One of the most common disabilities in the US, hearing loss, is reported by The National Institutes of Health to affect approximately 36 million Americans. One of the major contributing factors to this loss in hearing is the loss of the sensory hair cells (HCs) within the cochlea. Also in the mammalian cochlea, six major groups of supporting cell (SC) subtypes reside in close proximity to HCs and may have the potential to regenerate HCs after damage. These subtypes include cells of the greater epithelial ridge, inner phalangeal/border cells, inner and outer pillar cells, Deiters’ cells, Hensen cells, and Claudius cells. During embryonic development, progenitor cells differentiate into HCs or one of the SC subtypes by Notch-mediated lateral inhibition. In the neonatal mouse cochlea, many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal undamaged cochleae, as well as in drug-damaged cochlear explants. This mechanism is also implicated during spontaneous HC regeneration that occurs in non-mammalian vertebrates. We and others have recently observed that spontaneous HC regeneration can also occur in the neonatal mouse cochlea. However, little is known about the molecular mechanism or the SC subtypes which act as the source of regenerated HCs. In the neonatal mouse cochlea, HCs were killed in vivo at birth using a genetically-modified mouse model to express a toxin in HCs. Subsequently, SCs formed new HCs by either direct transdifferentiation, where no cell division occurred, or by mitotic regeneration. My dissertation investigated the role of Notch signaling in the ability of SC subtypes to regenerate HCs after damage. My central hypothesis is that after HC ablation is induced at birth, Notch signaling is partially eliminated and therefore lateral inhibition is lost in neonatal SCs in a subtype specific manner, which allows some SCs, but not others, to differentiate into and regenerate HCs. Aim 1 focused on changes in the Notch signaling pathway in response to HC damage during the window of spontaneous HC regeneration. Changes in the expression of genes in the Notch pathway were measured using real time qPCR, immunostaining, and in situ hybridization. The Notch effector HeyL was increased in the apical one-third of the cochlea while other Notch players are decreased. The most notable example is the Notch effector Hes5, which is directly responsible for inhibiting HC fate, and was reduced in outer pillar cells and Deiters’ cells, but not in other SC subtypes. From this we conclude that Notch signaling is reduced differentially among SC subtypes. In Aim 2 we investigated whether inhibition of Notch signaling is required for spontaneous HC regeneration to occur by maintaining active Notch signaling in all SCs in the context of HC damage. We hypothesized that maintaining active Notch signaling after HC damage will prevent SC-to-HC conversion thus preventing HC regeneration. We found significantly fewer regenerated HCs while maintaining Notch expression compared to controls with HC damage and no manipulation of Notch signaling. Therefore we conclude loss of Notch mediated lateral inhibition is required for the majority of spontaneous HC regeneration. In Aim 3 we investigated the ability of different SC subtypes to regenerate HCs by fate-mapping SC subtypes during the HC regeneration process. Since fate-mapping creates a permanent label in targeted cells, we can track their potential change in cell fate or reentry in the cell cycle after HC damage. We hypothesized that pillar cells and Deiters’ cells are the source for spontaneously regenerated HC within the neonatal mouse cochlea based on our results from Aim 1. We used three CreER mouse lines to fate-map distinct groups of SC subtypes during the HC damage and regeneration process. More pillar and Deiters’ cells regenerated HCs after damage than other SC populations. We found that outer pillar cells and Deiters’ cells are capable of downregulating the cell cycle inhibitor, p27Kip1, after HC damage. Therefore we investigated the ability of SC subtypes to mitotically regenerate HCs by including a mitotic tracer along with fate-mapping. A larger proportion of mitotically regenerated HCs came from pillar and Deiters’ cells. From these experiments, we conclude that outer pillar and Deiters’ cells are the source for the majority of spontaneously regenerated HCs in vivo. This knowledge will allow targeted investigation into outer pillar cells and Deiters’ cells that maintain regenerative plasticity at postnatal ages. Understanding how these cells change with age will inform efforts to induce HC regeneration in more mature cochleae. Additionally, understanding how Notch signaling regulates this regenerative plasticity will lead to the development of potential targets for the treatment of hearing loss.

