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Effects of nitric oxide on novel soybean cystatin gene expression under salt stress in soybeanSilulwane, Nasiphi Loyola January 2012 (has links)
>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.
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The effects of nitric oxide on soybean superoxide dismutase activity during osmotic stressJack, Babalwa Unice January 2012 (has links)
>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.
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Characterization of the role of single domain soybean cystatins in regulating drought responses in soybeanKarriem, Zaheer January 2015 (has links)
>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)
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Mechanism of Action of ERBB Decoy Cancer Therapeutic Peptide SAH5Makhani, Kiran, Makhani, Kiran January 2017 (has links)
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.
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Reactive Oxygen Species (ROS) Up-regulates MMP-9 Expression Via MAPK-AP-1 Signaling Pathway in Rat AstrocytesMalcomson, Elizabeth January 2011 (has links)
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.
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The Roles of Nitric Oxide, Oxidative Stress, and Angiotensin II Type 1 Receptor in Regulating Cutaneous Blood Flow and Sweating During Prolonged Exercise in the Heat with and without Fluid ReplacementMcNeely, Brendan January 2017 (has links)
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.
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Copper homeostasis and Salmonella pathogenicity : interplay with resistance to nitrosative stressPointon, Thomas January 2014 (has links)
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.
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Potamanautes warreni biomarker assays to monitor silver nanomaterial contaminants in aquatic environmentsWalters, Chavon Rene January 2016 (has links)
Philosophiae Doctor - PhD / There has been extensive growth in nanoscale technology in the last few decades to such a degree that nanomaterials (NMs) have become a constituent in a wide range of manufactured commercial and domestic products. This surge has resulted in uncertainties regarding their environmental impact, due to the significant increases in the amount of NMs released into the environment (Dowling et al., 2004) through intentional and unintentional releases. Like many other toxins, the aquatic environment is particularly vulnerable as it acts as a sink for nanoparticles (NPs) (Scown et al., 2010). The escalating growth of NMs has not advanced without efforts to understand its properties. Despite the dramatic advances in both the
production and application of NMs, very little is known regarding their interaction with and effects on environmental and human health. Given the lack in scientific knowledge, particularly under various environmental conditions, it is often difficult to accurately assess the potential exposure pathways to ecological receptors of all NMs, silver nanoparticles (AgNPs) are the most widely used NPs, present in several consumer products mainly because of their anti-bacterial properties. It is estimated that the annual production exceeds 1000 tons/year (Piccinno et al., 2012). The increase uses of AgNPs in consumer products (e.g. textiles, cosmetics and personal hygiene), household appliances (e.g. washing machines and vacuum cleaners) and medical equipment have led to their increase release into the environment, thereby posing an environmental risk and human health concern. Silver NPs are known to induce the production of Reactive Oxygen Species (ROS) (Ahamed et al., 2010; Levard et al., 2012; Piao et al., 2011). Also since AgNPs are oxidized to ionic Ag (Ag+), it is still unclear whether the effects of ROS can be attributed to Ag+ release or to the AgNP itself (Fabrega et al., 2009; Miao et al., 2009). The behaviour of AgNPs is collectively influenced by inherent (nanoparticle size, shape, surface area, surface charge, crystal structure, coating, solubility/dissolution) and environmental factors (temperature, pH, ionic strength, salinity, organic matter). Climate change predictions indicate that the frequency, intensity and duration of extreme natural events (such as temperature elevations) will increase in the future (IPCC, 2001; IPCC, 2007). Global warming and climate change could increase atmospheric temperatures by 2.4 – 6.4 °C (IPCC, 2001; IPCC, 2007). The main feature associated with global climate change is the anticipation of wetter winters (i.e. increased flood events) and drier, warmer summers (i.e.extreme temperatures). These changes are likely to affect the inputs of contaminants into the environment as well as affect their behaviour, fate and transport, and toxicity in aquatic environments. It is known that the current temperature predictions in climate change scenarios could directly affect aquatic ecosystem communities (Carpenter et al., 1992), since temperature is also regarded as an important abiotic factor influencing growth and production of primary producers (i.e. algae, macrophytes etc.), and may also affect species distribution. For example, Liu et al. (2010) reported higher dissolution rates of AgNPs with increased temperature. Similarly, sudden hydrographic activity like high flood conditions may cause resuspension and redistribution of sediments. Few