana_M.pdf: 5833117 bytes, checksum: 9ef22e0c9d416945c3eddacc3a3bb437 (MD5) Previous issue date: 2012 / Resumo: Diversos estudos mostram que o sulfeto de hidrogênio (H2S) exerce importante papel em diferentes aspectos da fisiologia e da patologia pulmonar. O objetivo deste trabalho foi investigar o efeito do tratamento com um doador de H2S no estresse oxidativo produzido pela inflamação alérgica pulmonar. Camundongos BALB/c foram sensibilizados com ovalbumina (OVA) e tratados com NaHS 30 minutos antes de cada desafio com OVA. Os animais sofreram eutanásia em 24h- 144h após o desafio com OVA, quando foi coletado o lavado bronchoalveolar, para contagem total e diferencial de eosinófilos, e retirados os pulmões. Os pulmões foram homogeneizados para análise da atividade das enzimas aconitase (que possui relação inversa com a produção de O2o- intracelular), fumarase, superóxido dismutase (SOD), catalase, glutationa peroxidase (GPx), glutationa redutase (GR) e para a verificação do conteúdo de substâncias reativas ao ácido tiobarbitúrico (TBARS) por método colorimétrico. Os níveis de glutationa reduzida (GSH) e glutationa oxidada (GSSG) foram determinados por cromatografia. Os resultados dos camundongos não tratados (controle) mostraram que o aumento do conteúdo de eosinófilos para os pulmões ocorreu como uma resposta bifásica entre 48h/96h e 144h após o desafio com OVA. Nestes horários, observamos no grupo controle redução na atividade da aconitase, que foi acompanhada do aumento da peroxidação lipídica (TBARS). A atividade da enzima SOO estava aumentada nestes animais a partir de 24h, seguida de uma redução em 144h, enquanto que as atividades das enzimas catalase e GR estavam aumentadas somente em 48h. Não foram observadas modificações na atividade da GPx, assim como nos níveis de GSH e GSSG, em nenhum dos períodos estudados. O tratamento dos camundongos alérgicos com NaHS reduziu o infiltrado de eosinófilos em 48h e 144h após o desafio antigênico, recuperou a atividade da aconitase e reduziu a peroxidação lipídica, quando comparado aos controles. Nestes horários, o tratamento com o NaHS também aumentou as atividades das enzimas SOD, GPx e GR e os níveis de GSH. Diferente destes resultados verificou-se uma redução da atividade da catalase em 48h, quando comparado ao grupo controle. Em conclusão, nossos resultados sugerem que o H2S exerce efeito benéfico na inflamação alérgica pulmonar ao inibir o recrutamento de eosinófilos para os pulmões. Do mesmo modo, produz um importante efeito no estresse oxidativo ao reduzir a peroxidação lipídica e a produção de O2o- intracelular (determinado pela atividade da aconitase) e ao aumentar a atividade da SOD. As atividades das enzimas GPx e GR também estavam aumentadas contribuindo para o aumento de GSH. Deste modo, o NaHS (e provavelmente outros doadores de H2S) podem apresentar potencialmente importante uso terapêutico para o tratamento de doenças pulmonares caracterizadas pela presença de células inflamatórias e desbalanceamento do equilíbrio oxidante/antioxidante / Abstract: Several studies show that the hydrogen sulfide (H2S) plays an important role in different aspects of lung physiology and pathology. The aim of this study was to investigate a H2S-donor treatment on the oxidative stress produced by pulmonary allergic inflammation. BALB/c mice were sensitized with ovalbumin (OVA) and treated with NaHS, 30 minutes before each OVA-challenge. The animals were sacrificed at 24h-144h after challenge, when bronchoalveolar lavage was collected for total and differential eosinophils counts and the lungs were collected. The lungs were homogenized for aconitase (that has inverse correlation with intracelular O2o- production), fumarase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR) enzymes analysis and thiobarbituric acid reactive substances (TBARS) measurement by colorimetric methods. The determination of reduced glutathione (GSH) and glutathione disulfide (GSSG) levels were done by chromatography. The results from untreated (control) mice showed that an increase of eosinophil influx to the lungs happened as a biphasic response between 48h/96h and at 144h after OVA-challenge. At the same hours we observed in this group a reduction in aconitase levels that were accompanied by lipid peroxidation (TBARS) increase. The SOD activity was increased in control animals at 24h followed by reduction at 144h, whereas catalase and GR activities were increased only at 48h. There were no changes in GPx activity, neither in GSH nor in GSSG levels at any time- studied. NaHS-treatment reduced eosinophils infiltration at 48h and 144h after antigen challenge when it was observed a recovery in the aconitase activity and a decrease in TBARS lung contents as compared to control mice. It was also observed in these times after antigen challenge increases in SOD, GPx as well as GR activities and in GSH levels. Differing from these results, NaHS-treatment reduced catalase activity at 48h, as compared to control group. In conclusion, our results suggest that NaHS exerts beneficial effect on allergic pulmonary inflammation by inhibiting the eosinophils recruitment to the lungs of mice after OVA-challenge. It also produces a significant effect on oxidative stress by reducing lipid peroxidation and intracelular O2o- production (measured by aconitase activities) as well as by increasing SOD activity. The GPx and GR activities were also improved, and that could account for GSH increase. Therefore, NaHS (and probably others H2S donors) may emerge as potentially useful therapeutic tools for treatment of lung diseases characterized by the presence of inflammatory cells and oxidant/antioxidant imbalance / Mestrado / Farmacologia / Mestra em Farmacologia

