We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase. Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.

京都大学 / 新制・論文博士 / 博士(工学) / 乙第13406号 / 論工博第4192号 / 新制||工||1762(附属図書館) / (主査)教授 松島 信一, 教授 竹脇 出, 教授 牧 紀男 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Reference samples are a vital part of the forensic analysis of deoxyribonucleic acid (DNA) evidence. Efficient processing and analysis of these sample types are required for comparative analysis of an unknown electropherogram (EPG) and forensic databasing purposes (12). These reference samples can be derived from blood swabs or cheek swabs, the latter also being known as buccal cell swabs (20, 22, 32). Buccal cells, or epithelial cells of the oral cavity, are the preferred cell type for known samples as their collection is non-invasive and painless (20-21). Buccal cell collection devices typically consist of a swab (cotton or foam) and a filter paper, commonly FTA paper (1). FTA paper contains proprietary chemicals that lyse cell membranes upon contact, trapping and stabilizing DNA for downstream processing (21, 34). FTA paper also inhibits bacterial and viral growth and protects against damage from UV radiation, nucleases and oxidation (21, 34). Some of the benefits of using FTA cards include the ability to store the cards at ambient temperature for years (21, 35, 37) and to perform direct amplification of the samples thereby removing the need to utilize DNA extraction and quantitative polymerase chain reaction (qPCR) (32, 37, 39). The EasiCollectTM (EC) and EasiCollectTM + (EC+) Buccal Sample Collection Devices (General Electric (GE) Healthcare Life Sciences, Buckinghamshire, UK) have FTA sample collection cards that contain a proprietary dye that changes color from pink to white, indicating where colorless fluids, such as saliva, were likely deposited (42). This study consisted of four phases. Phase 0 determined the optimal amplification conditions, including number of polymerase chain reaction (PCR) cycles and an appropriate capillary electrophoresis (CE) injection time for high template, single source samples obtained from FTA cards using the EC and EC+ buccal cell collection devices. Samples were obtained from EC FTA cards with a Harris 1.2-mm Uni-Core Punch and amplified using the GlobalfilerTM Express PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA) using the manufacturer’s protocol with 26, 27 or 28 PCR cycles (28). Fragment separation was achieved on an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA) with 5, 15 and 25 second (s) 1.2 kiloVolt (kV) injections. Samples were analyzed on GeneMapper® ID-X v1.4 (Applied Biosystems, Foster City, CA) with an analytical threshold of 150 RFU (relative fluorescence units) (31). It was determined that amplification with 26 PCR cycles was optimal for high template, single source samples from FTA cards in this laboratory. The three injection times were utilized in the remaining phases and no other parameters were changed. In Phase 1 of this study, the optimal collection method for the EC+ device from various processes was assessed using the following collection variables: 1) a dry or saliva-wet swab; 2) a circular or up-down/side-to-side motion; 3) 2, 3 or 4 motions; and 4) swabbing of one or both cheeks. This resulted in a total of 24 distinct collection processes. We found that collection techniques that involved wetting the foam head of the EC+ device provided higher peak heights, improved heterozygote balance (Hb) and minimized the rate of drop-out in EPGs. When swabbing two cheeks versus one, the median peak heights increased, indicating an increase in transfer of cellular material onto the FTA surface. The motion of swabbing - circular or up-down/side-to-side - did not have an effect on the overall quality of the EPG data. During Phase 2a, the distribution of cellular material was assessed for two collection processes that involved swabbing of two cheeks with a wet swab four times; the variation among the methods being the motion (circular or up-down/side-to-side). Two punches taken surrounding the original punch assessed during Phase 1 showed similar average peak heights (i.e. ca. 3500 RFU at a 5 s injection on the ABI 3500 Genetic Analyzer) for both collection processes. No allelic drop-out was observed with either collection technique. Phase 2b compared the EPG signal of the EC and EC+ collection devices. The EC+ collection process used for this comparison involved rubbing a wet swab across two cheeks using four circular motions as this produced no allelic drop-out and fewer samples which saturated the CE laser detector. Therefore, this method provided more data for analysis. Samples from both devices produced comparable peak heights and PHRs above 0.6 with no allelic drop-out and stutter ratios below the thresholds set by the manufacturer (28). The EC+ device was found to be robust and provided full profiles using a minimalist sample collection method. However, the probability of drop-out increased as both the number of motions and the number of cheeks decreased. Based on this study, a collection using four circular motions divided between two cheeks with a wet swab is recommended with a 5 s, 1.2 kV injection on an ABI 3500 Genetic Analyzer, since full DNA profiles were obtained with balanced heterozygote loci, expected stutter ratios, and acceptable levels of minus A artifact. Further, it was determined that this recommended collection method resulted in high-fidelity DNA signal for up to three punches. Thus, the EC+ device is reliable, easy-to-use and non-invasive for the collection of buccal cells for known reference samples. A sample obtained from the area of transfer on an FTA card from the EC+ device can produce an EPG of the quality required for the comparison of known samples to an evidentiary profile as well as for input of the genotype into a national forensic database.

