The effects of various catalyst synthesis parameters on the photocatalytic degradation kinetics of aqueous methyl orange dye are presented. The four factors investigated were: i) InVO4 concentration, ii) nickel concentration, iii) InVO4 calcination temperature, and iv) ballmilling time. Three levels were used for each factor. Due to the large number of possible experiments in a full factorial experiment, an orthogonal-array experimental design was used. UV-vis spectrophotometry was used to measure the dye concentration. The results show that nickel concentration was a significant parameter, with 90% confidence. The relative ranking of importance of the parameters was nickel concentration > InVO4 concentration > InVO4 calcination temperature > milling time. The results of the orthogonal array testing were used to make samples of theoretically slowest and fastest catalysts. Curiously, the predicted-slowest catalyst was the fastest overall, though both samples were faster than the previous set. The only difference between the slowest and fastest catalysts was the milling time, with the longer-milled catalyst being more reactive. From this result, we hypothesize that there is an interaction effect between nickel concentration and milling time. The slowest and fastest catalysts were characterized using energy-dispersive spectroscopy (EDS), scanning electron microscopy (SEM), x-ray powder diffractometry (XRD), BET surface area analysis, and diffuse-reflectance spectroscopy (DRS). The characterization results show that the fastest catalyst had a lower band gap than the slowest one, as well as a slightly greater pore volume and average pore diameter. The results indicate that fast kinetics are achieved with low amounts of nickel and a long ball milling time. Under the levels tested, InVO4 concentration and the calcination temperature of the InVO4 precursor were not significant.

We describe here for the first time an efficient high yield production method for clinical grade recombinant human Oncofetal Antigen/immature laminin receptor protein (OFA/iLRP). We also demonstrate significant antitumor activity for this protein when administered in liposomal delivery form in a murine model of syngeneic fibrosarcoma. OFA/iLRP is a therapeutically very promising universal tumor antigen that is expressed in all mammalian solid tumors tested so far. We have cloned the human OFA/iLRP cDNA in a bacterial expression plasmid which incorporates a 6x HIS-tag. Large scale cultures of the plasmid transformed Escherichia coli were performed and the crude HIS-tagged OFA/iLRP was isolated as inclusion bodies and solubilized in guanidine chloride. The protein was then purified by successive passage through three column chromatography steps of immobilized metal affinity, anion exchange, and gel filtration. The resulting protein was 94% pure and practically devoid of endotoxin and host cell protein. The purified OFA/iLRP was tested in mice for safety and efficacy in tumor rejection with satisfactory results. This protein will be used for loading onto autologous dendritic cells in an FDA approved phase I/II human cancer vaccine trial in OFA/iLRP-positive breast cancer patients.

The goals of this research project were the design and construction of a carbon nanotube (CNT) reactor based on the plasma enhanced chemical vapor deposition (PECVD) principle and the development of a method for directed assembly of CNTs by catalyst patterning. PECVD was selected as the growth method due to the requirement of a catalyst for the growth process, thereby facilitating directed assembly and controlled diameter CNT growth at well-defined locations. The reactor was built in accord with horizontal flow design using standard ultra high vacuum components. The controllable parameters of the reactor include sample temperature, DC plasma intensity, chamber pressure, gas flow ratios, and total gas flow. The most favorable parameters for growing CNTs of well defined length, diameter, and separation were obtained by initially using parameter values obtained from literature, then optimized by changing a parameter and noting the effect on CNT growth. Catalyst patterns for the directed assembly of CNTs were prepared by electron-beam lithography (EBL). Experiments were performed that demonstrated the feasibility of using lithographic methods to achieve directed assembly of carbon nanotubes for the manufacture of CNT devices. Experiments focusing on growth interruption and regrowth of CNTs were conducted to investigate methods of introducing tailored branching points into carbon nanotubes during the growth process. These experiments clearly demonstrate that growth interruption increases the occurrence of CNT branching. An analysis of the relationships between CNT diameter, branching points, and the number of growth steps was conducted.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第17773号 / 理博第3896号 / 新制||理||1562(附属図書館) / 30580 / 京都大学大学院理学研究科化学専攻 / (主査)教授 丸岡 啓二, 教授 大須賀 篤弘, 教授 時任 宣博 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17978号 / 医博第3842号 / 新制||医||1001(附属図書館) / 80822 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 河本 宏, 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

