京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20982号 / 医博第4328号 / 新制||医||1027(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 川口 義弥, 教授 瀬原 淳子, 教授 野田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21335号 / 医博第4393号 / 新制||医||1031(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 髙折 晃史, 教授 濵﨑 洋子, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

第一章 緒論 說明本文為因應生物科技之發展，除了新技術的衝擊外更加上了” 人性尊嚴”的基本上位觀念，故於科學研究上，從胚胎研究階段此涉及了物化之價值觀，而出生之後更涉及了血統身份的法律關係。故希望將目前最熱門也最有爭議之「胚胎幹細胞」及「複製人」以生物發育之過程加以串聯，並以生技產業及人文法學兩個角度加以探討。 第二章 胚胎複製之概說與流程 藉著一個細胞新生命的生長過程，而展開本論文之主軸；由一個複製胚胎細胞開始，接著發育成6-8個胚囊內組織，此後即可取出作為胚胎幹細胞之來源；然若直接植入子宮而發育出生，此時即有複製人之法律關係。 第三章 胚胎複製細胞 於第一階段之「胚胎複製」階段之研究，首先就需探討「胚胎細胞」此一生命體之法律地位及科學研究之適法性。本文將於第一節由生技產業面之實際技術方法加以說明，再於第二節依宗教面、道德觀、法理學，法律面綜合加以探討。 第四章 治療性複製 ( 胚胎幹細胞 ) 於治療性複製 (therapeutic cloning)上，亦分為生技產業及法律建制兩方面來觀察。 其中在「生技產業」面下，又再細分1.基礎技術： 複製技術及2.臨床醫療: 幹細胞產業。於此將由基礎技術中來介紹幹細胞之分類，如胚胎幹細胞、成人幹細胞、全能及多能幹細胞，及在臨床醫療下來介紹最重要的於醫療實益。 於「法律建制」面下，則先由宗教道德面，再論及法律面。於道德爭議上主要為1.複製有機體之地位:2.造人再殺人?。於各國比較法制方面則包含各國民俗背景及法律發展，如英國法 、德國法 、美國法 、及國際法等。最後論及我國現有法制，包含了憲法位階之學術自由，法律位階之優生保健法，職權命令之人工協助生殖技術管理辦法；及行政指導性質之胚胎幹細胞研究的倫理規範。而待審法案則有人工生殖法草案。 第五章 生殖性複製 ( 複製人 ) 在生殖性複製 ( reproductive cloning)上，亦分為生技產業及法律建制兩方面來觀察。 故首先於第二節討論「生技產業面」，亦分為第一項之基礎技術研究及第二項的臨床醫療應用 兩方面加以探討。對於複製人之研究，此即使在醫學界亦有正反兩說，否定說強調目前科學技術上並未成熟，常常存在尚未明瞭的缺陷，例如早期衰老及發育殘障，動物實驗上成功率甚低。然而肯定說則強調醫療科技之進步，複製技術終將改良。 其次於第三節討論人文面之「道德法律面」。第一項首先論及宗教與道德之爭議，於宗教面自然不容科學家違反自然而取代神的地位，道德則涉及人性尊嚴而不可物化，及何謂「人」與「物」之主客體地位之區分。第二項則著重在法律面的探討，雖然研究人員常強調學術自由，參與複製者亦自認生殖方式之自由不容剝奪，然而目前在實際上，無論各國之比較法之研究，或是我國之法規命令皆不容許複製人之誕生。第三項則涉及然若複製胎兒一旦出生，則有「人」之主體地位及身份關係的問題。原則上，出生後其為「權利主體」地位之保障，自無庸置疑，然而其與原複製者之身分關係，究竟如何定位，則值得由民法親屬編及相關法規命令來加以討論。 第六章 結論 總結上述各章，並提出相關看法與建議

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23745号 / 理博第4835号 / 新制||理||1691(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 / DGAM