Lipid peroxidation is well established to contribute to the etiology of many deteriorative conditions including neurodegeneration, cardiovascular disease, cancer, aging, and recently in ferroptosis—a regulated, necrotic modality of cell death that results from the accumulation of lipid hydroperoxides. Recent high-throughput screening efforts have uncovered ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as two premiere inhibitors of ferroptosis. We propose that these compounds function as radical trapping antioxidants. We employ a systematic methodology of evaluating inherent radical trapping antioxidant (RTA) activity of Lip-1, Fer-1, and various aryl amine and aryl nitroxide analogues to put forward a biologically relevant mechanism of action based on our previous work in the field. Joining these observations with the efficacy of tetrahydronaphthyridinols (THNs), the results support a clear role of autoxidation in the execution of ferroptosis. Next, we expand the THN repertoire by targeting the payload towards the engine of our cells, the mitochondria. Decades of research have implicated mitochondrial dysfunction brought about by the peroxidation of mitochondrial membranes and the leaking of downstream oxidants, in the death of their symbiotic host cells. Isolated successes in the field have been demonstrated academically, though viable drugs remain to be developed, partially due to the lack of effective diagnostic tools. We endeavor to address some of these issues by investigating mitochondrially-targeted THNs (MitoTHNs) as a targeted chain-breaking antioxidant of unparalleled potency. Furthermore, we advance development of the THNs towards therapeutic applications by demonstrating their biodistribution in mice.

Cyanobacteria are notorious for their potential to produce toxins with human health effects, particularly the hepatotoxic microcystins (MCs), but cyanobacteria also produce other bioactive compounds. A wide variety of oligopeptides including aeruginosins, cyanopeptolins and cyanobactins may be as toxic as MCs. To investigate the production of these compounds, an UPLC QTOF-MS/MS method was developed to compare the metabolomic profiles of various strains of a common bloom-forming and toxigenic species, Microcystis aeruginosa, as well as those obtained from lakes with mixed cyanobacterial assemblages. Although many compounds could not be confirmed, MCs were rarely the dominant secondary metabolite in any sample. Since the biological role of MCs remains unknown, I tested the hypothesis that MCs provide protection against oxidative stress as induced through exposure to the herbicide atrazine and UV radiation in pure cultures of toxic vs non-toxic strains. Results were inconclusive and varied between strains suggesting other mechanisms exist to counter oxidative stress.

Despite its numerous side effects, morphine and the opioids derived from this drug, belong among the only effective options for treatment of pain linked to oncological illness. The effect of opioids on the efficiency of cytostatics in vitro has been the subject of many papers but the results are often contradictory, which could be probably caused by the great variability of experimental models and approaches. Some recent studies indicate that the consequences of activation of opioid signaling in astrocytes display certain differences from other cell types. Glioblastoma multiforme, the tumor derived from astrocytes, belong among those with the worst prognosis, mostly for the frequent resistance to cytostatics. In this thesis we focused on the effect of morphine, methadone and DADLE on the efficiency of cytostatics of temozolomide, doxorubicin and vincristine on the cell lines C6 and CCF-STTG1 derived from glioblastomas. Also, we examined the effect of the above mentioned opioids on the level of oxidative cellular stress and using N-acetylcysteine, a ROS scavenger, we verified the role of oxidative stress in cellular systems activated by the effect of the mentioned opioids on the efficiency of cytostatics. We also assessed opioid receptors and the receptor TLR4 in the examined cell lines. The...

