Lee, Chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 124-145). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 槪要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of pharmacogenetics --- p.2 / Chapter 1.2 --- Clinical importance of nitrogen oxidations --- p.8 / Chapter 1.3 --- Characteristics of trimethylamine (TMA) --- p.9 / Chapter 1.4 --- Genetic polymorphism of trimethylamine N-oxidation --- p.12 / Chapter 1.5 --- Enzyme systems mediating trimethylamine N-oxidation --- p.15 / Chapter 1.6 --- Aims and objectives --- p.19 / Chapter Chapter 2 --- Development of an Analytical Method for Trimethylamine & Trimethylamine N-Oxide --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials --- p.26 / Chapter 2.3 --- Methods --- p.27 / Chapter 2.3.1 --- Preparation of stock solutions --- p.27 / Chapter 2.3.2 --- Preparation of GLC glass column --- p.28 / Chapter 2.3.3 --- Optimizing GLC conditions for the separation of trimethylamine --- p.29 / Chapter 2.3.4 --- Sample pretreatment for GLC analysis --- p.29 / Chapter 2.3.5 --- Construction of calibration curve --- p.31 / Chapter 2.3.6 --- Determination of the required amounts of titanium (III) chloride for trimethylamine N-oxide reduction --- p.31 / Chapter 2.3.7 --- Intra- and inter-assay variations --- p.31 / Chapter 2.3.8 --- "Equations used in the determinations of free TMA, total TMA, and percentage TMA excreted as TMAO" --- p.32 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- GLC chromatogram --- p.33 / Chapter 2.3.2 --- Construction of calibration curve --- p.33 / Chapter 2.3.3 --- Determination of the required amounts of titanium (III) chloride for trimethylamine N-oxide reduction --- p.33 / Chapter 2.3.4 --- Intra- and inter-assay variations --- p.37 / Chapter 2.4 --- Discussion --- p.37 / Chapter Chapter 3 --- Trimethylamine N-Oxidation in Chinese --- p.41 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Experimental protocols --- p.43 / Chapter 3.2.1 --- Whole day urine collections --- p.43 / Chapter 3.2.2 --- Spot urine collections --- p.44 / Chapter 3.3 --- Results --- p.44 / Chapter 3.3.1 --- Whole day urine collections --- p.44 / Chapter 3.3.2 --- Spot urine collections --- p.46 / Chapter 3.3.3 --- Comparison between whole day urine collections and spot urine collections protocols --- p.50 / Chapter 3.3.4 --- Comparison between smokers and non-smokers --- p.52 / Chapter 3.3.5 --- Comparison the results obtained in between four individual periods of whole day urine collections --- p.52 / Chapter 3.4 --- Discussion --- p.56 / Chapter Chapter 4 --- Effect of Age and Diet on Trimethylamine N-oxidation --- p.64 / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.2 --- Experimental protocols --- p.73 / Chapter 4.2.1 --- Effect of age on TMA N-oxidation --- p.73 / Chapter 4.2.2 --- Effect of diet on TMA N-oxidation --- p.73 / Chapter 4.2.3 --- Effects of control diet on TMA N-oxidation --- p.75 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1 --- Effect of age on TMA N-oxidation --- p.76 / Chapter 4.3.2 --- Effect of diet on TMA N-oxidation --- p.80 / Chapter 4.3.3 --- Effects of control diet on TMA N-oxidation --- p.83 / Chapter 4.4 --- Discussion --- p.89 / Chapter Chapter 5 --- Effect of Disease on Trimethylamine N-Oxidation --- p.101 / Chapter 5.1 --- Introduction --- p.102 / Chapter 5.2 --- Experimental protocols --- p.108 / Chapter 5.3 --- Results --- p.109 / Chapter 5.4 --- Discussion --- p.116 / Chapter Chapter 6 --- General Discussion --- p.119 / References --- p.124 / Appendices / Chapter Appendix A / Chapter A.1 --- Sample record sheet for food intake and activity (English) --- p.A-l / Chapter A.2 --- Sample record sheet for food intake and activity (Chinese) --- p.A-3 / Chapter Appendix B --- Food intake and activity record for volunteer Ain controlled diet experiment --- p.B-1

