京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22738号 / 医博第4656号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 中川 一路, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Average human life expectancy has progressively increased over many decades largely due to improvements in nutrition, vaccination, antimicrobial agents, and effective treatment/prevention of cardiovascular disease, cancer, etc. Maximal life span, in contrast, has changed very little. Caloric restriction (CR) increases maximal life span in many species, in concert with improvements in mitochondrial function. These effects have yet to be demonstrated in humans, and the duration and level of CR required to extend life span in animals is not realistic in humans. Physical activity (voluntary exercise) continues to hold much promise for increasing healthy life expectancy in humans, but remains to show any impact to increase maximal life span. However, longevity in Caenorhabditis elegans is related to activity levels, possibly through maintenance of mitochondrial function throughout the life span. In humans, we reported a progressive decline in muscle mitochondrial DNA abundance and protein synthesis with age. Other investigators also noted age-related declines in muscle mitochondrial function, which are related to peak oxygen uptake. Long-term aerobic exercise largely prevented age-related declines in mitochondrial DNA abundance and function in humans and may increase spontaneous activity levels in mice. Notwithstanding, the impact of aerobic exercise and activity levels on maximal life span is uncertain. It is proposed that age-related declines in mitochondrial content and function not only affect physical function, but also play a major role in regulation of life span. Regular aerobic exercise and prevention of adiposity by healthy diet may increase healthy life expectancy and prolong life span through beneficial effects at the level of the mitochondrion.

In this thesis, I have explored the role of three genes related to p53, namely p21, MDM2 and MDMX, in regulating ferroptosis, a form of non-apoptotic cell death. Ferroptosis, an iron-dependent mechanism that leads to cell death due to lipid peroxidation, has a large potential to be used as a cancer therapy. My results indicate that p21, the effector of p53-mediated cell cycle arrest, can suppress ferroptosis possibly through its interaction with CDKs. Further, that MDM2 and MDMX, the negative regulators of p53, can act as pro-ferroptosis agents and that this role is independent of p53. Using various approaches to alter their activity, I found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. They were found to alter the cellular lipid profile to prevent the cells from mounting an adequate defense against lipid peroxidation. For example, inhibition of MDM2 or MDMX lead to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q₁₀, an endogenous lipophilic antioxidant. Moreover, I found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis. My findings also suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Further, that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers. Therefore, I believe that this thesis project has successfully identified several new regulators of ferroptosis and this knowledge can aid better design of therapies centered around ferroptosis.

Apoptosis is a type of programmed cell death which plays a fundamental role in maintaining homeostasis in multi-cellular organisms. The Bcl-2 family has been identified as the central players in regulation of apoptosis. It consists of anti-apoptotic proteins (e.g Bcl-XL) and pro-apoptotic proteins which are further classified as BH3 proteins (e.g Bim, Bid) and effector proteins (e.g Bax, Bak). BH3-proteins regulate apoptosis by activating the pro-apoptotic proteins Bax and Bak to permeabilize mitochondria, and/or by inhibiting anti-apoptotic proteins such as Bcl-XL and Bcl-2. In this study, we employed fluorescence spectroscopy and functional assays with full-length Bim and showed that major Bim isoforms have similar activities in vitro. Bim preferably activates Bax over Bak while Bid preferably activates Bak. Bim displayed a unique binding to Bcl-XL so that the Bim-Bcl-XL complex is resistant to BH3-mimic drug ABT-263 treatment while Bid does not. A Bcl-XL enhancer BH3 TCTP was also shown to interact with Bim-Bcl-XL and Bid-Bcl-XL complex differently, where a single mutation abolished its enhancement of Bid-Bcl-XL but not Bim-Bcl-XL. Closer investigation of the dual apoptotic functions of the BH3-protein Bim revealed that the C-terminal membrane binding domain (MBD) is unexpectedly also involved both in binding of Bim to Bax in solution and in activating Bax. Multiple mutations in this domain reduced or abolished binding to membranes but did not affect binding to Bax or correlate with Bax activation. Deletion eliminated binding to and activation of Bax, but not binding to or inhibition of Bcl-XL. Thus MBD mediates binding to both membranes and Bax separately. On the other hand, although the MBD is not the determining factor for interactions with Bcl-XL, our data demonstrates Bim MBD also plays a major role in binding to Bcl-XL. The C-terminal MBD was shown to be contributing to the ABT-263 resistance of Bim-Bcl-XL complex by directly interacting with Bcl-XL. We discovered additional interactions between the MBD of Bim and both Bcl-XL and Bcl-2. Our data suggested a novel topology and mechanism for the Bim-MBD that positions the central hydrophobic residues of the MBD appropriately for binding to Bcl-XL. / Thesis / Doctor of Philosophy (PhD) / Apoptosis is a type of programmed cell death which plays a fundamental role in maintaining homeostasis in multi-cellular organisms. The Bcl-2 family has been identified as the central players in regulation of apoptosis. It consists of anti-apoptotic proteins (e.g Bcl-XL) and pro-apoptotic proteins which are further classified as BH3 proteins (e.g Bim, Bid) and effector proteins (e.g Bax, Bak). Here we showed that Bim is constitutively active in vitro and preferably activates Bax, distinguishing itself from Bid which preferably activate Bak. We showed that the C-terminus membrane binding domain (MBD) of Bim plays a crucial role in reguating binding and activation of Bax, as well as providing extra binding support to Bcl-XL, independent of its membrane binding feature.

