Ataxia-telangiectasia mutated kinase (ATM), a serine/threonine kinase primarily located in the nucleus, is typically activated in response to DNA damage. Individuals with mutations in ATM gene develop a disease called Ataxia telangiectasia (AT). These individuals are more susceptible to ischemic heart disease and metabolic disorder. Our lab has previously shown that ATM plays a critical role in β-adrenergic receptor (β-AR) - and myocardial infarction (MI)-stimulated myocyte apoptosis and cardiac remodeling. This study tested the hypothesis that ATM plays a critical role in cardiac autophagy and glucose metabolism following MI and ischemia, respectively. Early during MI (4 hours after its onset) and 4 hours post-treatment with ATM inhibitor KU-55933, ATM deficiency resulted in autophagic impairment in the heart and in cardiac fibroblasts, respectively. Such autophagic changes in the heart and in cardiac fibroblasts associated with the activation of GSK-3β and mTOR, and inactivation of Akt and AMPK. ATM deficiency also augmented autophagy in the infarct region of the heart 28 days post-MI as well as in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 24 hours. Autophagic changes in the infarct region during ATM deficiency associated with enhanced Akt, Erk1/2, and mTOR activation. Additionally, the lack of ATM accelerated glycolysis and gluconeogenesis and augmented TCA cycle metabolism under non-ischemic conditions. Following a 20 minute global ischemic period, the glycolytic pathway, not the gluconeogenic pathway, was down-regulated during ATM deficiency which was found to be associated with alterations in TCA cycle metabolism. Such metabolic rearrangements associated with changes in the phosphorylation of Akt, GSK-3β, and AMPK alongside alterations in Glut4 protein expression. Thus, ATM deficiency impairs autophagy early after the onset of MI and in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 4 hours. In contrast, ATM deficiency appears to augment autophagy late post-MI in the infarct region of the heart and in cardiac fibroblasts treated with ATM inhibitor KU-55933 for 24 hours. Lack of ATM alters glucose and TCA cycle metabolism with and without ischemia. Such findings implicate ATM as a key player in autophagic changes in the heart in response to MI as well as in glucose metabolism under non-ischemic and ischemic conditions.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells. / Yorkshire Cancer Research

Cryopreservation is currently the only method allowing for the long-term preservation of hematopoietic stem and progenitor cell (HSPC) grafts until their use. However, cryoinjuries reduce cell viability and potency of HSPC. New cryoprotectant (CPA) solutions have recently emerged that have not yet been investigated that may improve the cryopreservation of HSPCs. The overarching hypothesis of the work described in this thesis, is that different CPAs have diverse impact on the key biochemical processes essential for HSPC homeostasis which influences post thaw cell viability and potency. To test this hypothesis, 4 CPAs were extensively characterized for their cryoprotective properties on cord blood (CB) HSPCs in comparison to DMSO control. CryoProtectPure (CPP) supported similar post thaw cell viability and engraftment as DMSO control, whereas pentaisomaltose (PIM) and cryonovo (CN) failed as CPAs for HSPCs. Subsequently, the impact of CPAs on key biological pathways was explored to identify potential biochemical pathways implicated in HSPC cryopreservation. The impact of CPAs on cell membrane integrity, oxidative phosphorylation, glycolysis, and autophagy was examined. CPP and DMSO had varying impact on glycolytic and mitochondrial respiratory activities of HSPCs post-thaw, whereas both CPAs as well as PIM and CN had negligible impact on cell membrane parameters prefreeze. Cryopreservation and thawing strongly induced autophagy in HSPCs. Importantly, early inhibition of autophagy with 3-Methyladenine (3-MA) reduced the recovery of functional CB HSPCs post thaw. Together, my findings provide new insights regarding the biological processes impacted by CPAs and cryopreservation of HSPCs and identify potential targets to improve cryopreservation of HSC grafts.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24134号 / 医博第4874号 / 新制||医||1060(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 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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