学位授与大学：京都大学 ; 取得学位: 博士(工学) ; 学位授与年月日: 2007-09-25 ; 学位の種類: 新制・課程博士 ; 学位記番号: 工博第2865号 ; 請求記号: 新制/工/1421 ; 整理番号: 25550 / Many conventional methods have been used to modify the wettability of the polymeric surfaces for the biomedical applications of the artificial bionic organ. Those methods are the chemical treatment, the ultraviolet (UV) irradiation, the plasma process and the ion implantation. Many artificial bionic organs, for example, an artificial heart, an artificial blood vessel, a device for prevention of thrombosis stent and an artificial endocranium have been developed for the physical or mental disability. For development of the high function of an artificial bionic organ, the data transmission between the brain neuron cells and the external electrical circuit, and the high biocompatible materials for the interface between brain and electrode are required. It is related to the technology of brain-computer interface (BCI), sometimes called a direct neural interface or a brain-machine interface. In case of the brain-controlled devices, the study of the brain memory is necessary. Then, the artificial pattern network of the brain cells cultured on the surface in vitro for simulation of the brain function is the concerned issue. The arrangement of a lot of neuron on a detection electrode is required. So, a formation method of the artificial neural network that arranged a neuron as technology for this purpose is demanded As for the neuron arrangement, there were the reports about the immobilization of neuron by fabrication of the three-dimension structure, and they could be divided into two methods from their manipulation. One is the arrangement with one-by-one manipulation and the other is the arrangement with self-assembly. The former method is the fabrication of many micro-structures and then arranged a neuron in a desired position with one-by-one manipulation to for a neuron network. For the brain memory stimulation, however, the neuron network from more than 10 millions of neurons is required. So, this method is not suitable. The latter is the fabrication of the carbon nanotube pillar to immobilize the neurosphere with self-assembly adhesion. Although this method could be formed the large neuron network, the neurosphere consists of several 1, 000 cells. So, it is very difficult to analyze the mechanism of data transformation. In contrast, by surface modification even if on the same surface to modify a geometric pattern, the cells can adhere along the modified pattern by using single culture on such surface. The neuron will migrate itself to adhere on the pattern. The self-assembly adhesion occur. This method is very useful for the neuron arrangement method. The surface modification of the polymeric materials to pattern the cell adhesion area as a network has been taken place by using many techniques such as the plasma process, the irradiations of UV and X-ray and the ion implantation. The ion implantation technique into the polymeric-material surface has more advantage than the other techniques since its abilities to control the micro-area, and to break down the tight bonding of polymer material. The ion implantation with positive ion without charge neutralization results in a charge-up problem due to the insulating properties of most polymers. This charge-up problem exerts a bad influence on the implantation control of ion dose and ion energy. The negative-ion implantation occurs almost “charge-up free” even if no external charge compensation. Then, the negative-ion implantation into polymeric surface has a very precise control to obtain very fine pattern. So, it is expected to control the adhesion size of about single cells (about several 10 μm). Since this study will be used for the application in the biomedical fields, the ion element should be considered to be harmless for the living body. Then, carbon is selected since it is main component of polymer materials and more familiar to cells. As above described, in this thesis, I use the carbon negative-ion implantation to modify the polymeric surface to obtain the pattern of the neuron with self-assembly-adhesion. As for the polymeric material in the biomedical fields, I selected polystyrene (PS) and silicone rubber (SR). In this research, the fundamental parameters for cell adhesion on the modified surface by carbon negative-ion implantation were described (Chapters 3, 4 and 5). As for the fundamental issue, the wettability relating to the atomic bonding state of the new functional group and the surface morphology (Chapter 3), the protein adsorption (Chapter 4), and also the adhesion of nerve-like cells on the pattern (Chapter 5) were examined. In these chapters, I clarified the relationship among them and the negative-ion implantation. Then, based on these phenomena, I have developed the new application techniques by negative-ion implantation for the adhesion patterning of neuron (Chapters 6 and 7). In the development of these techniques, I have proposed two methods since the neuronal cells required the special base surface to adhere. One is degradation method of the special base surface by which I tried to make an artificial neuron network (Chapter 6). The other is the patterning of the stem cell adhesion and differentiation into neuron with maintaining the adhesion position. So, the neuron patterns were formed on the pattern (Chapter 7). The obtained results are summarized as the following. In Chapter 3, the surfaces of the PS and SR were implanted by carbon negative ions at the energies of 5 – 20 keV and the doses of 1×1013 – 3×1016 ions/cm2. After the implantation, the change in the physical surface properties, relating to the adsorption properties of adhesive proteins, was described. The new atomic bonding, the surface morphology and the wettability were studied by XPS analysis, AFM and contact angle measurement, respectively. XPS analysis showed the formation of new oxygen function groups of hydroxyl and carbonyl on the implanted surfaces from the adsorption of the oxygen in the residual gas and in the moisture in the air on the ion-induced defects. These new bonds refer to the hydrophilicity for the wettability. The ion implantation sputtered and changed the surface morphology of surface roughness in order of several nm that dose not interfere to the protein adsorption and to cell culture. The wettability properties of the C¯-implanted surfaces of SCPS and SR were evaluated by measuring the change in contact angle. At first, the angles were measured by the water drop method. The contact angles of PS measured by water drop method decreased from 91° to 86° for the non-implantation to the implantation, respectively. Those of SR also decreased from 100° to 86°for the non-implantation to the implantation, respectively, even if the main chain bonds in