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High concentrations of intracellular glutathione enhance the in vitro production of porcine embryos. Six experiments were conducted to study the effects of varying concentrations of different supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and in vitro culture (IVC), and evaluate subsequent embryo viability. In Exp. 1, 2, 3, and 4, porcine oocytes were matured in either 3.3 mM cysteine, 150 μM cysteamine, 3.3 mM cysteine and 150 μM cystemaine; 1.0 mM glycine, 2.5 mM glycine, 5.0 mM glycine; 1.0 mM L-glutamate, 2.5 mM L-glutamate, 5.0 mM L-glutamate; and 3.3 mM L-α-aminobutyrate, 25 μM β-mercaptoethanol, 3.3 mM cysteine and 25 μM β-mercaptoethanol, or 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol. After IVM (44 h), concentrations of intracellular glutathione (GSH) were determined using a colorimetric assay based on absorbency. The supplements that elicited significantly (P < 0.05) the greatest increase in GSH concentrations were 3.3 mM cysteine, 1.0 mM L-glutamate, 3.3 mM L-α-aminobutyrate, and 3.3 mM L-α-aminobutyrate with 25 25 μM β-mercaptoethanol. In Exp. 5, oocytes matured with 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol had a significantly less (P < 0.05) occurrence of polyspermy and greater occurrence of MPN formation during IVF compared to the other treatment groups and a significantly greater percentage (P < 0.05) of embryos reaching the 2 cell developmental stage by 48 h post-IVF and blastocyst stage of development by 144 h post-IVF compared to the other treatment groups. In Exp. 6, treatment had no effect on the time of cell death. The times at which embryo mortality was significantly the greatest (P < 0.05) were located within the middle of IVC. The approximate time of the onset of cell death occurred between 24 to 42 h post-IVF with the greatest occurrence around 36 h. These results suggest that supplementing 3.3 mM L-α-aminobutyrate and 25 μM β-mercaptoethanol into the IVM medium 1) increases intracellular GSH concentrations by the end of IVM, 2) decreases the occurrence of polyspermy during IVF, 3) increases the MPN formation during IVF, and 3) increases embryo development parameters during IVC. Supplementation to the maturation media does not have an effect on cell death during embryo development. The onset of cell death appears to occur between 24 to 42 h post-IVF with the greatest occurrence around 36 h post-IVF. In order to increase the success of in vitro derived porcine embryos and offspring, the basic fundamentals of the system need to be fully understood. / Master of Science

Yau, Kwok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 64-71). / Abstracts in English and Chinese. / Chapter Chapter 1: --- Introduction --- p.1 / Trichosanthin --- p.2 / Apoptosis --- p.10 / Herpes Simplex Virus --- p.16 / Conclusion --- p.28 / Chapter Chapter 2: --- Materials and Methods --- p.29 / Cell lines and virus --- p.30 / Infectivity assay --- p.30 / Treatment of cells and virus infection --- p.32 / MTT assay for cytotoxicity --- p.34 / Preparation of cell lysate --- p.35 / Bradford assay for protein concentration --- p.36 / Western blot analysis --- p.37 / ELISA for quantification of HSV-1 antigen --- p.38 / Statistical analyses --- p.39 / Chapter Chapter 3: --- Results --- p.40 / Cytotoxicity and anti-herpetic activity of TCS and CHX --- p.41 / Selective cytotoxicity of TCS toward HSV-1 infected cells --- p.44 / Selective cytotoxicity is implicated in the antiviral activity of TCS --- p.50 / The effect of selective cytotoxicity on TI value --- p.53 / Chapter Chapter 4: --- Discussion --- p.55 / References --- p.64

Thesis (M.Sc.)--Memorial University of Newfoundland, 2001. / Restricted until June 2004. Bibliography: leaves 123-154.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves : 176-210). Also available on the Internet.