>Magister Scientiae - MSc / Salt stress is a major abiotic stress that accounts for huge agricultural losses worldwide, which in turn threaten food security and sustainable agriculture. Salt triggers the excessive production of reactive oxygen species (ROS) which accumulate to levels that become toxic to plants, resulting in cell death and reduced plant growth. Part of the plant’s mechanisms to counteract ROS-induced cell death involves the scavenging ability of the antioxidant defense system to maintain redox homeostasis. Gallic acid (GA) is an antioxidant that has been shown to reduce salt-induced ROS in legume plants. However, its effects on wheat plants have not been elucidated. This study thus investigated the role of exogenous GA (250 μM) on the physiological responses and antioxidant system of wheat plants under salt stress (150 mM). In addition, this study also investigated how GA and salt stress influenced changes in the membrane proteome of wheat plants using LC-MS proteomic analysis. / 2022

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

Preliminary studies were undertaken to investigate whether or not added iron (0, 35, 880 ppm of iron as ferrous fumarate) and fat type (corn oil, beef tallow, or menhaden oil ) influenced the oxidation of the rat diet during storage . Iron level affected thiobarbituric acid (TBA) values only in the menhaden oil diets. Storage for 4 d did not affect TBA values of the diets . Neither food intake nor body weight of the rats was affected by the different diets, suggesting all diets were equally acceptable to rats. The effects of iron supplementation and fat type on in vivo lipid peroxidation in rat colon were studied. Semi-synthetic diets were formulated to contain 15% (wt/wt) total fat, an amount comparable with human diets, as either 15% corn oil (C), 1% corn oil + 14% beef tallow (B), or 1% corn oil + 14% menhaden oil (M). Diets of each fat type were formuIated with ferrous fumarate to contain 35 ppm iron, a level sufficient to meet the requirement of the rats, or 880 ppm iron, a level similar to that found in iron-fortified breakfast cereals. During a 6-wk study, each of 6 groups of 10 male weanling Sprague-Dawley rats was fed one of the 6 diets (C35, C880, B35, B880, M35, M880). Lipid peroxidation products in the colon mucosa and in the feces were measured as thiobarbituric acid reactive substances (TBARS), and other possible physiological changes were monitored by measuring body weight, and iron levels of the feces and colon mucosa, and by observing the histology of the colon. At the beginning of the trial, each group of rats had similar body weights and TBARS in the feces. After the feeding trial, groups of rats remained similar in body weight, and no histological changes were observed in the colon. However, rats fed the different dietary fats had different (p < 0.05) TBARS in the feces and colon mucosa (BThus, the type of dietary fat was a significant determinant of in vivo lipid peroxidation, independent of dietary iron level. Rats fed the high iron diets had higher TBARS in both the feces and colon mucosa. When compared by dietary fat type, rats fed high iron diets had higher TBARS in the mucosa only if they were also fed the menhaden oil diet. Thus, dietarv iron was a significant determinant of in vivo lipid peroxidation only in combination with menhaden oil. The long-term intake of iron-fortified foods with high menhaden oil may lead to significant increased in vivo lipid peroxidation.

The nonapoptotic form of regulated cell death known as ferroptosis is an attractive target for combating numerous diseases. Ferroptosis is an iron-dependent death of cells by lipid peroxidation. Pharmacological inhibition of anti-ferroptotic pathways is a promising therapeutic avenue for treatment of cancer, and death by ferroptosis has been implicated in numerous neurodegenerative and ischemia-reperfusion-driven diseases. Therefore, demystifying the dynamics of lipid peroxidation in this cell death process opens a window to understanding disease processes and how to treat them. This dissertation makes use of ferroptosis-modulating compounds as chemical probes to elucidate the roles of different subcellular membranes in ferroptotic lipid peroxidation. Chapters two and three explore the structure-activity-distribution relationship of fatty acids and the ferroptosis inducer FINO2, respectively, and together demonstrate the endoplasmic reticulum as a driver of lipid peroxidation in ferroptosis. Chapter two makes use of stimulated Raman scattering imaging, while chapter three uses confocal fluorescence imaging. Chapter four shifts gears to focus on development of FINO2 as a drug lead, performing structure activity relationship analysis to increase the potency and pharmacological properties of the analogs. Altogether, this work answers questions about how cells die by ferroptosis, and provides footwork for how we can better modulate ferroptosis against cancer and other illnesses.

