Orientador: Eliana Cotta de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T21:51:52Z (GMT). No. of bitstreams: 1 Sodre_FabioLima_D.pdf: 2215244 bytes, checksum: d5f9f9a36b242a13e3536696c1a618e6 (MD5) Previous issue date: 2009 / Resumo: A aterosclerose é uma doença crônica degenerativa das artérias, caracterizada pelo acúmulo de lipídios na parede arterial. Na década de setenta, Zilversmit postulou que o fenômeno pósprandial era promotor da aterosclerose. O mecanismo que conecta o estado pósprandial com a doença cardiovascular (DCV) inclui a disfunção endotelial, estresse oxidativo e inflamação. No presente estudo, foi estabelecida a composição química das subfrações das lipoproteínas de alta densidade (HDLs) em uma população adulta normolipidêmica brasileira e, comparada esta com outras populações. Além disto, as modificações na composição química desta partícula em indivíduos saudáveis normolipidêmicos foram descritas após a ingestão de uma dieta rica em lipídios. Estes dados demonstraram uma diminuição do conteúdo de ésteres de colesterol e aumento de triglicérides nas partículas de HDL, os quais refletem o aumento da atividade da enzima transferidora de ésteres de colesterol (CETP). Estas mudanças levam a uma atividade anti-oxidante reduzida da HDL. Por fim, este estudo evidenciou que a geração intracelular de espécies reativas ao oxigênio (ROS) está reduzida durante o período pósprandial. A diminuição do consumo de oxigênio celular e suas correlações com a geração de ROS indicam que a mitocôndria participa ativamente deste fenômeno. Disfunção endotelial foi outro achado durante este período. No mundo ocidental, o período pósprandial representa uma parte significativa do dia, ressaltando a importância deste estado no desenvolvimento da aterosclerose. A completa elucidação dos mecanismos envolvidos pode melhorar as intervenções dietéticas e levar a efeitos benéficos no combate ao estresse oxidativo e disfunção endotelial no período pós-prandial. / Abstract: Atherosclerosis is a chronic degenerative disease of the arteries, characterized by an accumulation of lipids in arterial walls. In 1970s, Zilversmit postulated that postprandial phenomenon was a promoter of atherosclerosis. The mechanisms that link the postprandial state with CVD include endothelial dysfunction, oxidative stress and inflammation. In present study, the chemical composition of high density lipoproteins (HDLs) subfractions in a Brazilian adult normolipidemic population was determinated and compared it with others populations. In addition, the modifications in chemical composition of this particle in healthy normolipidemic subjects after an intake of a fat-rich meal, was described. This data presents a diminished core content of cholesteryl ester (CE) and elevated triglyceride (TG) of postprandial HDL particles, which reflected enhanced activity of cholesteryl ester transfer protein (CETP). These changes lead to an impaired antioxidative activity of dense HDL. Finally, this study provides evidence that intracellular reactive oxygen species (ROS) generation is reduced during the postprandial period. The reduction of oxygen consumption and it correlation with ROS generation suggests that mitochondria plays a pivotal role in this phenomenon. Endothelial dysfunction was also found during this period. In western societies, a significant part of the day is spent in the postprandial state, further emphasising the importance of this period in the development of atherosclerosis. The complete elucidation of the involved mechanisms may improve the appropriateness of such dietary intervention supported by beneficial effects on postprandial oxidative stress and endothelial dysfunction. / Doutorado / Clinica Medica / Doutor em Clínica Médica