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The 1,1-dichloroethene (1,1-DCE) cometabolic transformation abilities of indigenous and bioaugmented microorganisms were compared in microcosms constructed with groundwater and aquifer solids from the Moffett Field site, CA. Microbial community structure in the microcosms and possible community shifts due to 1,1-DCE transformation stress was evaluated by terminal restriction fragment length polymorphism method (T-RFLP). An existing biotransformation model was used to simulate the experimental data using parameter values determined by Kim et al. (2002) and Rungkamol (2001) with small adjustments to the parameter values. The laboratory microcosm studies showed that both indigenous and bioaugmented butane utilizers were capable of transforming 1,1-DCE when fed butane as a primary substrate. A butane-grown enriched culture was bioaugmented into the microcosms and exposed to several repeated additions of butane and/or 1,1-DCE, ranging from 7.1 to 76 ��mol and from 0.17 to 1.99 ��mol, respectively. The bioaugmented butane-utilizers showed a reduced lag period compared to the indigenous butane-utilizers. The greatest ability to transform 1,1-DCE was observed in bioaugmented microcosms, simultaneously exposed to butane and 1,1-DCE. Very little 1,1-DCE was transformed in the bioaugmented microcosms that were not fed butane, presumably due to lack of reductant supply and/or product toxicity of 1,1-DCE transformation. Microbial community analyses revealed similar results for replicate microcosms and differences in the community structure in microcosms subjected to different patterns of substrate addition and 1,1-DCE cometabolism. 1,1-DCE transformation resulted in temporal fluctuations in specific bacterial groups in the bioaugmented microcosms. It could be inferred that microorganisms, correlated with the T-RFL of 183 base pair (bp) were generally predominant in butane-fed bioaugmented microcosms simultaneously exposed to 1,1-DCE. Bioaugmented microcosms that were pre-exposed to 1,1-DCE for 29 days in the absence of growth substrate, followed by the addition of butane showed a significantly different microbial community from bioaugmented microcosms fed butane and 1,1-DCE simultaneously. Microorganisms with T-RFL of 179 or 277.8 bp dominated in these microcosms. These differences were possibly the result of extensive 1,1-DCE transformation product toxicity during the pre-exposure phase of the tests. A model developed by Kim et al. (2002) was used to mathematically describe the rate and extent of butane utilization and the cometabolic transformation of 1,1-DCE in the microcosm tests. Using the kinetic parameter values previously determined by Kim et al. (2002) and Rungkamol (2001), heuristic fits were obtained between the experimental data and model simulations. The model successfully predicted the trend of the butane utilization and 1,1-DCE transformation. The model outputs were statistically quantified for their fit to the experimental data by estimating Standard Error of Estimate (SEE). A reasonable fit between model predictions and experimental observations was achieved. A significant contribution of this study was developing the laboratory methods to evaluate the microbial abilities to cometabolize 1,1-DCE and determining the communities of microorganisms correlated with those biotransformation activities. Furthermore, the model comparison to experimental data indicated that there was a potential in using the existing model to predict and improve bioremediation strategies. The results showed the successful bioaugmentation of a butane-utilizing culture to improve transformation performance. / Graduation date: 2003

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Five strains (9L, 642A, 714, 5688T and 3384Y) of Histophilus ovis were investigated with respect to iron acquisition. All strains used ovine, bovine and goat, but not porcine or human, transferrins (Tfs) as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine Tf, total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine and goat) were bound by the same receptor(s). An affinity isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. A gene encoding a Pasteurella multocida TbpA homologue was shown to be present in each of two representative strains (9L and 3384Y); these genes were sequenced and determined to be the structural genes encoding the 78-kDa Tf-binding polypeptides. The identification of a fur homologue and a Fur box within the promoter region of tbpA in both strains indicated that Fur (and iron) is responsible for the iron-repressible nature of Tf-binding activity. Although tbpA transcripts were detected by reverse transcription (RT)-PCR with RNA isolated from strains 9L and 3384Y grown under iron-restricted conditions, with strain 3384Y, and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was isolated from cells grown under conditions of iron-restriction in the presence of Tf. The presence of an additional G in the tbpA gene of strain 3384Y grown under iron-replete conditions, compared to organisms grown under iron-restricted conditions plus bovine Tf, is

Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells. / By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function. / Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism. / Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins. / Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.

Productivity in large areas of the world's oceans is limited by low concentrations of dissolved iron in surface waters. Phytoplankton have adapted to persist in these environments by reducing their requirements for iron (Fe) in key metabolic pathways, in some cases by replacing Fe-containing catalysts with their iron-free functional equivalents. This thesis examines the requirements and biochemical roles for copper (Cu) and manganese (Mn) in Fe-limited centric marine diatoms. A major finding of my research is that diatoms have elevated requirements for Mn and Cu when grown in Fe-deficient seawater. Iron deficiency induces oxidative stress and increases the cellular concentrations of toxic oxygen radicals and damage products in Thalassiosira pseudonana. The increased Mn-requirement is used, in part, to activate Mn-containing isoforms of the antioxidant enzyme superoxide dismutase. Cultures co-limited by Fe and Mn exhibit high levels of oxidative stress and an inefficient detoxification pathway that further reduces cell growth. Diatoms isolated from the metal poor open ocean require more Cu to divide than related species from metal-rich coastal waters. This pattern is in stark contrast to all other known nutritive trace metals. One part of the diatom Cu requirement that is independent of provenance is for efficient Fe transport. The additional Cu requirement of oceanic species appears to be due to the constitutive expression of a Cu-containing electron transport protein, possibly plastocyanin. Coastal species, which have higher Fe-requirements for growth, retain the Fe-containing functional homologue cytochrome c6. By employing metals other than Fe within photosynthesis and antioxidant pathways, marine diatoms are able to increase their fitness in Fe-deficient environments. However, Mn and Cu also occur in low concentrations in the open ocean and thus may co-limit growth of natural populations of phytoplankton. Metal enrichment experiments i

Typescript (Photocopy) / xxx, 296 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agricultural Biochemistry, 1983

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.