The olfactory system exhibits remarkable regenerative ability in it’s neuronal population. The success of continuous neurogenesis is thought to be due, at least in part, to its unique glia – olfactory ensheathing cells (OECs). OECs bear characteristics of both peripheral and central glia, and serve to ensheath, guide and promote growth of olfactory receptor neurons (ORNs) throughout both development and adult life. Brain lipid binding protein (BLBP) is most highly expressed by radial glia during embryonic development. It is largely down-regulated in the adult CNS, but BLBP expression is retained in the adult by special subpopulations of glia, including OECs. BLBP expression is induced in radial glia via Notch signaling, but it is not known if these same mechanisms regulate BLBP expression in the adult CNS. Axonal-glial signaling is a dynamic process whereby closely apposed neuronal and glial cells regulate the growth, maintenance and plasticity of one another through direct cell-cell signaling. Delta/Notch-like EGF-related receptor (DNER) is a transmembrane protein expressed by Purkinje cells which has been implicated in the regulation of BLBP in Bergmann glia during cerebellum development through Notch1 deltex-dependent non-canonical signaling. We have found that DNER is expressed in more mature ORNs, and other exclusive subpopulations of cells within the CNS. OECs in close apposition with DNER-expressing ORNs in vivo appear to maintain the highest BLBP expression found in the nervous system through development and adulthood. Immunofluorescence shows that this close relationship between BLBP expressing cells and DNER expressing cells also appears to be retained in specialized areas such as the hippocampus, retina and spinal cord, throughout mouse CNS development as well as in the mature system. Removing DNER or axonal input in vivo decreases the robustness of OEC BLBP expression, and the number of cells in OEC culture expressing BLBP decreases rapidly with time. OEC co-culture with a DNER expressing monolayer increases the number of OECs in vitro which express BLBP, providing evidence for the regulation of BLBP expression in OECs by DNER expression in apposing ORNs. / Medicine, Faculty of / Graduate

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with a dismal prognosis. Few patients present with disease amenable to resection and chemotherapy is not curative. The incidence of ICC is rising worldwide and new therapeutic approaches are urgently required. Notch signalling is critical for the embryological development and regeneration of the biliary tree in the mammalian liver. Dysregulation of Notch is known to drive tumorigenesis in a range of solid and haematological malignancies and the aim of this work was to define its contribution to the pathogenesis of ICC. Transgenic overexpression of Notch1 has been described to result in the formation of biliary lineage tumours in the liver. I have used resected human tissue, a chemically-induced model of ICC in rat and a novel transgenic murine model in which the tumour suppressor p53 is conditionally deleted from biliary epithelia, to demonstrate that endogenous Notch signalling is acting via the Notch3 receptor to drive tumorigenesis. I use multiple independent methods of Notch3 blockade to establish that Notch3 promotes epithelial cell survival and self-renewal in ICC and demonstrate that Notch3 inhibition significantly attenuates tumour growth in vivo. My data suggest that Notch3 promotes activity through the PI3K/AKT cell survival cascade via a mechanism independent of the effector of canonical Notch, RBPJκ. Given the significant toxicity associated with gamma-secretase inhibitors these findings offer a novel and specific target for further investigation and future therapeutic development in ICC.