>Magister Scientiae - MSc / With a decrease in water availability and arable land, and the ever-increasing reports of toxic chemical pollutants, it is crucial to elucidate plants’ mechanisms of adaptability to these abiotic stressors. South Africa alone accounts for approximately 30% of global Zirconium (Zr) production. However, reports on Zr-induced stress in plants are sparse. Increased mining activity leads to soil contamination which subsequently has harmful effects on crop plants. Under normal conditions B. rapa crop plants flourish, they are rapid in their cycling and circumvent the seed dormancy stage which enables them to have high yields over relatively short periods. However, when unfavourable conditions arise, such as exposure to toxic chemicals and metal ions like Zirconium, the development and growth of B. rapa L., much like other crop plants is affected. More specifically, the damaging effects of Zr is not only attributed directly; as with substitutions of biometals [like Iron (Fe)] in various biomolecules rendering them inactive, but more as a consequence of the production of toxic molecules such as reactive oxygen species (ROS) and methylglyoxal (MG). ROS such as superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂ ) are known to have signalling roles in plants with reports on their involvement in alleviating seed dormancy and seedling development. However, the signalling roles of MG are not known with regards to plant cells and have been reported more so in animal cells; playing vital roles in fat signalling in diseases such as diabetes. Furthermore MG, in plant and animal cells, directly converts oxygen (O₂) to O₂⁻ and thus increases the cell’s oxidative imbalance, leading to cell damage if O₂⁻ is not rapidly dismutated to H₂O₂ and H₂O by superoxide dismutase (SOD). In turn, H₂O₂ is more stable than O₂⁻ and consequently is more toxic to cells over time. Therefore, H O must be removed as well by a collection of enzymes, such as ascorbate peroxidase (APX) and catalases (CAT). In this study, possible stress-signalling of MG in seedlings under normal conditions and Zr-stress were investigated to establish whether MG at a low dose (6 μM) would benefit seedling growth and development, via a proposed preinduction of the B. rapa L. antioxidant system. Therefore, it was proposed that ROS accumulation due to the exogenous application of MG, would incite the activation of antioxidants and thus mitigate the effects of Zr stress. Physiological tests to determine dry weights (figure 3.2.3) and germination percentage (figure 3.2.2) revealed that MG-treated seedlings yielded an improved biomass and early development compared to Zr-treated seedlings and the control. Membrane damage as assessed by lipid peroxidation viz. Malondialdehyde [MDA] (figure 3.2.4) and conjugated dienes [CD] (figure 3.2.5) also indicated less damage in MG-treated seedlings compared to the Zr-treated set. The chlorophyll content observed was prominent (table 3.1). MG-treated seedlings exhibited a 40% and 15.5% increase compared to Zr-treated seedlings and the control respectively. Moreover cell viability had improved in MG-treated seedlings compared to the control, and in MG+Zr-treated seedlings only a slight increase in cell death occurred despite Zr being present. O₂⁻, H₂O₂ and •OH (figure 4.2.1 – 4.2.3) were investigated in B. rapa L. seedlings in response to Zr and MG by spectrophotometric biochemical assays, as well as their scavenging enzymes, MG accumulation and Gly-I activity. Furthermore, BrGLY1 gene expression and Zr-uptake by ICP-OES were performed. Seedlings treated with MG and Zr respectively showed an increase in ROS. However, all of the ROS observed in MG+Zr-treated seedlings were markedly lower compared to Zr-treated seedlings. SOD and CAT activity observed in MG+Zr-treated seedlings had decreased compared to Zr-treated seedlings, whereas APX activity had increased. Gly-I activity and BrGlyI gene expression had increased across all treatments, showing an elicited response to oxidative stress, due to the observed upregulation, as a result of the accumulated MG. The observed Zr-uptake in MG+Zr-treated seedlings was inhibited by 5-fold compared to Zr-treated seedlings. Clear signs of stress were evident in seedlings treated with Zr compared to the control and MG- treated seedlings, the MG-supplemented (MG and MG+Zr) seedlings displayed a vast improvement comparatively. Modulation of antioxidant activity observed in this study is indicative of an incited response to oxidative stress (figure 4.2.4 – 4.2.6). MG revealed distinct involvement in stress-signalling, ROS levels had increased, although not as severely as with Zr-treated seedlings, but seemingly enough to activate antioxidants without eliciting damage. Furthermore, the proposed early-onset activation of antioxidants has been observed in B. rapa L. seedlings of this study, and as such has resulted in improved growth, development and seed germination. The results of this study has therefore negated the previous reports on MG-toxicity (at high concentrations), and has shed light on further properties of this ubiquitous and inevitably-occurring metabolite at low levels. / National Research Foundation (NRF)

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15728号 / 工博第3342号 / 新制||工||1505(附属図書館) / 28273 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

>Magister Scientiae - MSc / Nitric oxide (NO) has been shown to orchestrate multiple defense responses to both abiotic and biotic stress. Importantly, elevation of nitric oxide content in plants by using nitric oxide generating compounds has been shown to enhance plant tolerance to abiotic stresses such as salt and drought via up-regulation of genes involved in the regulation of plant responses to abiotic stress. In this study, the effect(s) of nitric oxide (generated from 10 μM of the nitric oxide donor DET/NO) on the expression of a novel soybean cystatin gene (Glyma20g08800), lipid peroxidation, caspase-like activity and cell death in salt (150 mM)-stressed soybean leaves, roots and nodules were investigated. Salt treatment resulted in elevated lipid peroxidation, caspase-like activity and increased cell death in organs studied while the observed detrimental effects of salt stress were reversed by NO treatment. Salt stress suppressed the expression of Glyma20g08800 while the levels of expression of Glyma20g08800 returned towards those of unstressed plants when the salt-stressed plants were supplemented with nitric oxide (DETA/NO). Furthermore, promoter sequences of GmCYS1p626 and three of its homologues (Glyma20g08800, Glyma14g04250 and Glyma18g12240) were analyzed for putative abiotic stress and/NO cisregulatory elements based on co-expression analyses using bioinformatics. Several abiotic stress induced transcription factors (TFs) were identified and were hypothesized to be co-acting either directly or indirectly through additional factors in the regulation of soybean cystatin expression in response to NO and abiotic stress. Taken together, these results highlight the possibility of using NO to drive high levels of expression of cystatins during salt stress and lead to accumulation of the cystatin to levels that are sufficient to inhibit salt stress-induced caspase-like activity, which will inhibit salt stress-induced cell death and thus enhance the tolerance of the plant to salt stress and possibly tolerance to drought stress as well.

>Magister Scientiae - MSc / Nitric oxide (NO) is a signaling molecule involved in mediating plant responses to various biotic and abiotic stresses. Major abiotic stresses (drought, salinity, cold) induce common cellular responses, causing osmotic stress in plants. This results in oxidative stress due to increased production of reactive oxygen species (ROS). The increased ROS levels simultaneously induce the antioxidative system (including antioxidant enzymes such as superoxide dismutase) that regulates ROS toxicity and enhance stress tolerance in plants. It is suggested that the scavenging of ROS by antioxidant enzymes can be controlled by NO. The aim of this study was to evaluate the role of exogenously applied NO on soybean (Glycine max L. Merr.) during osmotic stress, with the purpose of determining the effects of NO on the superoxide dismutase (SOD) activity in response to osmotic stress. This study also aimed at identifying and characterising SOD isoforms induced in soybean in response to osmotic stress and exogenous NO. To achieve these aims, soybean plants were treated with sorbitol (to induce osmotic stress), an NO donor [2,2'-(hydroxynitrosohydrazono)bis-ethanimine, DETA/NO] and its respective control (Diethylenetriamine, DETA). The results showed that exogenous NO alleviated osmotic stress-induced damage by reducing the superoxide radical content, lipid peroxidation levels and also maintaining cell viability in soybean leaves, nodules and roots. Only two SOD isoforms i.e. manganese SOD (MnSOD) and copper/zinc SOD (CuZnSOD) were identified and characterised in soybean leaves and roots, iron SOD (FeSOD) was not induced. The isoforms identified exhibited low SOD activity in response to osmotic stress, with the exception of a few isoforms that had increased activity. The SOD activity was regulated by exogenously applied NO. The enzymatic activity of SOD isoforms was up-regulated by exogenous NO, except for a few SOD isoforms that were not responsive to NO. The results also showed that the increased SOD activity was associated with reduced lipid peroxidation levels. The results obtained from this study suggest that exogenous NO improves osmotic stress tolerance in soybean by regulating and increasing the SOD activity of only specific isoforms. The increased SOD activity maintains the redox homeostasis balance by detoxifying and controlling the superoxide radical levels, subsequently reducing lipid peroxidation and maintaining cell viability.

>Magister Scientiae - MSc / This study investigated the effects that drought stress imposed on the growth and development of soybean plants. Soybeans were initially observed at the whole-plant level in order to identify the physical changes that had taken place in response to drought. Further investigation of the effects of drought stress on Soybean plants were quantified at the molecular level. Physical changes of soybeans in response to drought stress were typified by the change in leaf morphology and pigmentation. At the molecular level, it was observed that drought stress resulted in the accumulation of hydrogen peroxide in soybean leaves, which was met by elevated levels of lipid peroxidation. The effects of drought on the modulation of (and interplay between cystatins) cysteine protease (caspase-like) activity and programmed cell death (PCD) were also investigated. Total caspase-like activity and cell death were enhanced in response to water deficit despite the up-regulation in gene expression of the cystatin Glyma14g04250. The cystatin Glyma18g12240 was not expressed in soybean leaves, whilst the gene expression of the cystatin Glyma20g08800 remained unchanged in response to drought. This study was aimed at the characterization of two single domain soybean cystatins, namely, Glyma14g04250 and Glyma20g08800 which could potentially be overexpressed in transgenic soybean plants in an attempt to alleviate the effects of drought stress. / National Research Foundation (NRF)

