Thesis (M.S.)--Wisconsin. / Includes bibliographical references (leaves 187-191).

Thesis (M.S.)--University of Wyoming, 2008. / Title from PDF title page (viewed on August 3, 2009). Includes bibliographical references (p. 60-64).

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This thesis developes computer modelling techniques, and their use in the investigation of biochemical systems, principally the photosynthetic Calvin cycle. A set of metabolic modelling software tools, "Scampi", constructed as part of this project is presented. A unique feature of Scampi is that it allows the user to make a particular model the subject of arbitrary algorithms. This provides a much greater flexibility than is available with other metabolic modelling software, and is necessary for work on models of (or approaching) realistic complexity. A detailed model of the Calvin cycle is introduced. It differs from previously published models of this system in that all reactions are assigned explicit rate equations (no equilibrium assumptions are made), and it includes the degradation, as well as the synthesis, of starch. The model is later extended to include aspects of the thioredoxin system, and oxidative pentose phosphate pathway. Much of the observed behaviour is consistent with experimental observation. In particular, Metabolic Control Analysis of the model shows that control of assimilation flux is likely to be shared between two enzymes, rubisco and sedoheptulose bisphosphotase (SBPase), and can readily be transferred between them. This appears to offer an explanation of experimental evidence, obtained by genetic manipulation, that both of these enzymes can exert high control over assimilation. A further finding is that the output fluxes from the cycle (to starch and the cytosol), show markedly different patterns of control from assimilation, and from each other. An novel observation in behaviour of the Calvin cycle model is that, under certain circumstances, particularly at low light levels, the model has two steady-states and can be induced to switch between them. Although this exact behaviour has not been described experimentally, published results show charecteristics suggesting the potential is there in vivo. An explanation of all the observed behaviour is proposed, based upon the topology of the model. If this is correct then it may be concluded that the qualitative behaviour observed in the model is to be expected in vivo, although the quantitative detail may vary considerably.

The PsbL, PsbM and PsbT subunits of photosystem II (PSII) are single-helix membrane-spanning proteins found at the monomer-monomer interface that may stabilize the dimeric complex. This study has characterised strains of Synechocystis sp. PCC 6803 where psbL, psbM and psbT have been interrupted by the insertion of antibiotic-resistance cassettes. The [Delta]PsbL strain exhibited slowed growth that correlated with a disruption in PSII assembly leading to an accumulation of CP43-less PSII monomers. Moreover, the [Delta]PsbL:[Delta]PsbM and [Delta]PsbL:[Delta]PsbT double mutants were not photoautotrophic. In contrast, the [Delta]PsbM and [Delta]PsbT strains grew photoautotrophically and supported oxygen evolution, albeit at reduced rates compared to wild type. S-state analyses showed that the removal of PsbM or PsbT did not affect the donor side reactions of PSII, which includes the oxidation of water, however, the removal of PsbT impaired electron flow between Q[A] and Q[B] on the acceptor side of PSII. Blue-Native PAGE revealed that removal of either PsbM or PsbT was insufficient to entirely disrupt dimer formation; however, the combined removal of PsbM and PsbT resulted in the predominance of monomeric forms of PSII in the [Delta]PsbM:[Delta]PsbT strain. Under high light (2 mE m⁻� s⁻� at 30�C), [Delta]PsbM and [Delta]PsbT cells were considerably more susceptible to photoinactivation than wild type; however, they were able to fully recover in a protein synthesis-dependent manner when returned to moderate light levels (0.03 mE m⁻� s⁻�). A requirement for Psb27 was found in the protein-synthesis-dependent recovery of photoinactivated [Delta]PsbT cells. More significantly, an absolute functional requirement was found for Psb27 in the [Delta]PsbM strain, where functional PSII complexes are not assembled in the absence of Psb27. These results suggest that Psb27 is critical for PSII assembly in the absence of PsbM, and also for the protein-synthesis-dependent recovery of PSII in the absence of PsbT. Moreover, in addition to Psb27, the PsbU subunit of the oxygen-evolving complex was also found to be an absolute functional requirement in the [Delta]PsbM strain, where functional PSII centres are not assembled when both PsbM and PsbU are absent. It appears, therefore, that PsbM has crucial functional interactions with specific extrinsic proteins located in the vicinity of the oxygen-evolving complex. Interestingly, the [Delta]PsbM strain was also found to have a high susceptibility to suppressor mutations, indicating it has important functional roles in the cyanobacterial cell.

Thesis--Leiden. / Summaries in English and Dutch. Includes reprints of papers previously published in various journals. Includes bibliographical references.