The development of complex metazoans depends on the integration of a handful of signalling pathways that eventually modulate precise patterns of gene expression. The fact that just a few pathways are involved in the generation of such complexity in different organisms, suggests that these are highly regulated and conserved processes. The accurate spatio-temporal coordination of the signalling pathways controls the assignation of different cell fates and their patterning into tissues and organs. The source of diversity relies on the different possible interactions between signalling pathways, such as, the combination of signals and the order in which they are received by the cell or crosstalk. Due to their importance in development, abnormal signalling through these pathways has been strongly associated with developmental disorders, cancers and other diseases. The Notch and Wnt signalling pathways are key components of the intricate network that controls gene expression during development, and genetic analysis in Drosophila has highlighted that interactions between these two signalling pathways are important during this process.This thesis investigates the cross-regulatory interactions between Notch and Wnt signalling pathways in mammals. Using transcriptional reporter assays and biochemical analysis, I have found two molecular mechanisms underlying the inhibitory crosstalk between Notch and β-catenin, the effector of Wnt signalling pathway, in mammalian cells. At the membrane Notch inhibits β-catenin transcriptional activity through Deltex mediated endocytosis of Notch and a component required for β-catenin activation. This is similar to results observed in Drosophila. In the nucleus, I have identified a novel mechanism by which NICD-dependent transcription of Hes/Hey family of transcription factors prevents the activation of Wnt signalling pathway. This mechanism involves the formation of a physical complex between Hey1 and β-catenin/TCF, which allows Hey to block Wnt transcriptional activation. Additionally, I have found that these two mechanisms are conserved across vertebrates.Together the findings of this thesis improve our understanding of the molecular mechanism underlying the Notch/Wnt crosstalk. In turn, this will give an insight into unravelling how a handful of signalling pathways can generate sufficient diversity in signalling output to specify the hundreds of different cell fates generated to make a mammal. Elucidating these signalling networks will also contribute to our understanding of diseases, both their aetiology, by knowing how changes in one signal can influence another, and their treatment as mimicking points of crosstalk is likely to generate very specific therapeutic agents.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13364号 / 論医博第2206号 / 新制||医||1045(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 高橋 淳, 教授 髙橋 良輔, 教授 斎藤 通紀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13384号 / 論医博第2216号 / 新制||医||1048(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 高橋 淳, 教授 渡邊 直樹, 教授 中川 一路 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Research aimed at optimizing diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses resulting in reproductive losses due to foetal death or persistently infected (PI) calves that usually die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of BVDV infection. Persistently infected animals that survive calfhood are at risk of developing mucosal disease in later life which is a severe and usually fatal condition. In addition, persistently infected calves that become replacement heifers in the herd may experience significant morphological changes that occur in the ovaries which can result in impaired reproductive performance. This poses important challenges to overall animal/herd health and causes losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected (PI) with BVDV may affect the ability of diagnostic assays to efficiently detect the virus. This study assessed the effects of 1) long-term storage of formalin-fixed samples at room temperature to detect BVD viral antigen with the aid of immunohistochemistry (IHC), 2a) long-term storage of fresh ear notch samples kept at -20°C, and 2b) long term storage of phosphate-buffered saline (PBS) ear notch supernatant kept at -20°C on the ability of an antigen-capture ELISA (AC-ELISA) to detect viral antigen. Previous studies have verified 100% sensitivity for both AC-ELISA on ear notch supernatant and immunohistochemical testing of ear notches to detect BVDV provided that samples are properly collected and stored. In this study, ear notch samples from seven animals were subjected to prompt formalin fixation and fresh samples to prolonged storage at -20°C. Frozen ear notches and ear notch supernatant yielded positive results on AC-ELISA for the duration of the study, i.e. 6 months, and OD values remained significantly within range. There was no significant difference between storing fresh ear notch samples and PBS ear-notch supernatant at -20°C. However, positive IHC staining on formalin-fixed ear notches started to fade away between day 17 and day 29 when stored at room temperature. We conclude that fresh ear notches could safely be stored at -20°C for a period of 6 months for detecting BVD viral antigen at a later stage. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19224号 / 医博第4023号 / 新制||医||1010(附属図書館) / 32223 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 稲垣 暢也, 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

Carbon fiber composites are continually seeing increased use in aerospace applications. It is necessary to understand their failure modes in order to properly design and perform analysis on structures constructed primarily from them. This thesis studies woven carbon fiber composites with and without double-edge notches in a series of static and fatigue tests performed on an Instron 8801 servo-hydraulic testing system. Specimens were constructed of Advanced Composites Group product # LTM45EL woven carbon fiber pre-preg/epoxy and were cut to approximately 9-inch in length and 1-inch in width. Notches were cut into some of the specimens using a slitting saw blade of 0.006-in. thickness. Ultimate strength, Young’s modulus and Poisson’s ratio for specimens were determined to be 119,418 psi, 7,149,000 psi and 0.05, respeictively. Fracture stress for specimens with notch depths of 0.10, 0.15, 0.20, 0.25, 0.30 and 0.35 was determined to be 93,481 psi, 88,193 psi, 86,968 psi, 81,112 psi, 84,197 psi and 81,955 psi, respectively. The results from these tests showed that the specimens followed Griffith’s model for brittle failure. Average number of cycles to failure was determined to be 6,600, 37,200, 94,300 and 293,400 for fatigue tests with maximum stresses of 72.5%, 75%, 77.5% and 80% of the ultimate strength. Fatigue tests performed on notched specimens produced data that was too scattered from which to draw a statistically significant result. Numerical modeling in Abacus showed comparable results to experimental tests for stress and strain.

No description available.