Breast cancer is the most prevalent type of cancer and second leading cause of death in women. Among others, the triple negative breast cancer (TNBC) is the most invasive as it has the highest recurrence and death rates with no targeted therapeutic available thus far. Epidermal Growth Factor Receptor (EGFR) is one of the important targets as more than fifty percent of the TNBC overexpress it but all the therapies designed against it have failed to show significant results. The juxtamembrane domain of EGFR has been explored comparatively recently and has been used to design a decoy peptide with the anticipation to affect the EGFR downstream functions. Previous research has shown it to cause cell death in cancer cells. This study is aimed towards deciphering the mechanism of action of the stapled form of this decoy peptide-SAH5. It presents evidence that the peptide leads to an immediate intracellular calcium release from the Inositol 1,4,5 triphosphate on the endoplasmic reticulum, an inhibition of which can rescue SAH5 induced cell death. The study also demonstrate that the peptide is able to increase the production of Reactive Oxygen Species (ROS) in mitochondria, part of which is triggered by the peptide-induced calcium release.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

The current study evaluated whether NO synthase (NOS) contributes to cutaneous vasodilation and sweating during prolonged exercise in the heat. In addition, we determined if prolonged exercise-induced increases in reactive oxygen species (ROS) and activation of angiotensin II type 1 receptors (AT1R) impair heat loss responses. On two separate days, eleven young men completed 90-min of continuous cycling at ~600W of metabolic heat production followed by 40-min of recovery in the heat (40ºC). To evaluate the role of excess fluid loss via sweating, participants completed a second session of the same protocol while receiving fluid replacement (FR) determined during the first session (No-FR). Cutaneous vascular conductance (CVC) and local sweat rate (LSR) were measured at four intradermal microdialysis forearm sites perfused with either: (1) lactated Ringer (Control); (2) 10 mM NG-nitro-L-arginine methyl ester (L-NAME, NOS inhibition); (3) 10 mM ascorbate (non-selective anti-oxidant); or (4) 4.34 nM Losartan (AT1R inhibition). Ascorbate treatment increased CVC at 60- and 90-min of exercise versus Control during the FR (P < 0.02), but not the No-FR condition (P > 0.31). CVC was reduced at the L-NAME treated site (P < 0.02), but was not different relative to Control at the Losartan treated site (P > 0.19) irrespective of condition. LSR did not differ between sites or as a function of condition (all P > 0.10). We conclude that NO regulates cutaneous vasodilation but not sweating, irrespective of fluid replacement, and ascorbate sensitive ROS impair cutaneous vasodilation during prolonged exercise in the heat with FR.

Salmonella enterica serovar Typhimurium is responsible for a variety of diseases in domestic animals and humans. The infection of mice causes similar disease progression to human typhoid fever, thus representing a model for this systemic disease. The ability of S. Typhimurium to reside in a macrophage phagosome is important for their survival and spread to different organs. The antimicrobial mechanisms in this compartment include reactive oxygen species, reactive nitrogen species and elevated copper levels. S. Typhimurium possesses two copper-exporting P1B-type ATPases, CopA and GolT, both of which contribute to copper resistance. A previous study has shown that copper export by CopA and GolT confers a survival advantage in resting macrophage phagosomes. In this study the role of copper resistance systems has been examined further. The reduced survival of ΔcopA/ΔgolT in macrophages is detected beyond 8 hours post infection and coincides with increased nitrite production by macrophages. We have established that ΔcopA/ΔgolT display some increased sensitivity to reactive nitrogen species. However, whilst treatment of macrophages with the iNOS inhibitor L-NMMA reduced macrophage bactericidal activity against wildtype S. Typhimurium, this was not the case for ΔcopA/ΔgolT. In contrast, survival of ΔcopA/ΔgolT was not impaired in macrophages treated with the copper-chelator BCS. Furthermore real-time PCR confirmed the expression of copA and golT is elevated during infection of macrophages treated with IFN-γ or L-NMMA, but is reduced during infection of BCS treated macrophages. This indicates that bactericidal activity in macrophages is associated with copper availability and this is unaffected by reactive nitrogen species released due to iNOS activity. In contrast to Escherichia coli Salmonella lacks a cus system associated with export across the outer membrane and hence the mechanism of copper export from the periplasm is not known. TolC was investigated as a potential outer membrane copper exporter based on clustering of TolC dependent systems to genes with sequence similarity to the S. typhimurium periplasmic copper chaperone CueP, across several bacteria. Mutation of tolC gave reduced copper tolerance and over-accumulation of copper at non-lethal concentrations under aerobic conditions. However TolC does not provide a role in copper tolerance or homeostasis under anaerobic conditions. TolC also does not provide tolerance or homeostasis to other divalent cations: Zn, Ni and Co. TolC therefore provides specific transport of copper under aerobic conditions in S. Typhimurium.