The final publication is available at http://dx.doi.org/10.1111/pcmr.12297. Natsuko Okamoto et al. A melanocyte–melanoma precursor niche in sweat glands of volar skin. Pigment Cell & Melanoma Research. Volume 27, Issue 6, pages 1039–1050, November 2014 / 京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12890号 / 論医博第2090号 / 新制||医||1007(附属図書館) / 31644 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 羽賀 博典, 教授 鈴木 茂彦 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

This master's thesis deals with deposition of cobalt nanostructures using Electron Beam Induced Depositon (EBID). The thesis firstly describes theoretical background regarding EBID. In the following experimental part, the deposition process is optimized via analysis of exposition parameters and their influence on the final shape of nanostructures. These are analyzed via Atomic Force Microscopy (AFM). Optimized exposure parameters are then applied to the deposition of functional 3-dimensional nanostrucutres. In particular, the deposition of cobalt spheres. The elemental composition is analyzed by the means of element composition using Energy-dispersive X-ray Spectroscopy (EDX) and the nanostructures of desired morphologies are studied with Magnetic Force Microscopy (MFM) and Magneto-Optical Kerr Microscope).

Sn (II) oxide finds numerous applications in different fields such as thin film transistors1, solar cells2 and sensors.3 In this study we present the fabrication of tin monoxide SnO by using Sn (II) ketoacid oximate complexes as precursors. Tin (II) ketoacidoximates of the type [HON=CRCOO]2Sn where R= Me 1, R= CH2Ph 2, and [(MeON=CMeCOO)3Sn]- NH4 +.2H2O 3 were synthesized by in situ formation of the ketoacid oximate ligand. The crystal structures were determined via single crystal X- ray diffraction of the complexes 1-3 revealed square planar and square pyramidal coordination environments for the Sn atom. Intramolecular hydrogen bonding is observed in all the complexes. Furthermore, the complexes were characterized by Infrared (IR), Nuclear Magnetic Resonance (NMR) and elemental analysis. From thermogravimetric analysis of 1-3, it was found that the complexes decomposed in the range of 160 – 165 oC. Analysis of the gases evolved during decomposition indicated complete loss of the oximato ligand in one step and the formation of SnO. Spin coating of 1 on silicon or glass substrate show uniform coating of SnO. Band gaps of SnO films were measured and found to be in the range of 3.0 – 3.3 eV by UV-Vis spectroscopy. X-ray photoelectron spectroscopy indicated surface oxidation of the SnO film. Heating 1 above 140 oC in air gives SnO of size ranging from 10 – 500 nm and is spherical in shape. The SnO nanomaterial is characterized by powder X-ray diffraction(XRD), Raman spectroscopy, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM).

Surgical skin flaps are frequently used in plastic and reconstructive surgery to repair acquired or congenital defects. Necrosis is a common complication associated with these flaps postoperatively as a result of inadequate blood supply. Stem cells are precursor cells with the potential to differentiate into more specialized cells. Angiogenic factors act to direct cellular differentiation and organization to form new vascular elements. Our theory was that the combination of angiogenic growth factors with stem cells derived from the subject preoperatively would augment neovascularization, thereby increasing blood supply to the flap, which may ultimately improve flap survival. In phase I, 40 Lewis rats were randomized into 4 groups of 10. Random dorsal skin flaps were elevated and treated at the same time. The first group was injected with only medium, the second with stem cells, the third with stem cells and angiogenic factors, and the fourth with angiogenic growth factors. Millimetric measurements of flap viability at 7 and 14 days did not show any statistically significant differences between the studied groups. In phase II, 24 rats were also randomized into 4 groups of 6, but this time were treated 1 week before flap elevation. The viability measurements showed an increased rate of viability in the group in which stem cells and the angiogenic factors were administered simultaneously (84.5% ± 3.2%) as compared with the unmodified control group (62.6% ± 7.3%) or to the groups in which only precursor cells (60.4% ± 7.9%) or angiogenic factors (62.3% ± 10.1%). Increased blood supply brought by these manipulations is believed translated to increased tissue flap survival. Punch biopsies showed that "green fluorescent protein"-labeled precursor cells was noted to form luminal structures in the treated flaps. The vascular cast of all flaps was filled with Mercox plastic resin. After euthanasia, the soft tissues of the harvested flaps were dissolved and the remaining vascular cast was weighted. The weight-based ratio of the vascular composition was determined. The flaps injected with both stem cells and angiogenic factors showed higher values. We conclude that the administration of bone marrow stem cells with angiogenic factors 1 week before flap creation improves the survival of ischemic random skin flaps.

This study consisted of three parts. We first used a video observation method and statistical analysis to identify benign behaviors that occurred before the problem behavior. These benign behaviors are known as precursor behaviors. We then used a precursor functional analysis to identify the function of the precursor behaviors. Lastly, we developed and implemented an intervention based on the results of the precursor functional analysis. The interventions effectively reduced problem behavior for the participants in the study. The participants engaged in minimal instances of problem behavior throughout the study. The results of this study may be useful to teachers and caregivers of children who engage in severe problem behavior that would be unsafe to reinforce in an assessment. Results may also help when working with children whose caregivers are concerned with their children engaging in excessive problem behavior. Further research is needed to look at modifying the precursor analysis. Future research should also look at the social validity of the precursor assessment.

