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Introduction. Free tissue transfer is the 'gold standard' of surgical care for patients requiring composite tissue reconstruction when local options are unavailable or unsuitable. It is a form of autologous transplant wherein composite tissue is harvested from a distant site and used to reconstruct the primary defect. The flap is rendered ischaemic following transection of its vascular pedicle until successful anastomosis with the recipient vessels is completed. Ischaemia depletes cellular ATP, lowers pH and strains cellular homeostatic mechanisms. The only way to halt the inevitable progression to cell death is by reperfusion. However, reperfusion per se initially worsens the injury through the influx of inflammatory cells and mediators. This biphasic injury is named ischaemia reperfusion injury (IRI) and is characterized by microcirculatory dysfunction primarily mediated by oxidative stress. This can lead to inadequate perfusion and ultimately tissue necrosis. IRI occurs in all transplants, is unavoidable and has no treatment. Preconditioning is an intervention performed before a known event that improves the outcome of that event. The elective nature of transplants permits such interventions to be executed. Haem-­oxygenase 1 (HO-­1) is a cytoprotective enzyme that is up-­regulated in response to diverse stressors including oxidative stress. Haem arginate (HA) is a potent inducer of this enzyme. Pharmacological preconditioning with HA has been shown to reduce IRI and improve clinical outcome in models of visceral IRI. Aim: to investigate whether HA could be used to improve outcome in myocutaneous flaps. Objectives: (1) to establish a reliable model of myocutaneous IRI (2) to assess the effects of pharmacological preconditioning with HA on clinical outcome measures and (3) to investigate the mechanisms underlying the effects of HA preconditioning demonstrated in the in vivo model by in vitro work. Methods. An in situ transverse rectus abdominis myocutaneous (TRAM) flap was developed. Forty male, Lewis rats were randomly assigned to receive IV: Control (NaCl); HA; HA + tin mesoporphyrin (SnMP, an HO-­1 inhibitor) and SnMP alone. Laser Doppler imaging (LDI) scans were performed to assess perfusion. Clinical outcome was assessed by percentage area flap necrosis and perfusion. In vitro adult human epidermal keratinocytes (HEKa) were treated in: Control medium; HA; SnMP and Desferrioxamine (DF) or combinations thereof. MTT and VialightTM plus ATP assays were used to assess cytotoxicity. Intracellular reactive oxygen species (ROS) concentration was determined by flow cytometry (CMH2DCFDA assay). Statistical analysis was performed by one-­way analysis if variance (ANOVA) followed by Tukey's test. Results. In vivo preconditioning with HA increased HO-­1 protein expression and level of bioactivity. This bioactivity was successfully inhibited by SnMP. In the skin, HO-­1 up-­regulation occurred in macrophages. HA based treatments resulted in significantly worse necrosis at 48 h: Control vs HA (p = 0.01). HA based treatments significantly decreased perfusion at: 24 h (Control vs HA, p = 0.0002) and 48 h (Control vs HA, p = 0.04). By contrast, SnMP did not affect either clinical outcome measure. In vitro preconditioning with HA was cytotoxic and increased intracellular ROS: both were reversed by co-­administration of DF but not SnMP. Conclusion. In contrast to data from visceral models, HA preconditioning proved deleterious in myocutaneous flaps. This is most likely due to the generation of ROS by free haem independent of HO-­1 up-­regulation.

Ischaemia-reperfusion (IR) injury is caused by the re-introduction of oxygen to organs, following periods of reduced blood flow (ischaemia). Whilst re-establishing blood flow (reperfusion) to the heart following myocardial infarction is vital for organ survival, this paradoxically leads to tissue damage. Mitochondria are at the heart of IR injury, with succinate dehydrogenase (SDH) a major player in orchestrating the damage. Succinate accumulates during ischaemia and is rapidly oxidised by SDH upon reperfusion, producing reactive oxygen species (ROS), leading to cellular death. I have investigated the development of drugs, aimed at targeting succinate metabolism to ameliorate IR injury. I firstly screened a range of compounds for their ability to inhibit SDH, having been chosen for their similar structures to succinate or the classical SDH inhibitor, malonate. Interestingly, only malonate and oxaloacetate showed potent SDH inhibition, thus were selected for further development. Malonate ester prodrugs with different properties were characterised. Hydrolysis rates of the esters differed greatly, with tuned, labile, malonate esters releasing malonate much more rapidly. Malonate esters were taken up into cells and hydrolysed to release malonate to different extents. Additionally, mitochondria-targetedmalonatemono and diesters were developed, each differing in mitochondrial and cellular uptake andmalonate release. Targeted and nontargeted malonate esters distributed into tissues in vivo, with preliminary in vivo work carried out on IR injury models, to assess for protective effects of the compounds. In addition, the physiological role of the tricarboxylic acid cycle metabolite, itaconate, was investigated. In lipopolysaccharide stimulated macrophages, itaconate has been reported to exert its effects by inhibition of SDH however, I found itaconate was a relatively poor SDH inhibitor, indicating other mechanisms of action. Current prodrugs of itaconate have many non-specific effects, not attributable to itaconate. I therefore characterised a novel itaconate prodrug and found it to be a much better surrogate, which could be subsequently used to elucidate roles for itaconate. Overall, I have shown the importance of ester selection for the prodrug delivery of dicarboxylate molecules and developed methods to improve their biological delivery.