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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23762号 / 医博第4808号 / 新制||医||1056(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 生田 宏一, 教授 川口 義弥, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Bronchial thermoplasty has recently been FDA approved as a novel therapy for use on adults suffering from severe asthma. The procedure uses radiofrequency energy to heat the airways to 65°C for 10 s. This has been shown in dogs to lead to a reduction of airway smooth muscle mass and in humans to improve quality of life and asthma control. Early cellular reactions to this treatment are unclear; as well, there is limited information regarding thermal sensitivity of airway smooth muscle when exposed to extreme temperatures (50-65°C). We examined the cellular impact of bronchial thermoplasty by investigating the response of airway smooth muscle to heat by immersing bovine tracheal strips and bronchial segments in heated Krebs. We confirmed dramatically decreased functionality over the temperature range 50-60°C at 1 h and 24 h in all tissues. TUNEL analysis noted significant cell death in all tissues heated to 65°C and limited cell death in bronchial tissues treated with <55°C. Immunohistochemical analysis showed an effect of temperature on caspase 3 activation in bronchi; tracheal strips demonstrated co-localization of caspase 3 and TUNEL at 55°C but not 65°C. These data suggests that cell death of airway smooth muscle contributes to the cellular effects observed following heating to 65°C; at lower temperatures, cell death may be limited. We conclude that bronchial thermoplasty (heat treatment to 65°C for ~30 seconds) leads to a number of structural and functional changes in the airway smooth muscle, which culminate in marked loss of function and cell death.</p> / Master of Health Sciences (MSc)

<p>Cancer occurs when cells acquire a number of mutations that trigger uncontrolled cell growth. The normal cellular response to this dysregulation of growth is the activation of programmed cell death. While focus in cancer research has been mainly concentrated in the mechanism of programmed cell death at the mitochondria, endoplasmic reticulum is slowly emerging as an essential platform for this regulatory mechanism.</p> <p>Bcl-2 is the founding member of the Bcl-2 family of protein, which contributes to the regulation of cell death at the mitochondria and at the endoplasmic reticulum. Previously in our lab, we have shown using MCF-7 cells stably expressing Bcl-2 targeted to the endoplasmic reticulum; they were protected from estrogen deprivation induced cell death. Thus the regulatory mechanism of Bcl-2 at the endoplasmic reticulum represents an interesting avenue to improve current cancer therapeutics.</p> <p>Two approaches were utilized to identify and characterize Bcl-2 and its interacting partners at the endoplasmic reticulum. Using an affinity tag fused to Bcl-2 that has been engineered to target the endoplasmic reticulum, tandem affinity purification was utilized to identify novel Bcl-2 interacting partners when estrogen receptor positive cells are treated with estrogen deprivation. Using fluorescent protein fused to the proteins of interest, Fluorescent Lifetime Imaging Measurement (FLIM) was used to characterize the interactions of Bcl-2 and its known interacting partner at the endoplasmic reticulum. The findings of this thesis verify the applications of the two aforementioned methods in the study of Bcl-2 interacting proteins at the endoplasmic reticulum.</p> / Master of Science (MSc)

The high metabolic demand of the heart makes it essential that an efficient and tightly controlled system be in place to regulate energy production. Contractility is mediated by a variable flux in intracellular calcium (iCa2+), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover, mitochondrial Ca2+ (mCa2+)-overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. However, the true function of cardiac mCa2+ in physiology remains unknown. Recent studies have reported that the Mcu gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for mCa2+ uptake (Baughman et al., 2011; De Stefani, Raffaello, Teardo, Szabo, & Rizzuto, 2011). To examine the role of mCa2+ in the heart, we generated a conditional, cardiac-specific knockout model and deleted Mcu in adult mice (Mcu-cKO). Loss of Mcu protected against myocardial ischemia-reperfusion (IR) (40 min occlusion of the left coronary artery (LCA) followed by 24h reperfusion) injury by preventing the activation of the MPTP. We observed a 45% reduction in infarct size per area-at-risk and a 65% reduction in cardiac troponin-I serum levels from 24h post-IR. In addition, while we found no baseline phenotype or change in baseline mCa2+ content, Mcu-cKO mice lacked contractile responsiveness to β-adrenergic receptor stimulation (isoproterenol infusion) as assessed by invasive hemodynamics, and, in parallel, were unable to activate mitochondrial dehydrogenases, thereby decreasing tricarboxylic acid (TCA) cycle flux and cardiac NADH. We found that Mcu-cKO mice had a 3-fold increase in pyruvate dehydrogenase (PDH) phosphorylation and a 50% decrease in PDH activity post-isoproterenol infusion. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in oxidative phosphorylation capacity) supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca2+-dependent metabolism during the ‘fight or flight’ response. However, questions still remain on how basal mCa2+ levels are regulated and if it contributes to cardiac disease. The mitochondrial sodium/calcium exchanger (mNCX) is hypothesized as the primary mechanism of mCa2+ efflux, but to date no study has confirmed its identity or function in an in vivo system (Palty et al., 2010). To investigate the role of mNCX in the heart, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene, Slc8b1, and crossed them with a tamoxifen-inducible, cardiomyocyte-specific, αMHC-Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Biophysical study of cardiomyocytes isolated from mNCX-cKO mice revealed a significant reduction in mCa2+ efflux rate. Tamoxifen-induced deletion of Slc8b1 in adult hearts caused sudden death with less than 15% of mice surviving after 10 days. Echocardiographic evaluation of mNCX-cKO hearts 3d post-tamoxifen revealed significant left ventricular (LV) remodeling, characterized by significant dilation and a substantial decrease in function. In addition, mNCX-cKO hearts exhibited increased reactive oxygen species generation when assessed by DHE imaging of live myocardial tissue and mitoSOX Red imaging in isolated adult cardiomyocytes. Using an Evan’s blue dye exclusion technique, we found that mNCX-cKO hearts displayed significant sarcolemmal rupture (~8% of all myocytes at a single time point 3d post-tamoxifen), indicative of cellular necrosis. To rescue the sudden death phenotype and acute loss of cells, we crossed our mNCX-cKO mice with the cyclophilin d (a mediator of MPTP-opening) knockout mice. mNCX-cKO x CypD-KO mice had a significant improvement in survival and LV-function. In addition, loss of MPTP activation also rescued mitochondrial pathology on the subcellular level. Since deletion of mNCX was detrimental on cardiac function, we thought that increasing mNCX could protect cardiomyocytes by reducing mCa2+-overload during cardiac disease. To test this, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to assess if increasing mCa2+ efflux would prevent cardiac injury in multiple pathological surgical models. mNCX-Tg and controls were subjected to in vivo IR injury followed by 24h reperfusion and myocardial infarction (MI) (permanent LCA ligation). mNCX-Tg mice displayed reduced cell death (a 43% reduction in infarct size 24h post-IR and a 33% reduction in scar size 4w post-MI), preserved LV function, a reduction in ROS generation, and a decrease in numerous HF indices. For the first time, we showed that mNCX is essential for maintenance of the mCa2+ microdomain in cardiomyocytes and that mNCX represents a novel therapeutic target in HF. / Biomedical Sciences