This dissertation explores the role of information frictions in the design of financial securities, the pricing of securities, and their business cycle implications. The first essay studies the risk- shifting problem between bondholders and shareholders, and the moral hazard problem between shareholders and the manager. Although, these two problems have been studied separately, my model is the first tractable frame-work to study these two frictions jointly. Using my model, I explore: i) How the presence of managerial moral hazard affects the risk-shifting problem, and ii) How optimal policies of the firm change in terms of leverage, managerial compensation, and investment decisions when the two problems are considered jointly. I show that the optimal amount of risk-shifting is amplified in the presence of managerial moral hazard. Moreover, my model delivers a non-monotonic relation between risk-shifting and leverage. This non-monotonicity has the potential to reconcile seemingly contradictory empirical evidence on the sign of this relation. The second essay (coauthored with Jianjun Miao) studies the design of an optimal contract for the manager when the shareholders are concerned about model misspecification. The model delivers counter-cyclical firm level equity premium, and has interesting implications for security design. The third essay incorporates accounting practices into models that generate business cycles through borrowing constraints. I show that the interaction of accounting frictions with the borrowing constraint has implications for the persistence and amplification of macroeconomic shocks.

The ligand activated transcription factor retinoid X receptor (RXR) forms a DNA binding heterodimer with pregnane X rseceptor (PXR) in response to foreign xenobiotics. In addition to RXR and PXR there are other proteins involved in the RXR/PXR signaling pathway. Many proteins involved in this pathway are still unknown. This study documents the production of RXR and PXR in a bacterial recombinant fusion system. These proteins were expressed in a system that allowed purification with six histidine residues. Once the proteins were expressed and purified from E. coli, they were solublized and tested for function. Different strategies were employed including temperature and inducer studies and denaturing and renaturing techniques to solublize PXR. Following the solubilzation of each protein, all proteins were subjected to a method of functional analysis. RXR function was assessed by electrophoretic mobility shift assay (EMSA) and proved to effectively form a DNA binding heterodimer with PXR. These studies involving RXR and PXR demonstrate that these proteins can be efficiently produced in a functional manner utilizing an inexpensive bacterial system. In addition, this study documents various strategies for combating "inclusion body" formation in the overexpression ofPXR. Also, it describes the production of plasmid pCMV-RXR for transfection into the HepG2 cell line to monitor the levels of cellular RXR in various tissue types.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18898号 / 医博第4009号 / 新制||医||1009(附属図書館) / 31849 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木原 正博, 教授 中川 一路, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00270/full / 付記する学位プログラム名：グローバル生存学大学院連携プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19619号 / 医博第4126号 / 新制||医||1015(附属図書館) / 32655 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 木原 正博, 教授 松林 公蔵 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels.

No description available.