Aims: Cancer is the first cause of death in the Peruvian population; searching alternative treatments of medicinal plants constitute a promissory field to find new anticancer drugs. The main objective in this study was to evaluate the phytochemical screening, total phenolic content, antioxidant and cytotoxic activity of ethanol extract of Chromolaena laevigata (C. laevigata) on human tumor cell lines. Study Design: The fresh leaves of C. laevigata were soaked with ethanol followed by phytochemical screening using standard methods. Place and Duration of Study: Laboratory of Applied Chemistry, Faculty of Pharmacy and Biochemistry, Universidad Nacional San Luis Gonzaga de Ica, Ica, Peru; Laboratory “Abraham Vaisberg Wolach”, Universidad Peruana Cayetano Heredia, Lima, Peru. Methodology: Phytochemical screening was assessed by using chemical reactives. Total phenolic content (TPC) was developed using Folin Ciocalteu reactive and the antioxidant activity was determined against DPPH and ABTS radicals by spectrophotometry. The cytotoxic activity was determined on human tumor cell lines followed as: MCF-7, H-460, HT-29, M-14, K-562 and DU-145. Results: Phytochemical study confirmed flavonoids and phenolic compounds in ethanol extract. TPC resulted 45.21 ± 3.5 mg of gallic acid equivalent/g of dried extract. The highest antioxidant extract for DPPH and ABTS radical scavenging tests were IC50 = 11.66 ± 1.0 μg/mL, IC50= 12.45 ± 0.50 μg/mL respectively. Ethanolic extracts (μg/mL) showed a low cytotoxicity on human tumor cell lines (CI50 > 20 μg/mL) for DU-145, HT-29, MCF-7 and M-14. Whereas, for H-460, and K562 tumor cell lines showed high cytotoxicity. Conclusion: In our findings, C. laevigata demonstrated a high antioxidant and total phenolic content. The ethanol extract exhibited better cytotoxic effect compared with 5-FU. Hence, This medicinal plant could be effective to prevent chronical diseases as cancer and oxidative stress disorders.

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

Campylobacter jejuni infection is one of the leading causes of gastroenteritis in humans worldwide. During colonization of the gastrointestinal tract, C. jejuni will be unavoidably exposed to reactive oxygen species (ROS) produced by the host immune system and other intestinal microbiota. Identification of defenses against ROS is therefore important for understanding how Campylobacter survives this environmental stress during infection. Construction of isogenic deletion mutants into genes encoding potential oxidative stress defense systems followed by phenotypic screening revealed genes important for oxidant defense within C. jejuni. Surprisingly, genes involved in motility were found to play an indirect role in resistance to oxidative stress. Deletion of the flagellar motor apparatus genes, motAB, resulted in increased sensitivity towards superoxide which could be restored by fumarate supplementation or tandem deletion of motAB with ccoQ (cytochrome c oxidase). This finding suggested that disruption of the proton gradient across the inner membrane resulted in increased superoxide production in non-motile flagellar mutants. Phenotypic screening of the mutant library also identified a novel gene (cj1386) specifically involved in hydrogen peroxide defense within the cell. Hydrogen peroxide detoxification within living organisms is predominantly carried out by catalase enzymes. Interestingly, cj1386 is located directly downstream from katA (catalase) in the C. jejuni genome and it was found that a ∆cj1386 mutant had reduced catalase activity relative to wild-type C. jejuni. Immunoprecipitation of KatA from ∆cj1386 revealed a significant reduction in hemin content associated with KatA suggesting a role for cj1386 in hemin trafficking to KatA. Hemin binding experiments with purified Cj1386 demonstrated the ability of Cj1386 to bind hemin with a 1:1 hemin-to-protein binding ratio. Furthermore, co-immunoprecipitation experiments revealed an interaction between KatA and Cj1386. Mutagenesis of conserved amino acids in Cj1386 demonstrated that tyrosine 57 plays an important role in hemin affinity and is required for proper hemin content of KatA within the cell. Overall, this work provides a global characterization of key oxidant defenses within C. jejuni and provides one of the first studies investigating hemin trafficking to KatA.

There is a need for a sensitive, specific, rapid and cost-effective assay that can be used as an early warning signal of contamination of aquatic ecosystems. The purpose of this work was to develop a sensitive stress-specific microbial bioreporter responsive to pro-oxidants. Furthermore, the bioreporter was designed to be applicable in environments possibly affected by metal processing activities. An E.coli bioreporter was developed containing a plasmid with the katG promoter sequence as the sensing sequence and with mCherry as the reporter protein. The bioreporter responded to metal pro-oxidants (Cd, As, Zn, Pb, Ag and Ag nanoparticles). A new assay growth-medium was developed and contributed to improve the sensitivity of our assay that has the best detection limit to inorganic pro-oxidants compared to other oxidative-stress sensitive bioreporters in the literature. The bioreporter detected pro-oxidants in environmental samples. The assay has a reasonable sensitivity, however, it still lacks sensitivity to detect pro-oxidants at concentrations lower than those shown to be toxic to many aquatic species. Within-lab reproducibility and robustness were determined to be acceptable. For stress-specific bioreporters to be incorporated in regulative legislations and industrial monitoring programs there is a need to improve the sensitivity of these assays, they need to be calibrated with other relevant pro-oxidants, inter-lab reproducibility needs to be established and robustness to environmental samples needs to be further tested. To further validate the sensitivity and ecotoxicological relevance of the bioreporter as a relevant predictive tool, stress-specific bioreporter assays need to be performed in parallel with traditional ecotoxicological assays using contaminated environmental samples.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.