The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

abstract: This study aims to address the deficiencies of the Marcus model of electron transfer (ET) and then provide modifications to the model. A confirmation of the inverted energy gap law, which is the cleanest verification so far, is presented for donor-acceptor complexes. In addition to the macroscopic properties of the solvent, the physical properties of the solvent are incorporated in the model via the microscopic solvation model. For the molecules studied in this dissertation, the rate constant first increases with cooling, in contrast to the prediction of the Arrhenius law, and then decreases at lower temperatures. Additionally, the polarizability of solute, which was not considered in the original Marcus theory, is included by the Q-model of ET. Through accounting for the polarizability of the reactants, the Q-model offers an important design principle for achieving high performance solar energy conversion materials. By means of the analytical Q-model of ET, it is shown that including molecular polarizability of C60 affects the reorganization energy and the activation barrier of ET reaction. The theory and Electrochemistry of Ferredoxin and Cytochrome c are also investigated. By providing a new formulation for reaction reorganization energy, a long-standing disconnect between the results of atomistic simulations and cyclic voltametery experiments is resolved. The significant role of polarizability of enzymes in reducing the activation energy of ET is discussed. The binding/unbinding of waters to the active site of Ferredoxin leads to non-Gaussian statistics of energy gap and result in a smaller activation energy of ET. Furthermore, the dielectric constant of water at the interface of neutral and charged C60 is studied. The dielectric constant is found to be in the range of 10 to 22 which is remarkably smaller compared to bulk water( 80). Moreover, the interfacial structural crossover and hydration thermodynamic of charged C60 in water is studied. Increasing the charge of the C60 molecule result in a dramatic structural transition in the hydration shell, which lead to increase in the population of dangling O-H bonds at the interface. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12948号 / 論農博第2820号 / 新制||農||1035(附属図書館) / 学位論文||H27||N4935(農学部図書室) / 32207 / (主査)教授 三芳 秀人, 教授 加納 健司, 教授 三上 文三 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Chapter 1: “Role of cyclic electron transport around photosystem I in the regulation of proton motive force” is based on the following paper. Caijuan Wang, Hiroshi Yamamoto, Toshiharu Shikanai, Role of cyclic electron transport around photosystem I in regulating proton motive force, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1847, Issue 9, September 2015, Pages 931-938, ISSN 0005-2728, http://dx.doi.org/10.1016/j.bbabio.2014.11.013.(http://www.sciencedirect.com/science/article/pii/S0005272814006586) / 京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19535号 / 理博第4195号 / 新制||理||1602(附属図書館) / 32571 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 教授 西村 いくこ, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

La bactérie Salmonella enterica sérovar Typhi est l’agent responsable de la fièvre typhoïde qui cause 20 millions de cas et plus de 200 000 morts annuellement. Elle possède plusieurs facteurs de virulence y compris 14 fimbriae, des structures protéiques que l’on retrouve à la surface de certaines bactéries. Plusieurs facteurs impliqués dans la chaîne de transport d'électrons (CTE) influencent l’expression du fimbriae fim, tel que Ndh, une NADH déshydrogénase (NADH-2), qui accepte et transmet les électrons du complexe I au pool de quinones entre le complexe II et III, et est un facteur majeur de la respiration aérobie chez les bactéries qui régit l'équilibre entrele NADH et NAD, et YqiC, une protéine multifactorielle et un facteur accessoire des ubiquinones, ont été identifiés comme activateur du fimbriae Fim. Notre hypothèse est que la CTE est impliquée dans la régulation du fimbriae fim. Nous voulions déterminer si la CTE régule l’expression du fimbriae fim de façon directe ou indirecte. Pour ce faire, nous avons déterminé le rôle du complexe I ; l’effet du stress oxydatif; et le rôle de l’accepteur final d’électron. Plusieurs mutants ont été créés par échange allélique et l’expression de fim chez ces mutants est quantifiée à l’aide d’un gène rapporteur, soit une fusion du promoteur fim avec le gène rapporteur lacZ. Pour déterminer le rôle du complexe I de la CTE, nous avons testé le mutant NDH-1 et NDH-2, qui s’avère à jouer un rôle important essentiel pour la régulation de fim. Pour évaluer l’effet du stress oxydatif, nous avons testé les mutants OxyR, SodB, la catalase KatG et les 3 peroxydases (Tpx, AhpC et TsaA) qui détoxifient les espèces réactives d’oxygènes (ROS) produites lors de la respiration. L’absence combinée de la catalase KatG et de la peroxydase AhpC empêche totalement l’expression de fim. Le rôle de l’accepteur final d’électron a été évalué par la présence ou absence d’oxygène, avec ou sans ajout de nitrate, nous suggérant un rôle important de YqiC pour la synthèse des transporteurs d’électrons, la génération d’ATP et le maintien du métabolisme bactérien en cas de stress oxydatif. Cette étude a permis d’identifier Ndh (NDH-2) comme une cible thérapeutique potentielle puisqu’elle ne se retrouve pas dans la chaîne d’électron de la mitochondrie chez les mammifères, donc chez les humains. Aussi, cette étude a permis de cibler les gènes détoxifiant essentiels à la régulation de fim, devenant eux aussi des cibles thérapeutiques potentielles. / The bacterium Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes 20 million cases and more than 200,000 deaths annually. It has several virulence factors including 14 fimbriae, protein structures found on the bacterial surface. Several factors involved in the electron transport chain (ETC) influence expression of the fim fimbriae, such as Ndh, an NADH dehydrogenase (NDH-2), which accepts and transports electrons from complex I to the quinone pool between the complex II and III, and is a major factor in aerobic respiration in bacteria that governs the balance between NADH and NAD, and YqiC, a multifactorial protein and an ubiquinone biosynthesis accessory factor, have been identified as an activator of Fim fimbriae . Our hypothesis is that ETC is involved in the regulation of the fim fimbriae. Our aim was to determine whether ETC regulates the expression of the fim fimbriae directly or indirectly. To do this, we determined the role of complex I; the effect of oxidative stress; and the role of the final electron acceptor. Several mutants were created by allelic exchange and fim expression in these mutants was quantified using a reporter gene, i.e. a fusion of the fim promoter with the lacZ reporter gene. To determine the role of ETC complex I, we tested the NDH-1 and NDH-2 mutants, which appears to play an important role essential for fim regulation. To assess the effect of oxidative stress, we tested mutants of OxyR, SodB, catalase KatG and the 3 peroxidases (Tpx, AhpC and TsaA) which detoxify reactive oxygen species (ROS) produced during respiration. When both the catalase KatG and the peroxidase AhpC were mutated, the fim expression was totally turned off. The role of the final electron acceptor was evaluated by the presence or absence of oxygen, with or without addition of nitrate, suggesting an important role of YqiC for electron carrier synthesis, the generation of ATP and the maintain bacterial metabolism in the event of oxidative stress. This study made it possible to identify Ndh (NDH-2) as a potential therapeutic target since it is not found in the electron chain of the mitochondria in mammals, therefore in humans. Also, this study made it possible to target the detoxifying genes essential to fim regulation, also becoming potential therapeutics targets.