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20315号 / 薬科博第84号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21786号 / 工博第4603号 / 新制||工||1717(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 村田 靖次郎, 教授 辻 康之, 教授 小澤 文幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Gene mit gerigem Einfluss auf einen untersuchten Phänotyp können durch den Ein- schluss einer genetischen Variation im Tierversuch untersucht werden. Adulte Neuro- genese, der Prozess der Neubildung und Integration von funktionellen Neuronen in das existierende neurale Netzwerk, wird von vielen solchen Genen mit geringem Effekt beeinflusst. All diese Gene im lebenden Tier zu untersuchen wäre mit einem hohen Arbeitsaufwand verbunden, und würde hohe Tierzahlen erfordern. Bereits publizierte Ergebnisse zeigen, dass diese Gene auch in der Zellkultur unter Verwendung von Zelllinien genetisch rekombinanter Tiere untersucht werden können (Kannan et al., 2016). Die hier verwendeten, ingezüchteten Mausstämme des so genannten BXD Panels stellen die Nachkommen der Kreuzung der beiden Mausstämme C57BL/6J und DBA/2J dar (Peirce et al., 2004), die sich in der Ausprägung von unterschiedlichen Neurogenese bezogenen Phänotypen bereits deutlich unterscheiden (Kempermann et al., 2006). Durch die Verwendung der BXD Tiere wird hierbei die Aussagekraft der genetischen Variation mit dem Zellkultursystem verbunden. Die Aussagekraft dieser Studie ist jedoch darin limitiert, dass aufgrund des verwendeten Protokolls nur eine Zelllinie pro Mausstamm generiert werden konnte. Daher präsentiere ich hier ein neues Protokoll welches es erlaubt eine Zelllinie aus nur einem einzelnen Tier zu generieren. Diese Methode kombiniert zwei bestehende Zellkultursysteme, die Neurosphärenkultur und die Monolayerkultur. Es stellte sich heraus, dass die Überlebensrate der einzelnen Zelllinien vom biologischen Hintergrund der Zellen beeinflusst wird. So ist die Überlebensrate von Zellen der DBA/2J Mäuse deutlich schlechter als die der C57BL/6J oder die der F1 Generation aus der Verpaarung der beiden Stämme. Es zeigte sich allerdings, dass diese Überlebensrate nicht ausschließlich von der vorhandenen Anzahl proliferierender Zellen abhängt, da B6D2F1 (F1 Generation mit einem C57BL/6J Muttertier) signifikant weniger proliferierende (Ki67 positive) Zellen in vivo aufweisen, jedoch keine geringere Überlebensrate der Zelllinien haben. Eine hoch standardisierte, umfangreiche Analyse der Zelllinien aller vier Mausstämme (C57BL/6J, DBA/2J, und die zwei reziproken F1 Nachkommen BDF1 und DBF1) zeigte eine hohe Varianz innerhalb genetisch identischer Linien, was die Be- stimmung eines Effektes, der durch den genetischen Hintergrund der Linien verursacht wird, beeinträchtigte. Die Zelllinien werden signifikant von äußeren Faktoren beeinflusst, wie z.B. durch das Einfrieren der Zellen. Dies gibt Hinweise darauf, dass Untersuchungen in der Zellkultur genau geplant, kritisch hinterfragt, sowie möglichst alle potentiellen Einflussfaktoren gleich gehalten werden müssen. Nur so können valide, aussagekräftige Ergebnisse mit der Zellkultur gewonnen werden. Automatische Zellkultursysteme, neue Mikroskopieverfahren, sowie besser definierte Langzeitstudien werden unser Verständnis von Zellen in der Zellkultur deutlich verbessern und dabei ihren Wert, sowie bestehende Limitationen, endgültig klären.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117 / Uncovering gene loci that assert only small effects onto a phenotype of interest, can be achieved by including genetic variation in animal research. Adult hippocampal neurogenesis, the process of the formation of new neurons and their functional integration into existing circuitry, is influenced by a broad range of such small effect genes. Analyzing all of these genes in vivo would be laborious and require a high number of animals. Previously published data merged the power of genetic variation with a cell culture system by using cell lines generated from the BXD recombinant inbred mouse strains (Kannan et al., 2016). These strains are inbred progeny of F2 crosses originating from the two mouse strains C57BL/6J and DBA/2J (Peirce et al., 2004), which already differ quite extensively in neurogenesis related phenotypes (Kempermann et al., 2006). As previous studies were limited by the number of strains that could be generated due to the demand for high numbers of animals, I developed a new method that allows the generation of a cell line from one single animal. For this new method, I combined the neurosphere culture with a subsequent monolayer culture. The survival of the resulting cell lines, is thereby greatly influenced by the genetic background. The survival rate of cell lines derived from DBA/2J animals is much lower as compared to C57BL/6J-derived lines or lines from the F1 generation of crossing the two strains. Whether or not a cell line survived did not seem to be solely influenced by the number of proliferating cells in vivo, as B6D2F1 (F1 progeny with a C57BL/6J mother) showed significantly less proliferative (Ki67 positive) cells in vivo while exhibiting a survival rate that exceeded both parental strains. An extensive study of the cell lines gained from all four mouse strains (C57BL/6J, DBA/2J, and the two reciprocal F1 progeny B6D2F1 and D2B6F1) in a highly standardized manner showed that the individual difference between single cell lines was rather high, hampering the successful detection of in-between strain differences. The standardized characterization of the generated cell lines, further allowed the identification of external factors, influencing the cells, as for example the freezing of the cells. This indicates that cell culture experiments need to be thoroughly planned and critically scrutinized, while all external factors should be kept as constant as possible to ensure the validity of the resulting data. Automated cell handling, new imaging technologies, as well as more defined long-term studies will greatly improve the understanding of cells in culture and thereby show their true values and limitations.