Vitamin E and selenium play key roles in preventing in vitro lipid peroxidation and free radical damage to retinal tissues. In this research, we studied the effects of hyperbaric oxygen on retinal structure in rats fed diets deficient in vitamin E and/or selenium. We also correlated any alterations in retinal structure with previously measured alterations in electroretinograms (ERGs). Age-matched rats were fed a basal diet deficient in both vitamin E and selenium (B diet), a basal diet supplemented with vitamin E alone (B+E diet), or selenium alone (B+Se diet), or with both micronutrients (B+E+Se). Half the rats in each group were treated (+ HBO) with hyperbaric oxygen (100% O2 at 3 ATA for 1·5 per hr day, 5 days per week) and half were not (-HBO). We previously found that the rats fed the B diet for 6 weeks and treated with HBO for 4 weeks (B+HBO group) had diminished a-wave ERG amplitudes. At this time point all rats in the B group and half of the rats in the B+E+Se group were killed for the structural studies reported here. Surprisingly, we found no evidence of photoreceptor cell necrosis [i.e. a decreased thickness of the outer nuclear layer (ONL)] in retinas from rats in the B+HBO group despite the diminished amplitude of the a-wave which arises from this retinal layer. Quantitative structural analyses of retinas from rats in the B+HBO, B-HBO, B+E+Se-HBO and B+E+Se+HBO groups also failed to reveal any significant differences in the cell height of the retinal pigmented epithelium (nasal, central or temporal regions) or the number of mitochondria, phagosomas or inclusion bodies in the central retinal pigment epithelium (RPE). The inner nuclear layer (INL) thickness was, however, consistently decreased in all retinal regions for the rats in the B+HBO group. Our previous work also showed that only rats fed the B+Se diet for 17 weeks and treated with HBO for 15 weeks (B+Se+HBO group) showed diminished a-wave and b-wave ERG amplitudes. At this time point rats in the B+E+Se, B+E, and B+Se groups were killed for structural studies reported here. Only rats in the B+Se+HBO group showed a significantly decreased (about 20%) thickness of the central ONL. This evidence of photoreceptor cell necrosis correlated very well with our previous observation of diminished a- and b-wave amplitudes only in the B+Se+HBO group (at week 17). Ultrastructural studies after 17 weeks of feeding the experimental diets revealed two different types of inclusion bodies in the central RPE. On the basis of morphological appearance we have termed these inclusion bodies 'electron-dense' and 'granulated'. The central RPE of rats in the B+Se+HBO and B+Se-HBO groups showed a larger number (P < 0·001) of 'granulated' inclusion bodies and a smaller number (P < 0·001) of 'electron-dense' inclusion bodies than rats in any other diet/treatment group at this time point. In marked contrast, there were no observable 'granulated' inclusion bodies and no significant differences in the number of electron-dense inclusion bodies found in the central RPE from rats in any diet/treatment group after 6 weeks of feeding the experimental diets. Our results are discussed with respect to the potential effects of lipid peroxidation on retinal morphology and on electroretinograms.

