Classical novae (CNe) are interacting binary systems whose outbursts are powered by a thermonuclear runaway in accreted material on the surface of a white dwarf (WD). The secondary stars in such systems fill their Roche lobe and material is transferred onto the WD primary star via an accretion disk. Recurrent novae (RNe) show many similarities to CNe, but have had more than one recorded outburst. They play an important role as one of the suspected progenitor systems of Type Ia supernovae (SNe) which are used as primary distance indicators in cosmology. Thus, it is important to investigate the nature of their central binary systems to determine the relation between the parameters of the central system and outburst type, and finally ascertain the population of novae that might be available to give rise to the progenitors of Type Ia SNe. The investigation looking for characteristics that may distinguish RNe from CNe systems, the selection of initial targets for detailed study, and results of the investigation are presented in this thesis. The proposal that RNe occupy a region separated from CNe in an outburst amplitude versus speed class diagram was adopted. Since the low amplitude results from the existence of an evolved secondary and/or high mass transfer rate in the quiescent system, RNe candidates should accordingly have low amplitude. The 93 novae with observed V amplitudes given in the literature and 43 novae with photographic amplitudes have been combined and plotted on an outburst amplitude versus rate of decline diagram from which 16 target novae suspected to be RNe candidates were selected for photometric and spectroscopic follow-up. Quiescent photometric magnitudes and spectra were obtained using RATCam on LT, FRODOSpec on LT, and RSS on SALT. Spectral type and luminosity class determined from the near-IR colour-magnitude diagrams were compared to those derived from the spectra. Determination of spectral types was accomplished by identifying specific lines and calculating indices from TiO bands, VO bands, and the Na atomic line for giants (finding 4 stars) and sub-giants/giants (3 stars). A spectral library template was used instead of the indices in cases of main-sequence stars (2 stars).Our investigation also confirmed the positions of AR Cir, V794 Oph and EU Sct where there had been some ambiguity previously. Ultimately, we suggest four prime RNe candidates (2 novae with giant secondaries - V3964 Sgr and EU Sct, and 2 novae with sub-giant secondaries - V794 Oph and V368 Aql) which are currently classified as CNe, to look for more than one outburst in archival plates or large sample sky surveys such as SMEI (see below). By introducing the high cadence full-sky space-based observational archive of the Solar Mass Ejection Imager (SMEI) which operated on the Coriolis satellite from 2003-2011, we derived light curves of one Mira (O Cet) as a general example and two novae with known outbursts during 2003-2011 (V2467 Cyg and V1187 Sco). The SMEI light curves potentially reveal more details than those given by ground-based observations. The pre-maximum halt was found in V2467 Cyg as well as oscillations in light curves found earlier than those found in previous studies. The precise date of maximum of each nova was provided. Four bright novae that are potentially RNe candidates (V4074 Sgr, V3964 Sgr, DK Lac and V368 Aql) were searched for second outbursts in the SMEI data, but none were found. Among the nova outbursts detected by SMEI, we found however unprecedented detail in first class data of the Recurrent Nova T Pyx in its 2011 outburst. We investigated the optical light curve of T Pyx during its 2011 outburst through compiling a database of SMEI and American Association of Variable Star Observers (AAVSO) observations. The SMEI light curve, covering t=1.5-49 days post-discovery, was divided into four phases based on the idealised nova optical light curve; the initial rise (1.5-3.3 days), the pre-maximum halt (3.3-13.3 days), the final rise (14.7-27.9 days), and the early decline (27.9 days, until the end of SMEI observations on day 49). The SMEI light curve contains a strongly detected period of 1.44+-0.05 days during the pre-maximum halt phase. These oscillations resemble those found in recent TNR models arising from instabilities in the expanding envelope. No spectral variations that mirror the light curve periodicity were found however. The marked dip at t~22-24 days just before light curve maximum at t=27.9 days may represent the same (shorter duration) phenomenon seen in other novae observed by SMEI and present in some TNR model light curves. The spectra of T Pyx from the 2m the Liverpool Telescope and the Small and Moderate Aperture Research Telescope System (SMARTS) 1.5m telescope were obtained from t=0.8-80.7 and 155.1-249.9 days, covering the major phases of development. The nova was observed very early in its rise where a distinct high velocity ejection phase was evident with derived Vej~4000 km/s initially. A marked drop at t=5.7 days, and then a gradual increase occurred in derived Vej to stabilise at ~1500 km/s at the pre-maximum halt. Here we propose two different stages of mass loss, a short-lived phase occurring immediately after outburst and lasting ~6 days followed by a more steadily evolving and higher mass loss phase. The overall spectral development follows that typical of a Classical Nova and comparison with the photometric behaviour reveals consistencies with the simple evolving pseudo-photosphere model of the nova outburst. Comparing optical spectra to X-ray and radio light curves, weak [Fe X] 6375A emission was marginally detected before the X-ray rise and was clearly present during the brightest phase of X-ray emission. If the onset of the X-ray phase and the start of the final decline in the optical are related to the cessation of significant mass loss, then this occurred at t~90-110 days.

