京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13067号 / 論医博第2122号 / 新制||医||1019(附属図書館) / 33218 / (主査)教授 長船 健二, 教授 松田 道行, 教授 岩井 一宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The failure of axonal regeneration is due to myriad reasons both cell intrinsic and extrinsic. In this thesis, I sought to investigate an intrinsic reason for regeneration failure in the CNS. Specifically, I investigated the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for two weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Interestingly, transection of axons within the sciatic nerve resulted in mitochondrial fission but did not result in the fragmentation of mitochondria. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDivi-1) blocked fragmentation. Additionally, it was determined that there is increased mitophagy in CST axons following spinal cord injury based on increased colocalization of mitochondria and LC3. In vitro models revealed that mitochondrial calcium uptake is necessary for injury induced mitochondrial fission, as inhibiting mitochondrial calcium uptake using RU360, a mitochondrial calcium uniporter inhibitor, prevented injury induced fission. This phenomenon was also observed in vivo. These studies indicate that following injury, both in vivo and in vitro, axonal mitochondria undergo increased fission, which may result in an ATP deficit that contributes to the lack of regeneration seen in CNS neurons. / Biomedical Sciences

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Cholesterol is known to affect biophysical quantities in one and two component membranes, overall increasing membrane thickness and orientational order and decreasing membrane fluidity. Although these effects are useful in the plasma membrane of cells where strength is a desired property, there is evidence suggesting that the appearance of cholesterol in other cellular membranes has adverse effects. Indeed, cholesterol is found at elevated levels in the mitochondria membranes of cancer cells and is associated with chemotherapy resistance. At the molecular level, the link between cancer and cholesterol seems to be that cholesterol interferes with apoptosis, or programmed cell death, specifically by inhibiting the insertion of the pro-apoptotic protein Bax in the mitochondria outer membrane. We studied the effects of cholesterol on a five component mitochondria-like membrane, in order to determine which of these are relevant for Bax membrane insertion. As expected we found, using x-ray and neutron scattering, that upon cholesterol addition: (1) the thickness of the mitochondria-like membrane increases, (2) the area per phospholipid decreases, and (3) the orientational order of the membrane increases. Interestingly, our data indicate that the ordering effect of cholesterol is less efficient for the five component mitochondria-like membrane than for a single component membrane. Finally, we determined that in spite of the relatively high degree of unsaturation of the lipids in the mitochondria-like membrane, cholesterol adopts a canonical orientation. At higher cholesterol concentrations, cholesterol's polar hydroxyl group moves outwards and comes in proximity with the phospholipid's carbonyl group, allowing hydrogen bonding between the two types of molecules. Any of the above effects could in principle be responsible for cholesterol's inhibition of Bax insertion, thus follow up studies are required to confirm which, or what combination of them are relevant for apoptosis. / Thesis / Master of Science (MSc)

Objective – Sepsis is a syndrome in which infection triggers a systemic inflammatory and procoagulant response. Cell-free DNA (CFDNA) is elevated in sepsis, and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. CpG motifs on bacterial and mitochondrial DNA can stimulate inflammatory responses via TLR9. CFDNA can activate coagulation via the contact pathway. CFDNA may thus play an important pathogenic role in sepsis. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and pro-coagulant pathways with relevance to sepsis. Results – Mitochondrial DNA as well as nuclear DNA are elevated in plasma from septic patients compared to healthy controls. Bacterial, but not mitochondrial or nuclear, DNA increased neutrophil IL-6 secretion. Both mitochondrial and bacterial DNA increased neutrophil viability. Nuclear, mitochondrial, and bacterial DNA increased thrombin generation in both platelet-poor plasma and platelet-rich plasma to a similar degree. This effect was reduced by addition of corn-trypsin inhibitor and in FXII-depleted plasma, and abolished in FXI-depleted plasma, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, DNA from all three sources was capable of causing activation of platelet integrin αIIbβ3. Conclusions – CFDNA from nuclear, mitochondrial, and bacterial sources have varying pro-inflammatory effects, although all three have similar pro-coagulant potential. The pathophysiological effects of CFDNA in sepsis may vary with the source of DNA. / Thesis / Master of Science (MSc)

Reactive oxygen species (ROS), and the resulting oxidative stress caused by these species, have long been attributed to be one of the causes for aging and age related disorders. NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate (NADP), provides a critical and essential buffer against cellular toxicity due to ROS. NADPH is one of the cells most powerful reducing agents, capable of regenerating other endogenous antioxidants such as glutathione from its oxidized form, glutathione disulfide. Consequently, it is hypothesized that declining NADPH levels with age results in a depletion in cellular capacity to respond to ROS induced damage, further accelerating the aging process. To study the importance of NADPH on the aging process as well as the molecular mechanisms involved, lifespan assays were performed using knockdown of various enzymes involved in the production of NADPH in Caenorhabditis elegans. Preliminary results indicate declining NADPH levels do have an effect on expected longevity. More interesting however, is a possibly important distinction between cytoplasmic and mitochondrial enzymes involved in the production of NADPH. These preliminary results suggest the existence of a previously undescribed molecular mechanism that is potentially important to the aging process. However, further experiments and analysis are required to further elucidate these mechanisms and to confirm preliminary findings.