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.

Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

Glutathione (GSH), a 3-amino acid compound is ubiquitously expressed in eukaryotic cells and is the most abundant low molecular weight thiol. The importance of GSH is highlighted by its multitude of effects. Within the vascular wall GSH plays a crucial role as an intracellular antioxidant and it possess the ability to act as a signalling intermediate and store for nitric oxide (NO). The importance of NO and its role in vascular wall homeostasis is well recognized. Within the coronary circulation, NO is the primary dilator of many of the large arteries and the smaller arterioles. In addition to controlling coronary vascular tone, the importance of NO is highlighted by its antithrombotic, antihypertrophic, and antriproliferative effects. During instances of cardiovascular disease and normal aging, increases in the production of reactive oxygen species occur. A portion of the deleterious vascular effects of reactive oxygen species are believed to be due to reduction in NO bioavailability as a result of increased ROS-mediated destruction of NO. Altered GSH production in humans has been demonstrated to reduce endothelial function. Conversely, supplementation with GSH augments endothelium-dependent dilation. The mechanisms by which these alterations in GSH influence vasomotor function have not been resolved. The purpose of the studies within this thesis was to examine the impact of chronic and acute GSH modulations on coronary vascular resistance (CVR) and endothelium dependent dilation. In all experiments vascular reactivity was assessed in the isolated perfused rat heart. The advantage of this technique is that it allows the global coronary vasomotor functioning to be examined. Hearts were allowed to stabilize for 30 minutes to allow for the development of spontaneous coronary vascular resistance, followed by a bradykinin (BK) dose-response curve to assess endothelium-dependent dilation. The coronary circulation was then maximally dilated using an endothelium-independent agonist. In all cases BK-mediated dilation is expressed as a percentage of the endothelium-independent dilation. Chapter 2 of this document examines the chronic nature of GSH depletion and examines whether GSH depletion augments the influence of natural aging. Animals (mean age 33 and 65 weeks) were randomized to receive L-Buthionine-(S,R)-sulphoximine (BSO) in the tap water in order to inhibit GSH synthesis, or regular tap water (normal controls). Following 10 days of BSO treatment, ventricular GSH content was reduced in the BSO group compared to the control (0.182±0.021 vs 2.022±0.084 nmol/mg wet weight, p<0.05) and there was increased ventricular H2O2 content (1.345±0.176 vs 0.877±0.123 pmol/µg PRO, p<0.05). Baseline CVR was significantly reduced in the older animals compared to the adult animals (3.92±0.34 vs 4.76±0.20 and 3.67±0.24 vs 5.12±0.37 mmHg/ml×min-1 in the control and BSO treated groups, p<0.05). Conversely, in the presence of LNAME there was a significant increase in CVR in the adult BSO group (14.15±0.99, p<0.05) compared to all other groups. In the absence of LNAME, maximal dilation (percent endothelium-independent response) was reduced in the older animals compared to the adult animals (77±10.3% vs 95.0±1.0% for older and adult control and 92.7±4.5% vs 98.6±0.6% for the older and adult BSO, main effect of age). In the presence of LNAME the adult BSO group had a significantly reduced sensitivity (EC50) compared to all other groups (-7.39±0.09 Log M, p<0.05). Additionally, adult BSO treated animals had an increase in eNOS protein content. These results demonstrate that chronic thiol depletion resulted in an increased reliance on NO in the adult BSO group only. In chapter 3 the beneficial effects of GSH supplementation on BK mediated dilation were examined. Acute GSH was administered in the perfusate at either 0 (control) or with 10 µM for 2 reasons, 1) this concentration does not reduce basal coronary vascular resistance, allowing for a similar baseline CVR across conditions and 2) the 10 µM concentration is a physiologically relevant concentration of plasma/extracellular fluid GSH. The sensitivity to the endothelial agonist bradykinin was enhanced in the presence of GSH (-8.70±0.16 vs -7.94±0.06 LogM, p<0.01). The GSH effect was not dependent on NO production or utilization by soluble guanylate cyclase (sGC) as the enhanced dilation in the GSH group was maintained despite NOS (LNAME) and/or sGC inhibition. When the hearts were supplemented with a ROS scavenger TEMPOL, enhanced dilation was seen in the control group, but was not further enhanced in the GSH group. The requirement for ROS was best demonstrated when both the CON and GSH groups were supplemented with both TEMPOL and LNAME. This condition resulted in similar sensitivity (-7.76±0.19 vs -7.75±0.17 LogM, p>0.05) and area under the curve (182.33±12.70 vs 170±13.86, p>0.05) between GSH and CON. Thus, it was concluded that the effects of GSH administration requires the presence of ROS and exerts its effect in the microvasculature. The study presented in chapter 4 examined the effects of acute thiol modulation (depletion) on CVR and endothelium-dependent dilation. Previous reports have suggested that a reduction in intracellular GSH causes impaired NO production, and functional data support this contention. However, a majority of the data regarding the effects of thiol manipulation are from endothelial-removed vessels. The following agents were used to reduce GSH: the glutathione reductase inhibitor, BCNU; the thiol oxidizing agent, diamide; the thiol conjugating agent, ethacrynic acid (EA); and a thioredoxin inhibitor (CDNB). Preliminary data revealed that only CDNB (11.46±0.71 mmHg/ml×min-1) and EA (8.61±0.36 mmHg/ml×min-1) caused an elevation in CVR compared to the control (6.73±0.24 mmHg/ml×min-1). Conversely, Diamide and BCNU did not significantly affect baseline CVR, or the BK mediated responses. In the presence of EA, there was an overall blunting of the BK-response curve as observed by reduced EC50 (-7.85±0.07 Log M) and maximal dilation (90.8±1.8 %, percent endothelium-independent dilation) compared to the control group (-8.42±0.08 Log M and 97.7±1.6%). In the presence of CDNB the maximal dilation was 74.4±1.9% and the EC50 was -8.83±0.28 Log M. In addition to altering BK mediated responses, acute thiol depletion with all agents resulted in an increased minimal CVR with significant increases observed in the presence of CDNB and EA. There was a significant correlation with GSH:GSSG ratio and baseline (-0.547, p<0.05) and minimal CVR (r=-0.581, p<0.05). This study demonstrates that modulation of the GSH:GSSG ratio using a variety of agents with diverse mechanisms elicits differential responses within the vasculature. Specifically conjugation of GSH and inhibition of thioredoxin significantly alters BK mediated response, where as BCNU and dimaide did not. These results suggest that a modulation in the GSH:GSSG ratio impairs endothelium-dependent dilation and alters total dilatory capacity (baseline-minimal CVR) and thus may have implications for adequate tissue perfusion. Across all studies there was significant correlation between GSH and GSSG with both baseline and minimal CVR. Therefore it is likely that changes in overall glutathione content plays a role in determining baseline and minimal coronary vascular resistance. These results demonstrate the complexity that manipulations of GSH have on both CVR and endothelium-dependent dilation, and provide mechanistic insight into how changes in GSH alter coronary vascular resistance and endothelium-dependent dilation.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 6 uppsatser.

