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In response to salinity and drought, some higher plants accumulate the secondary metabolite glycinebetaine which functions as a compatible osmolyte (Rhodes and Hanson, 1993). Choline, a precursor of glycinebetaine (Rhodes and Hanson, 1993), is also a component of a primary metabolite phosphatidylcholine, an ubiquitous membrane phospholipid (Moore, 1982). In leaves of the glycinebetaine accumulator spinach, choline is synthesized as phosphocholine (PCho) and PCho is synthesized by three sequential N-methylations of phosphoethanolamine (PEA) ~ phosphomethyl-EA (PMEA) ~ phosphodimethyl-EA (PDEA) ~ PCho. The methyl group donor is Sadenosyl- L-methionine, SAM (Summers and Weretilnyk, 1993). The enzyme SAM: PMEA N-methyltransferase (PMEAMeT) is suggested to N-methylate PMEA ~ PDEA ~ PCho (Weretilnyk and Summers, 1992). A four-step strategy was developed for the partial purification of PMEAMeT on the basis of PMEA-dependent methylations (PMEAMeT activity) that involved the extraction of soluble leaf protein, ammonium sulfate precipitation, and column chromatography on DEAE Sepharose, Phenyl Sepharose and High Q Anion matrices. PDEA-dependent methylating activity co-purified with PMEAMeT activity which suggested PMEAMeT may N-methylate PMEA ~ PDEA ~ PCho. PMEAMeT was purified 43-fold and has specific activities of 14.7 and 18.0 nmol•min-1•mg-1 protein with PMEA and PDEA as substrates, respectively. Thin layer chromatography was used to identify the reaction products formed during the 30 minute assay incubation: with PMEA as the substrate, PDEA and PCho were detected in a ratio of 9: 1 as products; and with PDEA as the substrate, PCho was detected as the only product. PMEAMeT was estimated to have a native molecular mass of 76 kDa by HPLC gel filtration chromatography. Both PMEA and PDEA N-methylating activities have an alkaline pH optimum between 8.5 and 9.0 in 0.1 M Tris-HCl buffer. Neither activity was iii affected by the omission of Na2-EDT A from the assay. The addition of 10 mM Mg2+ to the assay inhibited PMEA and PDEA-dependent methylation by approximately 49% and 32%, respectively; whereas, the addition of 1 and 10 mM Mn2+ to the assay completely inhibited both activities. Both activities were inhibited by the reaction products S-adenosyl-Lhomocysteine by over 90% at 0.2 mM and PCho by approximately 80% at 10 mM. Of the products ofPCho hydrolysis, choline inhibited PMEA-dependent methylation by 10% at 10 mM; whereas, Pi inhibited PMEA and PDEA-dependent methylation by 38 and 19%, respectively at 10 mM. The compatible osmolyte glycinebetaine inhibited PMEA and PDEA-dependent methylation by between 20 and 30% at 140 mM; however, the inibition of PMEA-dependent methylation can be partly accounted for by the presence of cr ions in the assay. If present at these concentrations in the same subcellular compartment, these metabolites could serve as regulators ofPMEAMeT activities in vivo. Study of PMEAMeT contributed to identifying the number of enzymes that Nmethylate PEA ~ PMEA ~ PDEA ~ PCho and possible regulatory metabolites for choline biosynthesis in vivo. These data are pertinent to basic research and also to genetic-engineering studies aimed at introducing the glycinebetaine-accumulating trait into crop plants as an approach to enhancing osmotic-stress resistance. / Thesis / Master of Science (MSc)

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The purpose of this study was as follows: 1. Elucidate the laboratory conditions under which Anabaena circinalis produces the noxious odor component or components in maximum concentration, 2. Isolate the major noxious odorous metabolite(s), 3. Structurally define the odorous metabolite(s), 4. Quantitate the amount of metabolite(s) that a given amount of the organism produced.

A positive correlation between the flux of TCA cycle and productivity of Chinese Hamster Ovary (CHO) cells has been reported. Earlier work in this laboratory revealed that supplementation with nutrients that enter the TCA cycle (combination of glucose (Glc), pyruvate (Py), aspartate (Asp), asparagine (Asn) and glutamate (Glu)) significantly increased maximum viable cell density and antibody production of recombinant CHO cells. Increased amounts of extracellular citrate was associated with feeding conditions. It was hypothesized that increased flux through the TCA cycle and related metabolism was linked to enhanced growth and/or productivity of CHO cells. Therefore, the aim of this thesis is to clarify these relationships to provide routes to improve the efficiency of CHO cells by nutrient supplementation and metabolic engineering. The relationship between growth, antibody production and metabolite profiles of CHO-LB01 cells was examined in response to individual supplementation with Asn, Asp, Glu, Py and β-hydroxybutyrate (HB). Feeding HB significantly increased antibody titre while Asn feeding increased maximum cell density but led to earlier cell death. Both nutrients increased the amounts of TCA cycle intermediates and decreased the amounts of lactate, glycerol, sorbitol and amino acids. Moreover, oxygen consumption rate was increased in the presence of Asn or HB. This finding inferred that increased production of the TCA cycle intermediates in cells fed Asn or HB correlated with enhanced flux of the TCA cycle leading to enhanced oxidative metabolism. Combination of Asn or HB with Glc further improved cell growth, increased antibody titre and enhanced metabolic responses to feeds (TCA cycle intermediates). Based on these results, inhibition of sorbitol production using chemical reagent (EPBC) and siRNA designed against Akr1b1 and overexpression of malate dehydrogenase II (MDH II) were undertaken in order to increased flow of carbon atoms to TCA cycle and/or increased flux in the TCA cycle, respectively. Inhibition of sorbitol production was achieved in the presence of EBPC but there was no improvement of cell culture performance and accumulation of TCA cycle intermediates remained the same. CHO cells transfected with exogenous Mdh2 did not show improved cell culture performance. Whilst stable clones exhibited variable MDH II expression at protein level (and antibody titre), overexpression of exogenous MDH II could not be confirmed by Western blot. One CHO-MDH II clone showed greater antibody titre and exhibited similar metabolite profiling with cells fed Asn or HB. This contrasted to the majority of clones that were low producers. Comparison by RNA-Seq transcriptomic profiling of high- and low-producing CHO-MDH II clones showed that the majority of differentially expressed genes were genes related to cytoskeleton-related element and cell signaling pathways. Overall, these results confirmed the relationship between increased the amount of TCA cycle intermediates and increased antibody production. Increased amount of TCA cycle intermediates could result in increased the flow of TCA cycle lead to enhance energy and antibody production. In addition, this work represents the first study on addition of HB offers a simple effective strategy to increase antibody production.

There is a strong need for powerful unified statistical methods for discovering underlying genetic architecture of complex traits with the assistance of omics information. In this paper, two methods aiming to detect novel association between the human genome and complex traits using intermediate omics data are developed based on statistical mediation modeling. We demonstrate theoretically that given proper mediators, the proposed statistical mediation models have better power than genome-wide association studies (GWAS) to detect associations missed in standard GWAS that ignore the mediators. For each ofthe modeling methods in this paper, an empirical example is given, where the association between a SNP and BMI missed by standard GWAS can be discovered by mediation analysis.