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We have characterised two biologically important protein complexes. Firstly, we have captured and analysed a complex of the biotin protein ligase (BPL) and the biotinylation domain (BCCPΔ67) of acetyl-CoA carboxylase (ACC) from the hyperthermophile Aquifex aeolicus. The genes encoding both BPL and BCCPΔ67 were overexpressed in E. coli and purified to homogeneity. Isolation of milligram amounts of both recombinant proteins allowed us to perform kinetic analysis of the BPL enzyme using steady-state techniques. Furthermore, a chemically crosslinked complex of BPL and BCCPΔ67 was isolated and a comprehensive mutational study has identified a salt bridge between the two proteins which is important for heterodimerisation. The role of a conserved ‘thumb-like’ motif in BCCPΔ67 was also investigated by mutagenesis. Our results suggest an interaction between the biotin moiety and the ‘thumb’ reduces the propensity of BPL and holo-BCCPΔ67 to heterodimerise. In a separate project we have investigate the relationship between the tertiary structure and biological activity of a novel β-defensin related peptide (Defrl). Defensins are cationic antimicrobial peptides which have a characteristic six cysteine motif and are important components of the innate immune system Defr1 is a polymorphism of mouse β-defensin 8 and contains only 5 cysteines. Against a panel of pathogens, we found that oxidized synthetic Defr1 had significantly higher activity than its reduced form, and the oxidized and reduced forms of its six-cysteine containing analogue (Defrl Y5C). Using non-denaturing gel electrophoresis and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) we observed Defrl and Defrl Y5C dimers.

Background: As the detrimental effects of sedentary behaviour on health and well-being are well documented and with stroke survivors having an increased risk of poor health outcomes and being identified as being more sedentary than healthy adults (English et al., 2014), it was deemed important to address the gap in sedentary behaviour and stroke research by focusing on those stroke survivors who have severe mobility disability. The thesis aimed to investigate and better understand sedentary behaviour in stroke survivors with severe mobility disability living at home and identify the next steps needed for research into sedentary behaviour in this stroke population. Methods: Two systematic reviews were undertaken to evaluate the existing literature. A Q-methodological study was conducted to explore sedentary behaviour from the perspectives of stroke survivors who have severe mobility disability, their carers and healthcare professionals. A second study investigated the energy requirements for activities of daily living in stroke survivors with severe mobility disability living at home using indirect calorimetry. Results: Keeping moving and reducing sedentary behaviour following their stroke was an integral part of their rehabilitation, with an intervention and strategies to reduce sedentary behaviour in this stroke population being welcomed by the stroke survivors, carers and healthcare professionals involved in their care. The thesis also identified higher energy requirements for activities of daily living in stroke survivors with severe mobility disability compared to healthy adults and that it may be possible to incorporate seated functional tasks involving the upper limb in strategies to reduce sedentary behaviour. Conclusions: The novel research presented in the thesis highlights the importance of inclusion of stroke survivors with severe mobility disability in research and the need for further research to build upon the baseline provided by the thesis in order to inform future interventions to reduce sedentary behaviour in this stroke population.

Background: Stroke survivors with communication difficulties have poor outcome in the longer-term and may benefit from the support typically offered by self-management approaches. Aim: To critically examine the suitability of self-management as an approach for stroke survivors with communication difficulties and to use this knowledge to design an intervention to support stroke survivors with communication difficulties in the longer-term. Methods: Medical Research Council guidance for the development of complex interventions provided a framework for designing the intervention (MRC, 2008). In line with this guidance the intervention was designed iteratively using a staged approach. The design of the intervention was informed by a mixed methods systematic literature review and qualitative fieldwork undertaken with key stakeholders (stroke survivors with communication difficulties, carers and speech and language therapists). The Behaviour Change Wheel (BCW) provided a theoretical framework to identify which behaviours the intervention should target and appropriate behaviour change techniques. A Delphi survey was conducted to gain an expert consensus on the design of the intervention. Results: Stroke survivors with communication difficulties need further support to manage the consequences of their condition in the longer-term. Self-management may provide a structured and clearly defined intervention to support this group of stroke survivors with the challenges they face. However, existing self-management interventions do not address the needs of stroke survivors with communication difficulties and a condition specific self-management intervention is required. A novel self-management intervention for stroke survivors with communication difficulties was designed using the BCW as a theoretical framework. Feedback from an expert panel was used to refine the design of the intervention. Discussion: To the authors knowledge this is the first theoretically informed self-management intervention designed specifically for stroke survivors with communication difficulties. Further research is needed to develop the intervention. Future plans for feasibility testing and large-scale evaluation of the approach are discussed.