During chronic and severe acute liver injury, regeneration is thought to occur through hepatic progenitor cells (HPCs). Understanding the regulation of HPCs may offer therapeutic opportunities to enhance liver regeneration. HPCs are associated with an increase in laminins in the extracellular matrix. Laminins are heterotrimeric proteins, composed of an alpha, beta and gamma chain. There are 5 alpha chains with different distributions and functions, but the relative contributions of these in HPC-mediated liver regeneration are not known. My aims were to describe the laminin alpha chains associated with the HPC response and to define the functional effects of specific laminin chains on HPCs. I examined the laminin alpha chains in two mouse models of HPC activation: a transgenic model using conditional deletion of Mdm2 in hepatocytes, and a dietary model using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). The laminin alpha 5 (Lama5) chain is significantly upregulated in both models and forms a basement membrane which surrounds the progenitor cells. I have also demonstrated Lama5 expression in the ductular reaction seen in human liver disease. Using primary mouse cell cultures, I have shown that Lama5 is produced predominantly by the HPCs themselves, rather than by stellate cells. The HPCs express the cell surface receptor alpha-6 beta-1 integrin, a binding partner of Lama5. I then studied the functional effects of matrix on cell behaviour in vitro using recombinant laminins and a line of spontaneously immortalised mouse HPCs. Compared to other laminin chains, Lama5 selectively promotes HPC adhesion and spreading. These effects are partially blocked by antibodies against beta-1 integrin. Lama5 also significantly enhances HPC migration, resulting in an increase in cell migration. Furthermore, only Lama5 enhances HPC survival in serum-free medium, with an increase in cell viability. Culturing HPCs on HPCs maintained in culture on plastic synthesise Lama5 chain. Knock-down of endogenous Lama5 production using siRNA results in reduced proliferation and increased hepatocytic differentiation, with increased albumin production. I then studied the effects in vivo using transgenic Cre-lox mouse strains that allow conditional knock-out of either laminin alpha 5 or beta-1 integrin in HPCs. The effects of gene deletion were examined in healthy mice and two dietary models of HPC activation: the DDC diet and a choline-deficient, ethionine-supplemented (CDE) diet. Although these experiments were limited by a low number of experimental animals and low recombination rates, there was a suggestion of impaired HPC expansion associated with loss of laminin alpha 5. There was also a significant increase in hepatocellular injury and fibrosis in response to the DDC diet seen with loss of laminin alpha 5 expression. Laminin alpha 5-containing matrix is deposited around HPCs during liver regeneration and supports progenitor cell attachment, migration and maintenance of an undifferentiated phenotype. This work identifies a novel target for enhancing liver regeneration.

Bone marrow (BM) transfer from flagellin-treated mice has been reported to improve the survival of lethally-irradiated mice. Although the mechanism for flagellin’s antiviral and antibacterial effects have been elucidated, there remains a gap in knowledge regarding its radioprotective effects. Here, we report that flagellin treatment results in a 5-fold increase in the proliferation of Lin-Sca-1+C-Kit+(LSK) cells, a heterogeneous stem and multipotent cell population in BM, with the most striking increase within the ST-HSC, MPP2 and MPP3 subpopulations. Furthermore, the presence of TLR5 but not NLRC4 on radiosensitive, non-LSK cells in BM was both sufficient and necessary for the observed phenomenon. Finally, adoptive transfer of MPP3 cells along with an insufficient amount of whole bone marrow cells (WBM) to lethally-irradiated mice significantly improved their survival, recapitulating the effects of Whole bone marrow from flagellin-treated mice.

Mitofusin 1 and 2 are outer-mitochondrial membrane proteins that have been shown to be involved in fusion. Mitofusin 2 has also been associated with apoptosis and development. When Mfn1 and Mfn2 were each conditionally knocked out from the cerebellum, Purkinje cells in Mfn2 deficient cerebellum during development had undergone neurodegeneration. Mutations in Mfn2 have also been associated with the Charcot Marie Tooth Type 2A (CMT2A). We want to asses the effect Mfn2 and Mfn1 might have on the development of other regions of the brain such as the telencephalon. We generated Mfn1 and Mfn2 conditional knockouts in the telencephalon by crossing them with Foxg1 Cre - a cre expressed in the telencephalon. We found that Mfn1 deficient mice have lost their corpus callosum at the midline, but survive over 6 months with a decrease in progenitor cells postnatally. Mfn2 deficient mice die between P9 and P12 with a decrease in progenitor cells postnatally and a decrease in number of neurons in the cortex. Therefore, our results suggest that Mfn1 and Mfn2 play a significant role in the development of the telencephalon.

