The thioredoxin system is the major cellular reductant system present in the cell, whose role is to maintain cellular redox homeostasis. It does this in part, by regulating the activity of many other enzymes including ribonucleotide reductase, which is essential for DNA synthesis. It also acts as an antioxidant, reducing destructive reactive oxygen species. The thioredoxin system is comprised of thioredoxin (Trx) which reduces target protein disulfide bridges by thiol-disulfide exchange and thioredoxin reductase (TrxR) which reduces Trx back to its active state. Thioredoxin reductase is a common target for many cancer drugs including cisplatin and auranofin. Recently we have shown that the Florida red tide toxin, brevetoxin-2 (PbTx-2) can inhibit mammalian TrxR1. Brevetoxin-2 has α, β-unsaturated aldehyde moiety that was proposed to inhibit the enzyme by forming a Michael adduct. Several compounds which are similar to brevetoxin in size and functionality have a similar effect on TrxR. These compounds include antitumor and antibiotics such as manumycin A, geldanamycin, rifamycin SV and thiostrepton and toxins such as brevetoxin-3, nodularin and microcystin-LR. Manumycin A behaves as a typical TrxR1 inhibitor while other compounds screened activate the reduction of small disulfides such as DTNB (5,5’-dithiobis-(2-nitrobenzoic acid)). Mammalian thioredoxin reductase is a homodimer with two redox center viz. N-terminal dithiol buried in the enzyme and C-terminal selenosulfide located on the flexible C-terminal tail. Modification of the C-terminal tail of TrxR by these test compounds can expose N-terminal redox thiol that could reduce DTNB. The C-terminal Sec, a nucleophile can form a Michael adduct with α, β-unsaturated carbonyl moiety of test compounds. Together with point-specific mutant enzymes (C-terminal tail truncated, dead tail and Cys mutant) and enzyme assays that are specific/dependent on C-terminal Sec were used to decipher the site-specific interaction between these test compounds and TrxR. Inhibition of TrxR at the C-terminal redox center produces a pro-oxidant known as SecTRAP (Selenium Compromised Thioredoxin Reductase-derived Apoptotic Proteins), which uses NADPH to produce superoxide radical anion as observed with manumycin A. Since many cancer drugs target TrxR the present study has the potential to discover new cancer drugs.

Doctor Educationis / Maize (Zea mays L.) is grown globally as an important grain crop in South Africa, United States, China and Brazil and plays a major role in the worldwide economy. In South Africa, the grain is utilised for food consumption, livestock feed, for malting purposes and bioethanol production. Maize contains approximately 72% starch, 10% protein, 4% fat and supplying an energy density of 365 Kcal/100 g. The production of grain crops in South Africa is restricted by various factors such as abiotic and biotic stresses. The fungal genus Aspergillus is one of the most important biotic stresses affecting maize in the country. Aspergillus flavus can contaminate a wide range of agricultural commodities either in storage or field. Hydrogen sulfide appears to have a potential in the mechanism of resistance against pathogen attack by Aspergillus flavus. / 2023

>Magister Scientiae - MSc / Agricultural practices are significantly affected by drought. Drought is one of the most important plant stresses, causing several physiological, morphological, biochemical, and molecular changes in plants. Drought stress is of great challenge for crop growth, development and yield. Zea mays (maize) is one of the important crops worldwide due to the nutritional profile and other uses such as human consumption, manufacturing and animal feed. Under unfavorable conditions, plants produce high amounts of reactive oxygen species (ROS). Excessive formation of ROS is harmful for plant survival and can induce cell death. Defense mechanisms activated in response to drought in plants include antioxidant enzyme activity and proline accumulation. There is evidence of the use of nitric oxide (NO) donors and hydrogen peroxide (H2O2) at low concentrations to enhance the activity of antioxidant enzymes in stressed plants. Hence, the aim of the study is to examine the role of NO and H2O2 in regulation of antioxidant enzyme activity in maize subjected to water deficit. / 2023

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19751号 / 工博第4206号 / 新制||工||1649(附属図書館) / 32787 / 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 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24141号 / 医科博第142号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 斎藤 通紀, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defence regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defence genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cystine/glutamate antiporter that exports glutamate while importing cystine to synthesize glutathione. In response to oxidative stress, the cells increase system xc- activity in order to provide cystine for glutathione synthesis. There is evidence that expression of xCT, the specific subunit of system xc-,is regulated by NRF-2. However this has not yet been demonstrated in human breast cancer cells, which is the focus of this project. Basal expression of NRF-2, KEAP1 and xCT was characterized in three breast cancer cell lines (MDA-MB-231, MCF-7 and T47D) and compared to two non-cancer cell lines (184B5 and MCF10A). Basal protein levels of NRF-2 and KEAP1 showed no differences between cell lines. Basal protein levels of xCT were increased in MCF10A cells than T47D cells. MCF-7 cells were treated with hydrogen peroxide (H2O2) resulting in NRF-2 protein accumulation in the nucleus. With H2O2 treatment, xCT mRNA levels increased in MCF-7 cells. Additionally, transient overexpression of NRF-2 increased extracellular glutamate levels in MCF-7 cells. These data support a model that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. / Thesis / Master of Science (MSc)