SR are stronger than that in PS. The hydrophilic surfaces of PS and SR were obtained by carbon negative-ion implantation. Then, the contact angles were measured by the air bubble method. The sample was dipped in the water and the bubbles were injected on the surface. Then, the angle was evaluated from the arc circular of the bubble. After dipping in the water for 24 h, the average value of the angles decreased to 64° and to 52° for PS and SR, respectively. The more clearly hydrophilic properties were observed. In Chapter 4, I checked the adsorption properties of the adhesive protein and the poly-D-lysine (PDL) on the implanted surface. Generally, in the cell adhesion, the adhesive proteins exist between the cell surface and the surface. On the cell membrane, cells have specific receptors that anchor to the specific protein. So, the adsorptions of the adhesive proteins are necessary for the cell adhesion. In nature, protein has both hydrophobic and hydrophilic groups. Thus, the ultra hydrophobic and ultra hydrophilic surfaces are not suitable for protein adsorption. The adhesive proteins for the cell adhesion generally prefer to be adsorbed on the hydrophilic surface, which the contact angle is in the range of 40° – 80°. I evaluated the adsorption properties of adhesive protein such as type-I collagen, fibronectin and laminin and that of PDL on the modified surfaces of PS and SR by detecting the nitrogen atom with using XPS analysis. As a result, the adsorptions of the adhesive protein were almost improved with 1.2 – 3.3 times by carbon negative-ion implantation. In Chapter 5, the nerve-like cells of PC12h (rat adrenal pheochromocytoma) were cultured on the C¯-implanted surfaces of PS and SR to find out the fundamental condition for the neuron network formation. As a results, PC12h cells and their neurite outgrowth showed the self-assembly adhesion along the implanted pattern on both of PS and SR. The suitable condition of the ion implantation for the adhesion patterning of PC12h cells was about 1×1015 – 3×1015 ions/cm2. Almost no effect of energy in the range of 5 – 20 keV on the cell adhesion was observed. The effective minimum line width of the implanted region for the adhesion of single cell-body and single neurite outgrowth were about 5 and 2 μm, respectively. In Chapter 6, the brain neuronal cells require the specific surface culture, such as PDL. So, in this chapter, I used PDL coating on the PS and degraded it by the carbon negative-ion implantation. Two kinds of brain neuronal cells were used. One is newborn mouse brain neuronal cells (1 day) and the other is rat embryo brain cortex neuronal cells (16 – 18 days). As a result, obtained the effective ion dose for degradation of the adhesion at 1×1014 ions/cm2. The adhesion patterning of brain neuronal cells on the unmodified pattern of PDL could be achieved by carbon negative-ion implantation. In Chapter 7, I cultured the adult stem cells of rat mesenchymal stem cells (MSC), which has the multipotential to differentiation into many kinds of cell lines, especially into neuron, on the pattern region of the C¯-implanted surfaces of PS and SR. As a results, MSCs showed the self-assembly adhesion along the implanted pattern of PS and SR. Comparing to the adhesion patterning of PC12h cells, the adhesion patterning of MSCs required a lower ion dose to implant on the polymeric surfaces. By culturing with the culture medium supplementing withβ-Mercaptoethanol (BME) at concentration of 1 mM, the MSCs were induced to differentiate into neuronal cells. The adhesion patterning of the neuron-differentiated cells maintained on the implanted region was observed. By staining with anti-neuron-specific enolase, these differentiated cells were neurons. From all investigation, I clarified the change in the physical surface properties after the carbon negative-ion implantation into the polymeric surface and the mechanisms mentioned above. I showed the surface modification to obtain the hydrophilic surface by the ion-induced effect. This hydrophilic surface improved the protein adsorption properties. By using nerve-like cells, the ion implantation affecting to the cell adhesion were clarified. By the implantation through the micro-pattern mask, the cells adhered along the implanted pattern. The cells could adhere on the implanted area that was smaller than the cell size and their neurite also could adhere on the narrowed implanted area. So, I can obtain the self-assembly separation pattern of cell body adhesion and neurite outgrowth. For the application of patterning of real neuron, I coated the special surface with PDL and degraded it from patterning the negative-charge site on it by using carbon negative-ion implantation through a micro-pattern mask. I could pattern and form the neuron network of the brain neuron on the unmodified PDL. On the other hand, for the MSC, I also achieved the adhesion patterning by using carbon negative-ion implantation through a micro-pattern mask, and I succeeded the patterning of the neuron-differentiated cells from the adhered MSC with maintaining their adhesion pattern. As a conclusion, from all these researches, I achieved the cell-self-assembly adhesion and the patterning of the neuron network formation on the polymeric surfaces by using carbon negative-ion implantation. / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13394号 / 工博第2865号 / 新制||工||1421(附属図書館) / 25550 / UT51-2007-Q795 / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 石川 順三, 教授 髙岡 義寛, 教授 小林 哲生 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17597号 / 工博第3756号 / 新制||工||1573(附属図書館) / 30363 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 田畑 泰彦, 教授 木村 俊作 / 学位規則第4条第1項該当

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21922号 / 生博第407号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 垣塚 彰, 教授 豊島 文子, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19146号 / 生博第329号 / 新制||生||44(附属図書館) / 32097 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 清水 章, 教授 稲葉 ｶﾖ / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

This research was originally published in The Journal of Biological Chemistry. May Nakajima-Koyama, Joonseong Lee, Sho Ohta, Takuya Yamamoto, and Eisuke Nishida. Induction of Pluripotency in Astrocytes through a Neural Stem Cell-Like State. The Journal of Biological Chemistry, 290(52), 31173-31188, 2015. © The American Society for Biochemistry and Molecular Biology / 京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13027号 / 論生博第13号 / 新制||生||47(附属図書館) / 32955 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 西田 栄介, 教授 米原 伸, 教授 上村 匡 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(生命科学) / 乙第13582号 / 論生博第29号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 今吉 格, 教授 見学 美根子, 教授 安原 崇哲 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM