Lee Kai Woo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Thesis Committee --- p.iii / Abstracts --- p.v / 論文槪要 --- p.vii / List of figures --- p.viii / List of abbreviations --- p.ix / Chapter Chapter one --- Introduction --- p.1 / Chapter 1.1 --- Drug metabolism and UGTs --- p.1 / Chapter 1.2 --- Natural substrates of UGTs --- p.4 / Chapter 1.3 --- Functions of UGT isoforms: roles of UGT polymorphisms --- p.6 / Chapter 1.4 --- Evolution of the UGT1 gene locus in vertebrates --- p.8 / Chapter 1.5 --- Multiple Variable First Exons: A Mechanism for Cell- and Tissue-Specific Gene regulation --- p.13 / Chapter 1.6 --- Evolutionary Origin of the Variable and Constant Genomic Organization --- p.14 / Chapter 1.7 --- Variable and Constant Genomic Organizations Exist in Mammalian UGTs --- p.20 / Chapter 1.8 --- The history of recombinant UGT expression --- p.20 / Chapter 1.9 --- UGT1A8 --- p.21 / Chapter 1.10 --- Licorice and its active component --- p.24 / Chapter 1.11 --- Enzyme induction in the liver --- p.25 / Chapter 1 12 --- Objectives --- p.28 / Chapter Chapter two --- Methods and Materials --- p.29 / Chapter 2.1 --- UGT1A8 induction studies --- p.30 / Chapter 2.1.1 --- Drug preparation --- p.30 / Chapter 2.1.2 --- Cell viability study with Neutral Red Assay Rat treatment --- p.30 / Chapter 2.1.3 --- Cell treatment --- p.31 / Chapter 2.1.4 --- Rat treatment --- p.31 / Chapter 2.1.5 --- RNA extraction from rat liver and cell culture --- p.31 / Chapter 2.1.6 --- Quantization of RNA --- p.32 / Chapter 2.1.7 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.1.8 --- Northern hybridization --- p.33 / Chapter 2.1.9 --- Probe for Northern Blotting --- p.34 / Chapter 2.1.10 --- Agarose Gel analysis and Northern Blot analysis --- p.34 / Chapter 2.2 --- Recombinant expression of UGT1A8 in E.coli JM109 --- p.35 / Chapter 2.2.1 --- cDNA synthesis --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction --- p.35 / Chapter 2.2.3 --- Agarose gel electrophoresis for DNA --- p.35 / Chapter 2.2.4 --- "Amplification of target gene, UGT1A8" --- p.36 / Chapter 2.2.5 --- Restriction enzyme digestion of plasmid and insert --- p.36 / Chapter 2.2.6 --- Ligation of plasmid and insert DNA --- p.37 / Chapter 2.2.7 --- Amplification of target plasmid --- p.37 / Chapter 2.2.8 --- Screening of target plasmid --- p.37 / Chapter 2.2.9 --- DNA sequencing --- p.38 / Chapter 2.2.10 --- Transformation of protein expression host --- p.38 / Chapter 2.2.11 --- Confirmation of transformation of protein expression host --- p.38 / Chapter 2.2.12 --- Protein expression --- p.39 / Chapter 2.2.13 --- Protein purification --- p.39 / Chapter 2.2.14 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.15 --- Confirmation of the protein --- p.40 / Chapter 2.3 --- Characterization of recombinant UGT1A8 --- p.41 / Chapter 2.3.1 --- UGT assay --- p.41 / Chapter 2.4 --- Routine experiment methods --- p.41 / Chapter 2.4.1 --- Determination of protein --- p.41 / Chapter 2.4.2 --- Nucleic acid purification --- p.42 / Chapter 2.4.3 --- Preparation of chemically competent bacterial cells --- p.42 / Chapter 2.4.4 --- Colony PCR --- p.43 / Chapter 2.4.5 --- Plasmid rescue by alkaline lysis --- p.44 / Chapter 2.4.6 --- Charging of His-tagged column --- p.44 / Chapter 2.4.7 --- Washing of His-tagged column --- p.45 / Chapter Chapter three --- Results --- p.46 / Chapter 3.1 --- UGT1A8 Expression in clone9 and H4IIE after treatment with licorice and 18 β glycyrrhentinic acid --- p.46 / Chapter 3.2 --- UGT1A8 induction in wistar and j/j rats after treatment --- p.63 / Chapter 3.3 --- Construction of pRset-UGT 1A8 Vector --- p.70 / Chapter 3.4 --- Purification of recombinant UGT1A8 --- p.75 / Chapter 3.5 --- Screening of substrate of the purified enzyme --- p.77 / Chapter Chapter four --- Discussion --- p.78 / Chapter 4.1 --- Effects of licorice and 18βglycyrrhetinic acid in the induction of UGT1A8 in different cell lines --- p.78 / Chapter 4.2 --- Comparison of wistar and j/j rats in the induction of UGT1A8 --- p.79 / Chapter 4.3 --- Comparison of licorice and 18(3 glycyrrhetinic acid in the induction of UGT1A8 in rats --- p.81 / Chapter 4.4 --- Comparison of in vivo and in vitro of drug treatment --- p.81 / Chapter 4.5 --- Expression of UGT1A7 after drug treatment in vitro --- p.82 / Chapter 4.6 --- Protein expression and purification --- p.83 / Chapter 4.7 --- Substrates of UGT1A8 --- p.83 / Chapter Chapter Five --- Conclusions --- p.86 / References --- p.90 / Appendix --- p.105

Thesis (Ph.D.)--Flinders University, School of Medicine, Dept. of Clinical Pharmacology. / Typescript bound. Includes bibliographical references: (leaves 334-391) Also available electronically.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / The photosynthetic protist Euglena gracilis synthesizes a storage carbohydrate named paramylon, a glucan consisting only of β-(1-3)-glycosidic linkages. The enzyme that produces paramylon is a glycosyltransferase commonly known as paramylon synthase (EC 2.4.1.34; UDP-glucose: 1,3-β-D-glucan 3-β-D-glucosyl transferase). This enzyme uses UDP-glucose as its main substrate. In 2001, Bäumer et al. isolated and partially purified paramylon synthase, but never presented any sequence information. Hence, the main aim of this project was to isolate and characterize the gene(s) coding for the paramylon synthase. Different approaches were taken in order to isolate and characterize the gene(s). In the first part of the study molecular techniques were used to try and identify the gene. The two methods used were library screening and PCR amplification. Different libraries were screened using either functional staining or an affinity probe. The second method concentrated on the use of degenerate oligonucleotides, based on the amino acid sequences of conserved regions from known β-(1-3)-glucan synthase genes from various organisms, to PCR amplify the gene sequence from Euglena. These approaches were not successful in the isolation of the gene(s). In the second part of the study protein purification techniques were used in an attempt to obtain de novo protein sequence from the purified paramylon synthase enzyme. Several protein purification techniques were tried with the most successful being preparative ultra centrifugation followed either by sucrose density centrifugation or product entrapment (a type of affinity purification). These resulted in partial purification of the paramylon synthase protein. The partially purified proteins were separated using polyacrylamide gel electrophoresis, and the polypeptides able to bind the precursor, UDP-glucose, were identified using a radiolabeled isotope of UDP-glucose. These polypeptides were subjected to LC-MS-MS in order to obtain sequence information from them. One tryptic fragment showed high homology to β-(1,3)-glucan synthase genes from different yeasts.

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京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12834号 / 論農博第2798号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4857(農学部図書室) / 31372 / (主査)教授 栗原 達夫, 教授 植田 和光, 教授 平竹 潤 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Hepatic UDP-GTs are partly responsible for metabolism of the thyroid hormone, thyroxine (T4), in mammals, but little is known of UDP-GT activity in birds. To determine the ontogenic pattern of UDP-GT activity in precocial birds, we measured activity in Japanese quail (Coturnix japonica) liver at days 12 and 14 of the 16.5-day incubation, 3 perihatch stages and <1, 1, 4, 6, 7, 20 and 42 days posthatch. We used an enzymatic reaction with para-nitrophenol (pNP) as substrate that was validated for quail tissue. The pattern of UDP-GT development included low embryonic activity, increased activity beginning in the perihatch period, a peak in activity at day 4 posthatch and a return to lower activity levels from day 6 to adults. The profile of UDP-GT activity, in relation to the ontogeny of circulating T4 and triiodothyronine (T3) in quail, is consistent with UDP-GT playing a role in regulating circulating T4 and with the perihatch peak in T3 stimulating the posthatch peak in UDP-GT activity. To examine the effects of polychlorinated biphenyls (PCBs) on UDP-GT in developing precocial birds, we dosed chicken (Gallus domesticus) eggs with concentrations of PCB 126 from 0 to 0.80 ng/g egg (in sunflower oil) prior to incubation. Tissues were sampled at day 20 of the 21-day incubation and assayed for plasma hormones and UDP-GT activity. Eggs also were dosed with 0 or 0.25 ng PCB 126/g egg or with 0 or 0.64 ng/g egg of the coplanar PCB 77, allowed to hatch, and sampled at 42 days posthatch. There was no consistent pattern of altered thyroid hormones or UDP-GT activity in developing chickens exposed to either of these coplanar PCBs although previous studies indicated developmental alterations from exposure to the higher doses. / Master of Science

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The young internodes of sugarcane are ideal targets for altering metabolism, through genetic manipulation, to potentially control known fungal diseases such as Smut or to increase sucrose yields in these regions that are currently being discarded. At present, no regulatory sequences that specifically drive transgene expression in young developing sugarcane tissues are available. The objective of this study was therefore to isolate and evaluate such a sequence. The promoter targeted for isolation in this study regulates the expression of UDP-glucose dehydrogenase (EC 1.1.1.22), an enzyme which catalyses the oxidation of UDP-glucose to UDP-glucuronic acid, a precursor for structural polysaccharides which are incorporated into the developing cell wall. A strong correlation between the expression of UDP-glucose dehydrogenase and a demand for structural polysaccharides in developing tissues could therefore be expected. The first part of this study addressed the general practicality of promoter isolation from sugarcane, a complex polyploid. A gene encoding UDP-glucose dehydrogenase was isolated from a sugarcane genomic library. The gene contains an open reading frame (ORF) of 1443 bp, encoding 480 amino acids and one large intron (973 bp), located in the 5’-UTR. The derived amino acid sequence showed 88 – 98% identity with UDP-glucose dehydrogenase from other plant species, and contained highly conserved amino acid motifs required for cofactor binding and catalytic activity. Southern blot analysis indicates a low copy number for UDP-glucose dehydrogenase in sugarcane. The possible expression of multiple gene copies or alleles of this gene was investigated through comparison of sequences amplified from cDNA prepared from different tissues. Although five Single Nucleotide Polymorphisms (SNP) and one small-scale insertion/deletion (INDEL) were identified in the aligned sequences, hundred percent identity of the derived amino acid sequences suggested the expression of different alleles of the same gene rather than expression of multiple copies. The finding that multiple alleles are expressed to provide the required level of a specific enzyme, rather than the increased expression of one dominant allele, is encouraging for sugarcane gene and promoter isolation. In the second part of the study the suitability of UDP-glucose dehydrogenase as a target for the isolation of a developmentally regulated promoter was investigated. The contribution of UDP glucose dehydrogenase to pentan synthesis, as well as the expression pattern and subcellular localisation of the enzyme in mature sugarcane plants was studied at the tissue and cellular level. Radiolabelling with positionally labelled glucose was used to investigate the relative contributions of glycolysis, the oxidative pentose phosphate pathway and pentan synthesis to glucose catabolism. Significantly (P=0.05) more radiolabel was released as CO2 from [6-14C]- glucose than [1-14C]-glucose in younger internodes 3, 4 and 5, demonstrating a significant contribution of UDP-glucose dehydrogenase to glucose oxidation in the younger internodes. In addition, there was significantly (P=0.05) more radiolabel in the cell wall (fiber) component when the tissue was labelled with [1-14C]-glucose rather than [6-14C]-glucose. This also demonstrates a selective decarboxylation of glucose in position 6 prior to incorporation into the cell wall and is consistent with a major role for UDP-glucose dehydrogenase in cell wall synthesis in the younger internodes. Expression analysis showed high levels of expression of both the UDP-glucose dehydrogenase transcript and protein in the leafroll, roots and young internodes. In situ hybridisation showed that the UDP-glucose dehydrogenase transcript is present in virtually all cell types in the sugarcane internode, while immunolocalisation showed that the abundance of the protein declined in all cell types as maturity increased. Results obtained confirmed that this enzyme plays an important role in the provision of hemicellulose precursors in most developing tissues of the sugarcane plant, indicating that UDP-glucose dehydrogenase was indeed a suitable target for promoter isolation. Lastly, the promoter region and first intron, located in the 5’-untranslated region (UTR) of this gene, were isolated and subsequently fused to the GUS reporter gene for transient expression analysis and plant transformation. Transient expression analysis showed that the presence of the intron was essential for strong GUS expression. Analysis of stably transformed transgenic sugarcane plants, evaluated in a green house trial, showed that the isolated promoter is able to drive GUS expression in a tissue specific manner under these conditions.

Au quotidien, nous sommes exposés à une multitude de xénobiotiques, des molécules exogènes à un organisme vivant et qui sont considérées toxiques pour ce dernier. Parmi tous ces xénobiotiques, le Bisphénol-A (BPA), un xénoestrogène, est l'un de ceux attirants le plus l'attention de la communauté scientifique. Des travaux effectués antérieurement par notre laboratoire ont mis en évidence des effets cytotoxiques sur les cellules placentaires (Benachour et Aris, 2009). À de très faibles concentrations, le BPA induit une augmentation significative de l'apoptose et de la nécrose des cytotrophoblastes. De plus, à ces mêmes concentrations, le BPA provoque une augmentation de l'expression et de la sécrétion de facteur de nécrose tumorale alpha (TNF-alpha), une cytokine clef dans les phénomènes d'inflammation. À un niveau cellulaire, la nécrose, l'apoptose ainsi que l'augmentation de la sécrétion de TNF-alpha sont des observations décrites dans les cas de prééclampsie (PE), de diabète gestationnel (DG) et de retard de croissance intra-utérin (RCIU). Ces travaux laissent croire à une plus grande accumulation du BPA chez les femmes ayant développé l'une de ces complications, ainsi qu'un rôle du BPA dans le développement physiopathologique de ces complications. Pour vérifier cette hypothèse, quatre groupes ont été formés, un groupe témoin où aucune pathologie n'est connue, un groupe de femmes ayant développé la prééclampsie, un groupe de femmes ayant développé un diabète gestationnel et finalement un groupe de femmes ayant eu une grossesse compliquée par un retard de croissance intra-utérin. Chacun de ces groupes était formé de 23 candidates sélectionnées dans une cohorte préexistante, la cohorte PÉRICARD. Pour chaque candidate, le Bisphénol A a été quantifié dans le sérum maternel, dans l'homogénat placentaire ainsi que dans le sérum foetal. Les dosages ont été effectués par chromatographie gazeuse couplée à un spectromètre de masse, l'une des méthodes les plus sensibles existantes pour la quantification du Bisphénol A. Nos résultats ont permis de mettre en évidence une accumulation différentielle du BPA dans le placenta de femmes ayant une grossesse avec prééclampsie et ayant mené à un retard de croissance intra-utérin. Le placenta de ces femmes a accumulé une plus forte quantité BPA que les femmes du groupe témoins. Ces résultats entérinent donc l'hypothèse d'une implication du BPA dans la prééclampsie et dans le retard de croissance intra-utérin.

This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...

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