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. We are interested in investigating the true substrate for methylation because it is known from our work and the work of others that fully assembled 50S subunits are not substrates for methylation. We have published a model for 50S ribosomal subunit formation where, the precursor particle that accumulates in erythromycin treated cells is a target for methyl transferase activity. Current studies are aimed at investigating the role of the precursor particle as substrate for ermE methyltransferase activity and the competition between this enzyme and erythromycin for the 50S precursor particle. Slot-blot hybridization experiments have identified the presence of 23S rRNA in the 50S precursor region. Quantitation of the 23S rRNA in these blots also revealed that the percentage of the precursor increased as the concentration of erythromycin was increased in the growth media. Ribosomal proteins of S. aureus were studied by two-dimensional gel electrophoresis. Protein content of the 50S precursor particle was analyzed by MALDI-TOF. These studies have identified 16 50S ribosomal proteins in the precursor region. Methyltransferase assays showed that 50S precursor particle was a substrate for ermE methyltransferase. Importantly, RNA that is already assembled into 50S subunits was not a substrate for the enzyme. Inhibition curves showed that macrolide, lincosamide, and streptogramin B (MLSB) drugs bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. Competition experiments suggested that the enzyme can displace erythromycin from the 50S precursor particle and that erm methyltransferase has a lower association constant for the precursor particle compared to that of the erythromycin. This suggests that higher concentrations of erythromycin are needed to combat erm induced resistance. These studies shed light on the interaction of ermE methyltransferase and erythromycin in the clinically important pathogen S. aureus.

Cerium oxide (Ceria) at nano scale has gained significant attention due to its numerous technological applications. Ceria in both doped and undoped forms are being explored as oxygen sensor, catalysis, protective coating against UV and corrosion, solid oxide fuel cell (SOFC) electrolyte and newly discovered antioxidant for biomedical applications. Therefore, there is an imminent need of a technology which can provide a cost effective, large scale manufacturing of nanoceria and its subsequent consolidation, specially using thermal spray. This dissertation aims to develop a scientific understanding towards the development of pure and doped ceria- based coating for a variety of technological applications, from SOFC applications to corrosion resistant coating. Atmospheric plasma spray (APS) and solution precursor plasma spray (SPPS) techniques for the fabrication of nano ceria coating were investigated. For feedstock powder preparation, a spray drying technique was used for the agglomeration of cerium oxide nano particles to achieve high density coating. Deposition efficiencies and coating porosity as a function of processing parameters were analyzed and optimized using a statistical design of experiment model. The coating deposition efficiency was dependent on the plasma temperature and vaporization pressure of the ceria nanoparticles. However, low standoff distance and high carrier gas flow rate were responsible for the improved density upto 86 [plus or minus] 3%.An alternative novel SPPS technique was studied for a thin film of cerium oxide deposition from various cerium salt precursors in doped and undoped conditions. The SPPS process allows controlling the chemistry of coating at a molecular level. The deposition mechanism by single scan experiments and the effect of various factors on coating microstructure evolution were studied in terms of splats formation. It was found that the precursor salt (nitrate of cerium) with lower thermal decomposition temperatures was suitable for a high density coating. The high concentration and low spray distance significantly improve the splat morphology and reduced porosity (upto 20%). The feasibility of the trivalent cations (Sm 3+ and Gd 3+) doping into cerium oxide lattice in high temperature plasma was discussed and experimentally studied. XRD analysis revealed the nano crystalline characteristic of the coating and lattice expansion due to doping. The extensive transmission electron microscopy, Scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and thermo gravimetric were conducted to evaluate the precursors, and coating microstructure. Due to facial switching between Ce4+ and Ce3+ oxidation state, the cerium oxide surface becomes catalytically active. Thus, the APS ceria coatings were investigated for their applicability under extreme environmental conditions (high pressure and temperature). The air plasma sprayed coated 17-4PH steel was subjected to high pressure (10 Kpsi) and temperature (300 oF) corrosive environment. The coated steel showed continuous improvement in the corrosion resistance at 3.5 wt% NaCl at ambient temperature for three months study whereas, high pressure did not reveal a significant role in the corrosion process, and however, one needs to do further research. The ceria coated steel also revealed the improvement in corrosion protection (by 4 times) compared to the bare steel at low pH, 300 oF and 4000 Psi environment. This study projects the importance of cerium oxide coatings, their fabrication, optimization and applications.