Autoxidation is a radical mediated chain-process that involves initiation, propagation, branching and termination reactions and is responsible for the spontaneous peroxidation of hydrocarbons, formally appearing as RH + O₂ → ROOH. Autoxidation is a consequentially damaging process in many domains, ranging from materials to automotive transportation to biology and medicine. One of the key intermediates in the propagation of autoxidation is the peroxyl radical (ROO•) which can be targeted by radical-trapping antioxidants (RTAs) that promote chain-termination, mitigating the damage of autoxidation. Chapter 1 lays out the fundamental chemistry of both autoxidation and RTAs as well as a history of the rational design of phenol and diarylamine-type RTAs. Lipid-peroxidation (i.e. autoxidation) is a key feature of ferroptosis which is a form of cell death that has been associated with many serious conditions such as ALS, Alzheimer's, Huntington's and Parkinson's disease, and lipid-soluble RTAs such as Vitamin E have been shown to acutely suppress ferroptosis. An aspect of RTA chemistry that has not been well studied/understood hitherto is their kinetic behaviour in phospholipid membranes, and we hypothesized that this would be a very relevant consideration for designing compounds that target lipid-peroxidation and ferroptosis. In Chapter 2 we systematically examine the kinetic behaviour for a series of hindered and unhindered phenolic RTAs in various mediums, particularly in phosphatidylcholine (PC) liposomes. The key chemical interaction in the PC membrane that fundamentally changed the observed kinetics of the phenolic RTAs is a very strong hydrogen-bonding interaction with the phosphate-diester headgroup that suppresses the phenols' ability to trap ROO•, an effect that was previously overlooked. In Chapter 3 we further expanded/validated the model by studying over 40 phenoxazine (PNX) and phenothiazine-based (PTZ) RTAs, which showed the quantitative/predictive capabilities of the H-bonding effect. By introducing a water-soluble co-antioxidant, Vitamin C (ascorbate), we were able to study many features of the PNX/PTZ radical intermediates with respect to their reactivity and dynamics. The PNX/PTZ were far more persistent than the Vitamin E analogue 2,2,5,7,8-pentamethyl-6-chromanol (PMC), meaning that they catalytically trapped lipid-peroxyls far more efficiently (i.e., higher turnover number). Additionally, there is strong evidence suggesting that the PNX/ascorbate synergism is a diffusion-controlled process. The study was further expanded to biological models. Ferroptosis in vitro was inhibited by every single one of these compounds, and there was a general positive correlation between RTA kinetics (kᵢₙₕ) and ferroptosis rescue potency (EC₅₀) as well as a positive correlation between lipophilicity (logP) and ferroptosis rescue potency. A lead PNX compound, 3-trifluoromethyl-8-tert-butylphenoxazine, was identified in this study on the basis of superior potency and metabolic stability. When used to treat mice with GPx4 deletion in kidneys, an in vivo model of ferroptosis, it was found to extend the life of the mice in a statistically significant fashion compared to the vehicle control. In Chapter 4 there is further elaboration on the dynamics of PNX/ascorbate synergy and a demonstration of the early works toward developing a drug-like-PNX ferroptosis inhibitor, based on the conclusions from the work in Chapter 3. In Chapters 5 and 6 the research is focused on the development novel RTAs for the application of inhibiting autoxidation in lubricants in high temperature environments. Heavy machinery and most transportation technologies require lubrication to aid safe and efficient movement, and these lubricants/greases are highly susceptible to autoxidation. Large quantities of RTA additives are expended to extend the service life of these materials and there is a constant appetite for innovation to find new and improved RTAs for improved economics and competitiveness. In Chapter 5 the behaviour of PNX and PTZ in a simulated high temperature lubricant autoxidations are analyzed, revealing that PNX is highly susceptible to direct O₂-mediated oxidation due to its rapid electron-transfer kinetics, while PTZ is far more resilient despite both compounds having nearly identical oxidation potentials. In Chapter 6, in this same context, previously unreported substituent effects are analyzed which significantly enhance the period of inhibition (tᵢₙₕ) for PTZ compounds. Particular alkyl substituents on the PTZ can increase the number of chains-trapped at high temperatures by fortuitous substituent oxidation that promotes termination, substantially improving their atom-economy. These findings prompt a broader critique of putative catalytic RTA mechanisms which have been taken for granted for nearly three decades.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24909号 / 農博第2572号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙野 俊幸, 教授 上髙原 浩, 教授 河本 晴雄 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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