The main goal of this study was to further explore the hypothesis that experience-dependent neural network activity and neurotransmission can modulate adult OPC proliferation and differentiation. More specifically, we used stereology to establish whether extensive reference memory training and system-wide administration of GABAergic agonists and antagonists could influence the proliferation and differentiation of adult OPCs, as well as the prevalence of OPC-neuron pairs. Analysis of the effects of reference memory training on OPC proliferation and differentiation corresponds to experiment 2, analysis of the effects of GABAergic agents on OPC proliferation and differentiation corresponds to experiment 3, and analysis of the effects of both reference memory training and GABAergic agents on OPC-neuron pairs, as well as an histological analysis of these closely apposed cells, corresponds to experiment 4.

Ageing poses one of the largest risk factors for the development of cardiovascular disease (CVD). The increased propensity towards vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells (EPCs) to contribute to vascular repair and regeneration. Among all current putative EPC populations, outgrowth endothelial cells (OECs) display the most features consistent with a human postnatal vasculogenic cell. Cell-surface heparan sulfate (HS) proteoglycans, by virtue of specific sulfated domains within the glycosaminoglycan chain, are able to bind and modulate the activities of a variety of proteins important for EPC mobilisation, homing and function at sites requiring neovascularization. This study aimed to determine if human OEC function is impaired with age, and to ascertain whether this is accompanied by changes in the fine structure of OEC HS.Using in vitro cell culture methods, OECs were isolated from healthy subjects across an age range and cell phenotype was verified by the demonstration of numerous endothelial, but not hematopoietic, cell characteristics. The functional capacity of peripheral blood derived OECs from young and old subjects, and comparative cord blood derived OECs, was assessed in terms of their susceptibility to apoptosis, proliferative, migratory and tube-forming capabilities. In vitro scratch and transwell migration assays revealed that the migratory capacity of peripheral blood derived OECs isolated from old subjects was impaired in comparison to those from young subjects and cord blood derived OECs. Structural analysis of HS by high performance liquid chromatography (HPLC) demonstrated a significant reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC HS with age (r = -0.847, p=<0.01). Moreover, a decline in the migratory response of OECs towards a gradient of VEGF significantly correlated with the percentage expression of this disaccharide (r = 0.840, p<0.01). Disruption of cell surface HS by pre-treatment with heparinase I and III was found to significantly reduce the VEGF-induced migratory response of peripheral blood derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Understanding the role of HS in regulating the directional migration of EPCs to sites requiring neovascularization and developing approaches to facilitate EPC migration may aid in the design of more successful strategies to optimise the regenerative capacity of these cells in the ageing vasculature.

MicroRNA (miRNA) function is required for normal animal development, in particular in stem cell and precursor populations. I hypothesize that miRNAs are similarly required for stem cell maintenance and appropriate fate commitment in the brain. To test the requirement for global microRNA production, I depleted the microRNA biosynthetic enzyme DICER in the developing mouse brain. I found that DICER loss in embryonic neural progenitor cells leads to embryonic lethality with microcephaly. By histological analysis, I found defects in both neural progenitor cell maintenance and cell differentiation. I also identified new candidate microRNAs for this phenotype by profiling miRNAs in DICER-depleted and control cells. Three microRNAs which are good candidates to modulate nervous differentiation are miR-23b, -182, and -34a. I describe the expression pattern and functional characterization of these candidates. In particular, miR-34a depletes neuron production after progenitor cell differentiation in culture, likely by modulating cell cycling and Notch pathway genes.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22639号 / 医博第4622号 / 新制||医||1044(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 林 康紀, 教授 伊佐 正, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

Physical or psychological chronic stress can suppress the immune system. However, the mechanisms remain to be elucidated. We investigated the effect of hematopoietic stem-progenitor cells (HSPCs) on chronic stress-induced the alterations of immune responses. We demonstrate that HSPCs prevents stress-induced lymphocyte apoptosis. Moreover, we also demonstrate that the protective effect of HSPCs on stress-induced lymphocyte reduction exerts by steroid hormones. Furthermore, we reveal that chronic stress-induced T cell-mediated immune responses contributes to the protective effect of HSPCs. These results indicate that HPSCs might offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.