The plant Senecio jacobaea (tansy ragwort) produces several macrocyclic pyrrolizidine alkaloids which cause irreversible liver cirrhosis. All of the pyrrolizidine alkaloids in Senecio jacobaea are macrocyclic diesters of the necine base retronecine, with the two most predominant being seneciphylline and jacobine. Unlike horses and cattle, sheep and goats are generally resistant to chronic pyrrolizidine alkaloid toxicosis due to metabolism of the toxic pyrrolizidine alkaloids by ruminal bacteria. In this study, metabolism of jacobine and seneciphylline by ruminal bacteria was investigated, focusing on two possible metabolic pathways. One pathway involved hydrolysis of jacobine and seneciphylline and the subsequent production of the necine base retronecine. For use in these studies, retronecine was isolated and labeled with deuterium. A method was developed for the determination of retronecine to 0.02 μg /mL. Significant hydrolysis of jacobine and seneciphylline was not observed in either ovine whole rumen fluid or with a mixed culture of anaerobic microbes derived from ovine rumen fluid which metabolizes jacobine and seneciphylline. Direct metabolism of retronecine independent of the macrocyclic pyrrolizidine alkaloids was also not observed. The second metabolic pathway studied involved the production of the necine base modified by the conversion of the 1,2-double bond to an external methylene group. Previously this conversion has been observed during metabolism of pyrrolizidine alkaloids by the organism Peptostreptococcus heliotrinreducens. In this study, P. heliotrinreducens was used to convert the pyrrolizidine alkaloids heliotrine and lasiocarpine to the known 1-methylene-pyrrolizidines. The mixed culture of ovine anaerobic microbes also metabolized heliotrine and lasiocarpine rapidly to identical methylene products. This mixed culture metabolized jacobine and seneciphylline rapidly with production of very low levels of the corresponding 1-methylene compounds. In contrast, metabolism of jacobine or seneciphylline by P. heliotrinreducens was not observed. The mixed culture has demonstrated the ability to metabolize a greater variety of pyrrolizidine alkaloids than P. heliotrinreducens. Although the metabolites of jacobine and seneciphylline were not conclusively identified, it was determined that hydrolysis of jacobine and seneciphylline is not occurring. The second pathway studied appears to be more probable, with the production of 1-methylene compounds as intermediates, although not as end-products. / Graduation date: 1998

Thesis (M.D.)--University of Hong Kong, 1959. / Type written copy. Includes bibliographical references.

Thesis--Ph. D., University of Hong Kong. / Typewritten. Also availalbe in microfilm.

With the emergence of "omics" techniques, it has become essential to develop tools to utilise the vast amount of data produced by these methods. Genome-scale metabolic models represent the mathematical essence of metabolism and can easily be linked to the data from omics sources. Such models can be used for various analyses, including the investigation of metabolic responses to changing environmental conditions. Legumes are known for their ability to form a nitrogen-fixing symbiosis with rhizobia, a vital process that provides the biosphere with the majority of its nitrogen content. In the present thesis, a genome-scale metabolic model for the legume Medicago truncatula was reconstructed, based on the annotated genome sequence and the MedicCyc database. A novel approach was employed to define the compartmentalisation of the plant's metabolism. The model was used to calculate the biosynthetic costs of biomass precursors (e.g. amino acids, sugars, fatty acids, nucleotides), and its capability to produce biomass in experimentally observed ratios was demonstrated using flux balance analysis. Further investigation was carried out into how the biosynthesis fluxes and costs change with respect to different nitrogen sources. The precise charge balancing of all reactions in the model allowed the investigation of the effects of charge transport over the cellular membrane. The simulations showed a good agreement with experimentaldata in using different sources of nitrogen (ammonium and nitrate) to minimise the charge transport of the membrane. To allow the investigation of the symbiotic relationship, two rhizobial models were used. The first model, for Sinorhizobium meliloti, was reconstructed from the MetaCyc database (MC-model); the second model was a recently published model for S. meliloti specialised for symbiotic nitrogen xation (SNF-model). Combined models were created for both rhizobial networks using a specialised nodule submodel of the plant model. Potential interactions were extracted from the literature and investigated, with the analysis suggesting that oxygen availability is the main limitation factor in symbiotic nitrogen fixation. Within the analysis the SNF-model appeared to be too restricted and lacking the potential for sufficient nitrogen xation; therefore, further analysis was carried out using the MC-model, upon which it was observed that the availability of oxygen can also influence how nitrogen is supplied to the plant. At high oxygen concentrations ammonia is the primary form of nitrogen supplied by the rhizobium. However, the simulations, in accordance with experimental data, show that at lower concentrations of oxygen, alanine takes precedence. The findings also support the concept of amino acid cycling as a potential way to improve nitrogen fixation. The more flexible MetaCyc based model has allowed other potential genetic engineering approaches for higher nitrogen fixation yields to be proposed.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

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