studies have linked the foreseeable climate change with contaminant release and ecosystem impacts. Similarly, few studies have analyzed the behaviour of NMs in the environment considering these predicted changes in mean temperatures. This thesis focuses on the effects of AgNPs on oxidative stress responses in the Cape River crab Potamonautes perlatus. The present work was undertaken to interpret the biological effects of AgNPs (< 100 nm) on P. perlatus, as well as to assess its effects under different environmental conditions. To understand the uptake, accumulation and biological effects of AgNPs, freshwater microcosms were produced to mimic a typical aquatic environment and temperature manipulated microcosms to which a commercially-available AgNP powder was added. Nanoparticles were characterized in the dry state and in suspension under different environmental conditions. Dissolution of total Ag was measured by inductively coupled plasma mass spectrometry (ICP-OES). Nanoparticle toxicity was assessed by measuring mortality and biomarkers of oxidative stress (CYP450, SOD, CAT, GST) evaluated in crab tissues. The overall results demonstrated that: (1) AgNPs may be transformed in both size and state under variable environmental conditions. The formation of smaller aggregates at higher temperatures suggests higher toxicity, (2) the release of free metal ions from NPs and NPs aggregates contribute to a higher toxicity towards aquatic organisms, (3) oxidative stress is a significant mechanism of AgNP toxicity and consequently enzymatic activation/inhibition with increasing AgNP concentration and temperatures, (4) oxidative stress responses to AgNPs particles were significantly modulated by temperature stress in P. perlatus, (5) mortality was observed from day 2 with maximum mortality achieved at day 7, (6) enzymes involved in detoxification, i.e. CYP450, has functional significance in the
haemocytes, (7) P. perlatus has proved to be a significant target for AgNP exposure and, furthermore, has proved to be a suitable species to assess the ecotoxicity of AgNP in the aquatic environment, (8) antioxidant enzymes activities (are valuable tools to assess the oxidative status of crab tissues co-exposed to AgNPs and temperature. Furthermore, the results obtained in this study contributed to the understanding of the behaviour, bioavailability, uptake and toxicity of AgNPs under variable temperatures. / National Research Foundation (NRF) Thuthuka Fund and CSIR
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Thioredoxin and Jab1 Control Estrogen- and Antiestrogen-Mediated Progression of the Cell Cycle Through p27 InteractionsPenney, Rosalind B 11 March 2011 (has links)
A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.
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The Investigation of Photocatalysts and Iron Based Materials in the Oxidation and the Adsorption of Toxic Organic and Chromium MaterialsJiang, Wenjun 13 November 2013 (has links)
The presences of heavy metals, organic contaminants and natural toxins in natural water bodies pose a serious threat to the environment and the health of living organisms. Therefore, there is a critical need to identify sustainable and environmentally friendly water treatment processes. In this dissertation, I focus on the fundamental studies of advanced oxidation processes and magnetic nano-materials as promising new technologies for water treatments.
Advanced oxidation processes employ reactive oxygen species (ROS) which can lead to the mineralization of a number of pollutants and toxins. The rates of formation, steady-state concentrations, and kinetic parameters of hydroxyl radical and singlet oxygen produced by various TiO2 photocatalysts under UV or visible irradiations were measured using selective chemical probes. Hydroxyl radical is the dominant ROS, and its generation is dependent on experimental conditions. The optimal condition for generation of hydroxyl radical by of TiO2 coated glass microspheres is studied by response surface methodology, and the optimal conditions are applied for the degradation of dimethyl phthalate. Singlet oxygen (1O2) also plays an important role for advanced processes, so the degradation of microcystin-LR by rose bengal, an 1O2 sensitizer was studied. The measured bimolecular reaction rate constant between MC-LR and 1O2 is ~ 106 M-1 s-1 based on competition kinetics with furfuryl alcohol.
The typical adsorbent needs separation after the treatment, while magnetic iron oxides can be easily removed by a magnetic field. Maghemite and humic acid coated magnetite (HA-Fe3O4) were synthesized, characterized and applied for chromium(VI) removal. The adsorption of chromium(VI) by maghemite and HA-Fe3O4 follow a pseudo-second-order kinetic process. The adsorption of chromium(VI) by maghemite is accurately modeled using adsorption isotherms, and solution pH and presence of humic acid influence adsorption. Humic acid coated magnetite can adsorb and reduce chromium(VI) to non-toxic chromium (III), and the reaction is not highly dependent on solution pH. The functional groups associated with humic acid act as ligands lead to the Cr(III) complex via a coupled reduction-complexation mechanism. Extended X-ray absorption fine structure spectroscopy demonstrates the Cr(III) in the Cr-loaded HA-Fe3O4 materials has six neighboring oxygen atoms in an octahedral geometry with average bond lengths of 1.98 Å.
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