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京都大学 / 新制・課程博士 / 博士(理学) / 甲第24081号 / 理博第4848号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 川口 真也, 准教授 船山 典子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Gliomas are the most common and difficult to treat tumors of the central nervous system. Current treatments often fail to slow progression of disease due to the high invasive nature of glioma leading to a high percentage of recurrence. Our previous studies have demonstrated that the levels of alpha; 9 beta; 1 integrin found on high grade glioma were significantly increased in comparison to normal brain tissue where the levels were negligible. We also found that interaction between alpha; 9 beta; 1 integrin and nerve growth factor (NGF) plays a major role in progression of experimental tumor. Another receptor for NGF the common neurotrophin receptor p75NTR is also overexpressed in high grade glioma. p75NTR forms a high affinity complex with the specific NGF receptor, TrkA leading to an increase in cell proliferation and survival. In the absence of an association, p75NTR is involved in transferring pro-apoptotic signals through the JNK pathway. We have found that the α 9 integrin subunit of α 9 β 1 forms a stable, cation independent complex with p75NTR on the cell membrane of glioma both in vitro using glioma derived immortalized cells lines and in vivo using glioma tissue. The co-expression of p75NTR with α 9 β 1 integrin led to optimization of integrin-dependent cellular activities such as cell survival, proliferation, and migration. Co-expression of p75NTR was also required for implanted glioma cells to migrate in a glioma-like perivascular manner away from the site of implantation as was seen in the in vivo quail chorioallantoic membrane assay. / Biology

The mammalian palate develops early in embryogenesis by way of a carefully orchestrated series of temporally and spatially regulated signaling events. Molecular signaling pathways that have been proven to be vital to the process of palatogenesis include TGF-βs, BMPs, FGFs, EGF, and Wnts. The absence of connective tissue growth factor (CTGF/CCN2) has been shown previously to cause failure of proper palatogenesis, i.e. cleft palate. However, the details about the phenotype of this model of cleft palate were scarce. Additionally, CCN2 is known to interact with TGF-βs, BMPs, FGFs, EGF, and Wnts, though information on how these pathways were impacted in the developing palate lacking CCN2 were also not available. In Chapters 2 and 3, through our use of gross specimen and histological examination combined with cell and organ culture, we produced the most detailed characterization of the CCN2 knockout (KO) model of cleft palate with identification of negatively affected signaling pathways that lead to the clefting phenotype. Collection and examination of gross and histological sections revealed at 100% penetrance of cleft palate in which development is impaired around the phase of palatal shelf elevation. Organ culture also revealed that when artificially apposed, the CCN2 KO model system also suffers from a fusion deficit. Finally, utilizing cells isolated from the developing palates, we found a reduction in proliferation, adhesion, and spreading with an enhanced migratory ability. Addition of recombinant CCN2 was able to rescue cell spreading but not proliferation. CCN2 as an immobilized substrate did not rescue adhesive ability. Decreased adhesion and spreading in the KO cells are attributed to the inability of the KO cells to activate Rac1 and RhoA. Examination of gene expression differences by mRNA-sequencing and qRT-PCR revealed numerous gene expression alterations between the wild type (WT) and the KO palates, most notably FGF4 and EGFR. Addition of FGF4 or EGF to cell culture was unable to promote increased proliferation in the KO cells while producing a response in the WT cells. Examination of downstream signaling revealed highly amplified and prolonged ERK1/2 signaling in the FGF4 treated palate cells indicating that FGF signaling is significantly altered in the absence of CCN2. Treatment of the cells with EGF produced a response proportional to EGFR expression differences indicating that EGFR signaling is not impacted beyond the receptor protein levels. The link between EGFR protein levels and FGF mediated ERK1/2 activation is a protein called Spry2. We found greatly reduced Spry2 mRNA levels in the KO palates and upon FGF4 stimulation at 24 hours of exposure indicating that in the absence of CCN2, proper inhibition of FGF signaling and EGFR degradation is negatively altered. Collectively, the data demonstrate that CCN2 is vital to palatogenesis by impacting proliferation, shelf elevation, and shelf fusion through increased FGF signaling and reduced EGFR signaling resulting partially from reduced Spry2 activity. / Biomedical Sciences

Soil nutrients are often heterogeneously distributed in space and time at scales relevant to individual plants, and plants can respond by selectively proliferating their roots within nutrient-rich patches. However, many environmental factors may increase or decrease the degree of root proliferation by plants. I explored how soil fertility, nitrogen (N) or phosphorus (P) limitation, and soil oxygen availability affected root system response to nutrient heterogeneity in forested wetland ecosystems of southeastern United States. Fine root biomass was not correlated with soil nutrient availability within wetland ecosystems, but was related to ecosystem-scale fertility. Root systems generally did not respond to P-rich patches in both floodplain (nutrient-rich) and depressional swamps (nutrient-poor) swamps, but results were inconclusive because the growth medium (sand) potentially hindered root growth. In floodplain forests, roots proliferated into N-rich patches but not P-rich patches, even though litterfall N:P ratios were > 15, which suggested that these ecosystems were P-limited. The combination of nutrient and oxygen heterogeneity affected root proliferation and biomass growth of three common floodplain forest species (Liquidambar styraciflua, Fraxinus pennsylvanica, and Nyssa aquatica) in a potted study, which was related to species' flood tolerance. My results suggest that the environmental context of plants can affect roots system response to nutrient heterogeneity in forested wetland ecosystems and highlights the need for field studies that investigate this phenomenon. Learning how environmental conditions affect plant response to nutrient heterogeneity at a fine-scale will provide better predictions of nutrient cycling, plant competition and succession, and forest productivity, which are important factors that determine carbon sequestration and timber production. / Ph. D.

Resources in the soil are heterogeneously distributed. We know that plant species differ in their root responses to nutrient patches and that these differences in foraging can influence plant competition. However, most studies of root-resource interactions overlook the potential top-down influence of root herbivores. While root herbivores can influence plant community structure, the extent to which they influence ecosystem-scale factors such as net primary production is unclear. In addition, little is known regarding root herbivore foraging behaviors and, more importantly, whether these foraging behaviors can actually influence species interactions. In this dissertation, I present a conceptual model of soil-root-herbivore interactions in which soil resource heterogeneity structures both root dynamics and the abundance and influence of root herbivores. I conducted two field and one greenhouse experiment examining this proposed model. The dissertation includes an introductory chapter (Chapter 1), a field study examining root responses to manipulations of soil fertility and root herbivory (Chapter 2), a greenhouse study that used plant species responses to heterogeneity to develop predictions about the role of root herbivores in mixed-species neighborhoods (Chapter 3), and a field study of planted communities examining soil fertility and fauna effects on above- and belowground structure and function (Chapter 4). In all cases, there were significant effects of root herbivores on community structure and components of net primary production. Resource distribution had a strong effect in studies conducted in sandy, nutrient-poor soils (Chapter 2 and 3), but had a reduced effect in the study conducted at Kentland Farm in loamy soils (Chapter 4). Interactions between resource availability and root herbivory were common. These results support the theory that the potential benefit of resource-rich patches may be constrained by root herbivores. This research complements recent findings that demonstrate other potential costs of species foraging behaviors (such as exposure to soil anoxia and increased drought stress), as well as potential effects of root herbivores and other soil fauna on plant diversity. / Ph. D.