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As students prepare to return to their programmes this month, Lee Sweetlove provides insight into the struggles and joys of being one of the first students on the new tech to paramedic apprenticeship pathway in the East of England in this month's education edition of Paramedic Roles.

Treatment of Arabidopsis cell culture for 16 h with H₂O₂ menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatmnets indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased > 3-fold in H₂O₂/ manadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H₂O₂ as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O₂ consumption by mitochondria was significantly decreased by H₂O₂ treatment.

The aim of this thesis was to explore the use of genome-scale metabolic models to predict metabolic fluxes in plant tissues. Results from this thesis showed that the application of constraint-based modelling, namely flux balance analysis, to an Arabidopsis genome-scale metabolic model gave accurate predictions of metabolic fluxes in heterotrophic cell culture and in photosynthetic leaves. Two major factors important for the accuracy of model predictions were highlighted from the study: 1) the inclusion of energetic costs for transports and cellular maintenance in terms of ATP and NADPH; 2) consideration of the interactions between light and dark metabolism in modelling photosynthetic leaves. This study began with the construction of a well-curated and compartmented genome-scale metabolic model of Arabidopsis. Using the model, cellular maintenance costs in a heterotrophic cell culture under control and two stress conditions were estimated in terms of ATP and reductant usage. The results suggested that the cells were not stressed under hyperosmotic conditions. Comparisons between model predictions and experimentally estimated flux maps showed that the inclusion of transport and maintenance costs was important for obtaining accurate model flux predictions. To model leaf metabolism over a day-night cycle, a diel modelling framework was developed which took into account the interactions between light and dark metabolism. Numerous known features of metabolism in a C3 leaf were predicted such as the nocturnal accumulation of citrate utilised for diurnal glutamate and glutamine synthesis and the operation of an incomplete TCA cycle during the day. Using the Arabidopsis genome-scale metabolic model and the diel modelling framework, the operation of the CAM cycle was predicted as a direct consequence of blocking the CO2 exchange with the external air during the day to simulate closure of the stomata. Comparisons between model predictions of C3 and various subtypes of CAM leaves suggested that photon and nitrogen use efficiencies are unlikely to be the driving forces for the evolution of CAM plants under the current atmospheric CO2 concentration. Finally, the model was utilised to predict the changes in metabolic fluxes, in particular fluxes through various routes of alternative electron flow, in a C3 leaf with varying light intensity, nitrogen availability and at different stages of leaf development. From the model flux predictions, it was shown that constraint-based modelling can be utilised to elucidate the distinct metabolic roles of enzymes in different subcellular compartments and the tissue-specific use of distinct forms of enzymes with different coenzyme specificities.