Mitochondrial ATP production is achieved using the electron transport chain (ETC), whereby the controlled oxidation of biomolecules is coupled to the activity of ATP synthase. ETC complexes organize into supramolecular structures called supercomplexes (ETC SCs). Protein factors regulating ETC SCs remain largely unknown despite their fundamental implications to mitochondrial respiratory function. Recent knock-out studies have delineated external ETC proteins HIGD1A and HIGD2A as assembly factors of ETC complexes III and IV, and their incorporation into SCs. In order to clarify the primary functions of HIGD1A and HIGD2A, as well as other previously uncharacterized HIG1 protein family members, stable overexpression (OE) models of each HIG1 protein were generated in HEK293t cells to preform comparative studies. We uncover a general dichotomy in the effects observed from HIGD2A vs. HIGD1A/1B/1C OE. Furthermore, we demonstrate that the previously unstudied protein family member HIGD1C is a negative regulator of complex IV SCs. A very limited number of protein factors specifically regulating the I1III2IV1 “respirasome” ETC SC have been identified. We propose a new framework where select complex I accessory subunits regulate respirasome assembly through protein-protein interactions between ETC complexes. Through specific point mutations to one such subunit, we generate a novel cell model with selective disassembly of the respirasome but otherwise functional individual ETC complexes. We demonstrate that respirasome disassembly limits respiration and modifies electron transfer pathways within the ETC. These findings to respirasome assembly and function may represent just a portion of higher order regulation that we are beginning to describe within eukaryotic metabolism.

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付記する学位プログラム名: 京都大学卓越大学院プログラム「先端光・電子デバイス創成学」 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24401号 / 理博第4900号 / 新制||理||1700(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 川上 則雄, 教授 石田 憲二, 教授 田中 耕一郎 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Organic methods development has long dictated the molecular scaffolds available to the pharmaceutical and fine chemical synthesis industries. Photoredox catalysis has emerged as a powerful platform to enable novel reactivity with visible light irradiation through triplet sensitization and single-electron transfer. New methods involving radical intermediates are now readily accessible from countless starting materials through the application of these catalysts. Much of my work has utilized established photoredox platforms to enable both nickel catalyzed remote cross-coupling of primary amines via 1,5 hydrogen-atom transfer (HAT) and formal [3+2] synthesis of γ-lactams through triplet sensitization. My further work focuses on the application of ligand-to-metal charge transfer catalysis with cupric chloride and ferric chloride salts towards the alkylation of alkanes through the catalytic generation of chlorine radical to enable HAT. These studies expand photoredox catalysis to inner sphere mechanisms with abundant base-metal salts to enable redox chemistry at reduced electrochemical potentials.