Philosophiae Doctor - PhD (Biotechnology) / High temperature stress is synonymous with the attenuation of plant growth, metabolism and eventually death resulting in major loss of crop productivity worldwide. Part of the metabolic perturbations associated with heat stress leads to the excessive formation of reactive oxygen species (ROS), which have highly deleterious effects on cellular homeostasis. Naturally, through millions of years of evolution and adjustment, plants have developed antioxidant enzymes that neutralize harmful ROS species offering a protective role in the annulment of oxidative damage in response to high temperature. The aim of this study was to measure the activity of several antioxidant enzymes in response to heat stress in Zea mays.

Vitamin C (ascorbate and dehydroascorbic acid) is vital for plants and found throughout the plant cell including in the apoplast. The structure of ascorbate was determined eighty years ago; however, many of its degradation pathways remain unclear. Numerous degradation products of ascorbate have been reported to occur in the apoplast but many still remained unidentified. Ascorbate is well known as an antioxidant, and acts to quench reactive oxygen species (ROS), such as hydrogen peroxide and ozone in the plant apoplast. The immediate oxidation product of ascorbate is dehydroascorbic acid (DHA), which may be quickly hydrolysed to diketogulonic acid (DKG). The further reactions of radiolabelled and non-radiolabelled DHA and DKG with various ROS have been investigated. Differences were observed in the products formed from the various ROS, allowing a unique fingerprint of oxidation products to be described for each ROS. Equally, different compounds were produced depending on the starting substrate; for example cyclic oxalyl threonate was only observed in the reactions of DHA and not DKG. A major oxidation product of DHA is OxT. A novel enzyme activity involving the transfer of the oxalyl group from OxT to an acceptor substrate such as a sugar has been detected. This enzyme activity could have potential cell wall modification roles, in the formation of oxalate cross-linkages between cell wall components. This would provide a novel role for ascorbate derivatives in cell growth. Vitamin C is also a vital component of the human diet, and most dietary ascorbate comes from plants such as salads. The degradation of ascorbate during post-harvest processing and storage of salad leaves has been investigated. Spinach leaves were found to be particularly prone to losing ascorbate during the industrial washing process. The use of radiolabelled ascorbate has allowed the determination that the major degradation product formed from ascorbate during spinach washing was oxalate.

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