Primary esophageal (E) and gastro-esophageal junction (GEJ) cancer xenografts have the potential to become useful pre-clinical models of disease. In this study, we determined that p16 negative tumors that have not been exposed to neo-adjuvant chemo-radiation have higher engraftment chances. Morphological features and expression of certain molecular markers (p53, p16, Ki-67, EGFR, Her-2/neu) suggest that no major changes occur between primary tumors and xenografts or between early passage and late passage xenografts. Global gene expression data supported these results but revealed that approximately 2000 genes differed significantly between passage one xenografts and human tumors. Most of these genes, however, might coincide with stromal signals present in patient tumors but absent in xenografts. Primary E/GEJ cancer xenografts also showed a wide range of chemosensitivities to cisplatin-paclitaxel treatment, confirming the usefulness of these models in drug testing. These models also revealed potential ways to interrogate tumor initiating cell (TIC) dynamics after chemotherapy.

Primary esophageal (E) and gastro-esophageal junction (GEJ) cancer xenografts have the potential to become useful pre-clinical models of disease. In this study, we determined that p16 negative tumors that have not been exposed to neo-adjuvant chemo-radiation have higher engraftment chances. Morphological features and expression of certain molecular markers (p53, p16, Ki-67, EGFR, Her-2/neu) suggest that no major changes occur between primary tumors and xenografts or between early passage and late passage xenografts. Global gene expression data supported these results but revealed that approximately 2000 genes differed significantly between passage one xenografts and human tumors. Most of these genes, however, might coincide with stromal signals present in patient tumors but absent in xenografts. Primary E/GEJ cancer xenografts also showed a wide range of chemosensitivities to cisplatin-paclitaxel treatment, confirming the usefulness of these models in drug testing. These models also revealed potential ways to interrogate tumor initiating cell (TIC) dynamics after chemotherapy.

Droplet microfluidics is a promising field since it overcomes many of the limitations of single phase microfluidic systems. The improved mixing time scale, the increase of number of samples and the isolation of droplets are some of its virtues. The core of droplet microfluidics is a two-phase flow condition that is subjected to scaling of the confining geometry. With the scaling the complexities of the flow phenomena arise. For that reason both the processes of droplet generation and transport are not fully understood for various flow and fluid conditions. The work in this thesis aims to experimentally examine droplet generation and transport in microchannels for flow and fluid conditions that are experimentally challenging to perform. Examination of droplet generation in a T-junction microchannel design was performed with a quantitative velocity field approach known as micro particle image velocimetry (μPIV). The studies on droplet generation focused on very fast generation regimes, namely transition and dripping that have not been studied for a T-junction design. This achievement was accomplished because of the development of a fast optical detection and triggering system that allowed for acquiring images of different identical droplets at the same position. μPIV results indicate that the quantitative velocity field patterns of different regimes share some similarities. The filling stage in the transition and dripping regimes had some resemblance in their velocity patterns. The velocity patterns for the start of droplet pinch-off were alike for the squeezing and transition regimes. Furthermore, the presence of a surfactant in the droplet phase above the critical micelle concentration (CMC) did not have an effect on the general velocity patterns as long as the capillary number Ca was matched with the no-surfactant condition. The studies of hydrodynamic properties of droplet transport were performed in hard materials to avoid cumulative error sources, such as material pressure compliance and swelling effects. The project had several parts: designing a microchannel network that allowed studying the hydrodynamic properties of small droplets, surface treatments of the channel material for stable droplet generation and examining the hydrodynamics of small liquid droplets with sizes that have not been reported in the literature. The studies examined effects of changing the interfacial tension, viscosity, and flow conditions on the transport of droplets. The experimental results from the hydrodynamic transport studies indicated that for the droplet sizes that were examined the pressure drop of droplets was affected by the capillary number Ca and length of the droplet Ld. Also, the presence of surfactants altered the hydrodynamic properties of droplets. At a high concentration of surfactants the droplets pressure drop was reduced significantly. Moreover, the type of surfactant affected the magnitude of the pressure drop. Experimental results indicate that if the concentration of surfactants was very low (below CMC) it did not have an effect on the droplet excess pressure. These findings are important to consider in designing droplet microfluidic systems with complex channel networks that involve droplet sorting, splitting, and merging for droplets that contain surfactants.

The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

The role of gap junctions in modulating the dynamics of axonal dysfunction in spinal cord white matter injury remains uncertain; hence, I examined the functional role and changes in expression of gap junctions following CNS injury. I hypothesized that inhibition of gap junctions improves axonal conduction during oxygen and glucose deprivation (OGD) in vitro. Carbenoxolone and octanol, gap junction blockers, did not change CAP amplitude in non-injured tissue, yet they significantly reduced the extent of its decline during OGD. No difference in mRNA expression of connexins 32, 36 was found. However, during OGD in the presence of gap junction blockers, expression of connexins 30, 43 was downregulated. Immunohistochemistry confirmed the presence of connexins in spinal cord slices: connexins 30, 43 overlapping with GFAP, connexin 32 with MBP and connexin 36 with CC1. Thus, blocking gap junctions enhances axonal conduction during OGD and promotes dynamic changes in connexin mRNA expression.

Fabrication of nanoscale Josephson junctions and Superconducting Quantum Interference Devices (SQUID) is very promising but challenging topic in the superconducting electronics and device technology. In order to achieve best sensitivity of SQUIDs and to reproduce them easily with a straightforward method, new fabrication techniques for realization of nanoSQUIDs needs to be investigated. This study concentrates on investigation of new fabrication methodology for manufacturing nanoSQUIDs with High Temperature Bi-Crystal Grain Boundary Josephson Junctions fabricated onto SrTiO3 bi-crystal substrates using YBa2Cu3O7-δ (YBCO) thin-films. In this process nanoscale patterning of YBCO was realized by using electron beam patterning and physical dry etching of YBCO thin films on STO substrates. YBCO thin films were deposited using RF magnetron sputtering technique in the mixture of Ar and O2 gases and followed by annealing at high temperatures in O2 atmosphere. Structural characterization of YBCO thin films was done by Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Spectroscopy (EDX). Superconducting properties of thin films was characterized by AC magnetic susceptibility measurements. Nanoscale structures on YBCO thin films were fabricated by one E-Beam Lithography (EBL) step followed by Reactive Ion Etching (RIE) and physical dry etching. First SiO2 thin film were deposited on YBCO by RF magnetron sputtering and it was patterned by EBL using Polystyrene (PS) as resist material and RIE. Then SiO2 was used as an etch mask for physical dry etching of YBCO and nanoscale structures on YBCO were formed.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23901号 / 工博第4988号 / 新制||工||1779(附属図書館) / 京都大学大学院工学研究科材料工学専攻 / (主査)教授 田中 功, 教授 杉村 博之, 准教授 野瀬 嘉太郎 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [104]-122).