Extensive research has been conducted demonstrating the utility of differential reinforcement as an effective intervention for self-injurious behavior. However, the majority of this literature requires teaching an alternative response to access reinforcement. Further evaluation of treating self-injurious behavior in individuals that already possess the repertories to contact reinforcement appropriately. Prior to initiating the study, functional assessments were completed for both participant that demonstrated high-rate bursts of mands served as a reliable precursor to self-injurious behavior. In the present study, we evaluated a waiting period and differential reinforcement of low rate behavior on reducing mands while keeping self-injurious behavior at or near zero levels. Results indicated that shorter waiting periods and DRL values were effective at reducing mands and maintaining near zero levels of self-injurious behavior.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19036号 / 農博第2114号 / 新制||農||1031(附属図書館) / 学位論文||H27||N4918(農学部図書室) / 31987 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙部 圭司, 教授 髙野 俊幸, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Evaporation of pure and binary liquid droplets is of interest in thermal sprays and spray drying of food, ceramics and pharmaceutical products. Understanding the rate of heat and mass transfer in any drying process is important not only to enhance evaporation rate or vapor-gas mixing, but also to predict and control the final morphology and microstructure of the precipitates. Acoustic levitation is an alternative method to study micron-sized droplets without wall effects, which eliminates chemical and thermal contamination with surfaces. This work uses an ultrasonic levitation technique to investigate the vaporization dynamics under radiative heating, with focus on evaporation characteristics, precipitation kinetics, particle agglomeration, structure formation and droplet stability. Timescale and temperature scales are developed to compare convective heating in actual sprays and radiative heating in the current experiments. These relationships show that simple experiments can be conducted in a levitator to extrapolate information in realistic convective environments in spray drying. The effect of acoustic streaming, droplet size and liquid properties on internal flow is important to understand as the heat and mass transfer and particle motion within the droplet is significantly controlled by internal motion. Therefore, the droplet internal flow is characterized by Particle Image Velocimetry for different dropsize and viscosity. Nanosuspension droplets suspended under levitation show preferential accumulation and agglomeration kinetics. Under certain conditions, they form bowl shaped structures upon complete evaporation. At higher concentrations, this initial bowl shaped structure morphs into a ring structure. Nanoparticle iv migration due to internal recirculation forms a density stratification, the location of which depends on initial particle concentration. The time scale of density stratification is similar to that of perikinetic-driven agglomeration of particle flocculation. The density stratification ultimately leads to force imbalance leading to a unique bowl-shaped structure. Chemically active precursor droplet under acoustic levitation shows events such as vaporization, precipitation and chemical reaction leading to nanoceria formation with a porous morphology. The cerium nitrate droplet undergoes phase and shape changes throughout the vaporization process followed by formation of precipitate. Ex-situ analyses using TEM and SEM reveal highly porous morphology with trapped gas pockets and nanoceria crystalline structures at 70oC. Inhomogeneity in acoustic pressure around the heated droplet can induce thermal instability. Short wavelength (Kelvin-Helmholtz) instability for diesel and bio-diesel droplets triggers this secondary atomization, which occurs due to relative velocity between liquid and gas phase at the droplet equator. On the other hand, liquids such as Kerosene and FC43 show uncontrollable stretching followed by a catastrophic break-up due to reduction in surface tension and viscosity coupled with inhomogeneity of pressure around the droplet. Finally, a scaling analysis has been established between vaporizing droplets in a convective and radiative environment. The transient temperature normalized by the respective scales exhibits a unified profile for both modes of heating. The analysis allows for the prediction of required laser flux in the levitator experiments to show its equivalence in a corresponding heated gas stream. The theoretical equivalence shows good agreement with experiments for a range of droplet sizes.

Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome but why some brain cells and structures are initially spared by the virus is unknown. Here, a novel murine model of ZIKV infection incorporating in utero electroporation with cell type specific promotors was used to identify the time course of ZIKV infection and to determine which neural precursor cells are initially infected or spared. In vivo time course studies revealed early presence of ZIKV in apical radial glial cells (aRGCs) while infection of basal intermediate progenitor cells climbed after three days of virus exposure. ZIKV-exposed fetal brains exhibited microencephaly as early as 1 day post injection, caused by apoptosis and reduced proliferation, and this change in brain size persisted until birth regardless of developmental age at infection. During infection, 60% of aRGC basal fibers were perturbed while 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To evaluate this heterogeneous vulnerability, we generated cell type-specific fate mapping plasmid probes using a previously published single cell RNA-Seq dataset on the E15.5 mouse neocortical wall. The results indicate that one class of aRGC preferentially expresses the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than the other aRGC subtypes with low AXL expression. Together, these data highlight important temporal and cellular details of ZIKV fetal brain infection and may be important for prevention strategies and for management of congenital Zika syndrome.

Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors.