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Lau San Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Title Page --- p.1 / List of Thesis Committee --- p.2 / Declaration Page --- p.3 / Acknowledgements --- p.4 / Table of Contents --- p.5 / Abstract --- p.10 / 論文撰要 --- p.12 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Drug Metabolism --- p.14 / Chapter 1.2 --- Glucuronidation --- p.16 / Chapter 1.3 --- UDP-glucuronosyltransferase (UGTs) / Chapter 1.3.1 --- Nomenclature --- p.18 / Chapter 1.3.2 --- Tissue Distributions of UGTs --- p.20 / Chapter 1.3.3 --- Genetics --- p.26 / Chapter 1.3.4 --- Evolution of UGTs --- p.28 / Chapter 1.4 --- UDP-glucuronosyltransferase related Human Diseases --- p.33 / Chapter 1.4.1 --- Hyperbilirubinemia --- p.33 / Chapter 1.4.2 --- Cancer --- p.37 / Chapter 1.5 --- Rattus norvrgicus UDP-glucuronosyltransferase 1A8 --- p.38 / Chapter 1.6 --- Aims of the Project --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 1. --- Rat liver mRNA Extraction --- p.43 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.43 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.43 / Chapter 4. --- Construction of bacterial expression vector --- p.43 / Chapter 5. --- Expression of recombinant protein in E.coli --- p.44 / Chapter 6. --- Purification of protein with Ni column --- p.44 / Chapter 7. --- Purification of protein with gel filtration column --- p.44 / Chapter 8. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 9. --- Concentration and Desalting of protein --- p.45 / Chapter 10. --- Enzyme activity of glucuronidation --- p.45 / Chapter 11. --- Near UV and far UV circular dichroism (CD) spectroscopy --- p.45 / Chapter 12. --- Fluorescent properties studies --- p.45 / Chapter 13. --- Western Blotting --- p.46 / Chapter 14. --- 3D modeling of UGT1A8 and interactions with ligands --- p.46 / Chapter 2.2 --- Methods / Chapter 1. --- Rat liver mRNA extraction --- p.46 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.47 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.48 / Chapter 4. --- Cloning of UGT1A8 PCR product into expression vector pRSet B --- p.49 / Chapter 5. --- Confirmation of the presence of insert in the plasmid --- p.51 / Chapter 6. --- Sequence checking for UGT1A8 gene in the pRSet B vector --- p.52 / Chapter 7. --- Expression of recombinant protein in E.coli JM109(DE3) cell strain --- p.52 / Chapter 8. --- Purification of recombinant protein by Ni-column --- p.53 / Chapter 9. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.53 / Chapter 10. --- Recombinant protein purification by gel filtration column --- p.54 / Chapter 11. --- Concentration or Desalting of Purified Protein --- p.54 / Chapter 12. --- Determination of Protein Concentration --- p.55 / Chapter 13. --- Far- UV Circular dichroism spectroscopy --- p.55 / Chapter 14. --- Intrinsic Fluorescence Studies of Proteins --- p.57 / Chapter 15. --- Chemical denaturation stability studies --- p.58 / Chapter 16. --- Glucuronidation protein activity assay --- p.59 / Chapter 17. --- Mutagenesis --- p.60 / Chapter 18. --- Western Blotting for the presence of protein --- p.61 / Chapter 19. --- Protein Modeling with Insight II / Chapter 19.1 --- Construction of substrate 1-napthol structure --- p.62 / Chapter 19.2 --- Obtaining UDP-glucuronic acid in PDB file --- p.63 / Chapter 19.3 --- Obtaining rat UGT1A8 model structure in PDB file --- p.63 / Chapter 19.4 --- Optimization of rat UGT1A8 structure --- p.63 / Chapter 19.5 --- Docking studies of interaction between ligands and protein / Chapter 19.5.1 --- Setting up a Grid --- p.66 / Chapter 19.5.2 --- Docking of 1-napthol to UGT1A8 --- p.67 / Chapter 19.5.3 --- Docking of UDP-glucuronic acid in the complex of UGT1A8 and1- napthol --- p.68 / Chapter 19.5.4 --- Definition of Subsets --- p.68 / Chapter Chapter 3 --- Results --- p.70 / Figure 3.1 The extracted RNA from rat liver tissue --- p.76 / Figure 3.2 DNA gel of PCR amplified gene product --- p.77 / Figure 3.3 Colony PCR of UGT1 A8-pRSetB transformed DH5 a bacteria --- p.78 / Figure 3.4 The alignment of amplified gene sequence with the rat UGT1A8 sequence on NCBI database --- p.79 / Figure 3.5 SDS-PAGE of cell lysates with different expression temperature and time duration --- p.82 / Figure 3.6 SDS-PAGE of bacterial cell lysates --- p.83 / Figure 3.7 SDS-PAGE of Ni-column eluted protein --- p.84 / Figure 3.8 Elution Profile of Gel Filtration Chromatography --- p.85 / Figure 3.9 SDS-PAGE analysis of UGT1A8 fractions from Ni-column and gel filtration column --- p.86 / Figure 3.10 Sequence Alignment of UGTs in the rat UGT1A family and 2D structure prediction of UGT1A8 --- p.88 / Figure 3.11 Circular Dichroism (CD) measurements on rat UGT1A8 --- p.89 / Figure 3.12 Western Blotting of UGT1A8 wild-type and mutant proteins --- p.91 / Table 3.1 The specific activity of wild-type and mutated proteins --- p.92 / Figure 3.13 Fluorescence spectrum of wild type and two charged-residue mutants ofUGTlA --- p.93 / Figure 3.14 Fluorescence spectrum of wild type and Trp mutants of UGT1A8 --- p.94 / Figure 3.15 Chemical denaturation of wild type and Trp-mutated UGT1A8 proteins --- p.95 / Figure 3.16 Resolved Stern-Volmer plot of UGT1A8 on acrylamide quenching --- p.96 / Figure 3.17 The 3D modeling structure of rat UGT1A8 --- p.97 / Figure 3.18 Modeling simulated the interaction between UDP-glucuronic acid and UGT1A8 --- p.98 / "Figure 3.19 Modeling simulated the interaction between UDP-glucuronic acid, 1-napthol and UGT1A8" --- p.99 / Chapter Chapter 4 --- Discussion / Chapter 1. --- Successful Expression of Rat UGT1A8 --- p.100 / Chapter 2. --- The recombinant rat UGT1A8 protein was properly folded and enzymatic functioning --- p.102 / Chapter 3. --- Purified recombinant rat UGT1A8 protein contained well-ordered structure --- p.103 / Chapter 4. --- "Relative positions of Trp38, Trp64, Trp98 and Trp208 in the protein" --- p.105 / Chapter 5. --- Contribution of Trp residues in the folding and stability of the protein --- p.106 / Chapter 6. --- Probing of substrate coupling region by mutagenesis --- p.108 / Chapter 7. --- Interaction studies of substrates and UDP-glucuronic acid with UGT1A8 by computer modeling and docking simulation --- p.109 / Chapter Chapter 5 --- Conclusion --- p.111 / Chapter Chapter 6 --- References --- p.113

Embora seja considerável a quantidade de materiais educacionais disponíveis na Internet, poucos são os estudos reportados na literatura que tratam de investigar a efetividade dos mesmos como ferramenta de ensino. Neste trabalho descrevemos a elaboração, uso e avaliação de um sítio produzido como material de apoio para a disciplina de Química Analítica. As facilidades disponíveis no sítio são: páginas de conteúdo com descrições textuais dos tópicos discutidos na disciplina, hiperlinks para sítio da Web que oferecem conteúdo relevante ao curso, um fórum eletrônico que permite aos estudantes postarem questões e aos instrutores responde-las, um glossário, uma sala de bate-papo e uma ferramenta para escrita e envio de relatórios. Participaram, voluntariamente, como sujeitos da pesquisa dois professores de química, um estagiário do Programa de Aperfeiçoamento de Ensino da Universidade de São Paulo e 32 alunos de graduação em química. A navegação de cada um deles no sítio foi monitorada. Dados foram também coletados a partir da realização de entrevistas semi-estruturadas com os professores e da aplicação de questionários aos alunos. Durante o semestre em que foi utilizado pelos alunos da disciplina de Química Analítica, o sítio foi acessado 560 vezes. O sítio foi usado, principalmente, para a escrita e envio de relatório e para consulta às páginas de conteúdo. Poucos alunos fizeram uso do fórum para entrar em contato com os instrutores ou para apresentar questionamentos. O uso que os estudantes fizeram do sítio sugere que são capazes de utilizar o material suplementar para sanar dúvidas sobre conteúdos específicos. As entrevistas com os professores mostraram que nenhum deles havia utilizado anteriormente a Web como ferramenta de apoio na disciplina de Química Analítica. Os questionários aplicados aos estudantes apontaram para a percepção de que o acesso a materiais educacionais disponibilizados via Web é útil e pode contribuir para o aprendizado dos mesmos. / Despite the proliferation of educational material on the Internet, few published studies document the strengths and weaknesses of using the World Wide Web as a teaching tool. This work describes the design, use, and evaluation of a site created as a supplement to the Analytical Chemistry course. The site included the following elements: content pages with textual descriptions of the concepts discussed, hyperlinks to Web that provide information about topics in chemistry that are relevant to the course, an eletronic forum that enables students to pose questions and instructors to answer them, glossary, a chatroom and a tool for writing and sending reports. The results presented here will allow us to determine how best to develop other WWW resources and how best to utilize the site discussed here. The research population consisted of two chemistry professors, one teaching assistant and 32 undergraduate chemistry students who voluntarily participated in this research. All navigation for each subject was recorded. Research tools also included semi-structured personal interviews with faculty and teaching assistant, and a student´s survey. During the semester of use, the site was accessed 560 times. The site was mainly used for writing and sending reports and reading content pages. Only a few students used the forum to contact instructors and ask them questions. The analysis of student use of the site suggests that they are able to identify the areas that cause them trouble and are able to use supplemental resources to help them in those areas. Interviews with professors indicated that none of them had used the Web as support to the teaching in the Analytical Chemistry course. Analyzing the student\'s survey, we found that they noted that access to Web-based learning materials was valuable and contributed to their learning as well.

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Critical values are laboratory values that represent a life-threatening condition for which there is a treatment available. Laboratories make immediate notifications to ordering providers when critical values are identified so that they may quickly act to initiate a treatment for their patient. The majority of laboratories apply the inpatient critical value list to the outpatient setting, although there are many differences between an acutely ill inpatient population and an ambulatory outpatient population. The goal of this study was to determine if providers responded to the critical values in the outpatient setting and to determine if there was a difference in outcome indicators when providers responded to notifications and when they did not respond to notifications. Data for 673 critical value notifications for PT/INR, Digoxin, and Glucose results were collected from Riverside Health System’s five laboratories. Analysis suggested that the inpatient critical value lists and thresholds may not be appropriate to apply to the outpatient setting. In this study of 637 critical value notifications, providers chose not to respond to 25.7% of critical value notifications. Providers were more likely to respond to PT/INR and Digoxin critical value notifications that glucose critical value notifications. None of the cases for either of the three tests that went without a provider response resulted in death or serious harm to a patient, indicating that the critical value thresholds do not meet the definition of a critical value in the outpatient setting. In the future, laboratories should explore the utilization of a different critical value list and thresholds for the outpatient setting based upon patient outcomes.

acase@tulane.edu / Relative Sea-Level (RSL) change influences surface processes and stratigraphic architecture of deltaic systems and has been studied extensively for decades. However, we still lack a quantitative framework to define what constitutes a small vs. large or short vs. long RSL cycle. We explore these questions with a suite of physical experiments that shared identical forcing conditions with the exception of sea-level. We utilize two non-dimensional numbers that characterize the magnitude and period of RSL cycles. Magnitude is defined with respect to the maximum autogenic channel depth, while the periodicity is defined with respect to the time required to deposit one channel depth of sediment, on average, everywhere in the basin. The experiments include: 1) a control experiment lacking RSL cycles, used to define autogenic scales, 2) a low magnitude, long period (LMLP) stage, and 3) a high magnitude, short period (HMSP) stage. We observe clear differences in the response of deltas to the forcing in each experiment. The RSL cycles in the HMSP stage induce allogenic surface processes and stratigraphic products with scales that exceed the stochastic variability found in the control stage. These include the generation of rough shorelines and large temporal gaps in the stratigraphy. In contrast, the imprint of LMLP cycles on surface processes and stratigraphy is found in properties that define the mean state of a system. These include the mean shoreline location and extraction of sediment inbound of the mean shoreline. This work demonstrates the effectiveness of defining RSL cycle magnitude and period through autogenic scales and provides insights for generation of forward stratigraphic models influenced by RSL change. / 1 / Lizhu Yu

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