Enzymes are specific and economical biocatalysts and as such are highly desirable synthetic tools. While there are many examples of successful applications of enzymes into industrial or commercial processes, the vast potential of enzyme technology is yet to be reached. Protein engineering, specifically directed evolution, is a powerful tool for generating enzymes tailored for specific industrial tasks. The ease of this process dictates the availability of enzymes and their diversity as biocatalysts. This thesis is concerned with the use of directed evolution to enhance the physical and catalytic properties of dienelactone hydrolase, a small monomeric alpha/beta hydrolase fold enzyme. Solubility is an important property dictating the suitability of enzymes as industrial biocatalysts in terms of both cost and functional utility. This thesis describes the adaption and use of the dihydrofolate reductase fusion reporter system to select for more soluble dienelactone hydrolase variants. The selection system was modified to incorporate a pre-culturing period, then used to identify mutations in solvent accessible locations that appeared to offer increased expression and solubility. While the original system was capable of selecting for improvements to the solubility of inherently insoluble proteins, the modified system can be used to select for further improvements. This work provides a solid foundation for the continued use of the modified dihydrofolate reductase fusion reporter system. This thesis also discusses linked directed evolution experiments that were designed to rapidly alter and enhance the substrate specificity of dienelactone hydrolase in favour of non-physiological p-nitrophenyl ester substrates. The best variants possessed in excess of 2000-fold improvements in kcat/Km compared to the native enzyme. Active site mutations were able to accumulate rapidly, despite most being detrimental to the overall stability of the enzyme, due to constant monitoring and maintenance of enzyme stability. The roles of six of the seven active site mutations were elucidated with the use of substrate docking and structural analysis. Additional work focused on the surface mutations that seemed to provide compensatory stabilising effects. These mutations were made individually and in combination to determine the roles they served in the evolution process. Two of the three mutations were shown to be thermally and chemically stabilising while the third was destabilising individually but exhibited epistasis in combination with the former mutations. This work is of interest due to the evolutionary strategy but more importantly for the insight into the evolution and relationship between sequence, structure and function of alpha/beta hydrolase fold enzymes. Work investigating and improving the stability of dienelactone hydrolase in the presence of organic co-solvents is also discussed. Eight rounds of directed evolution yielded a variant with 7 surface mutations that provided increased thermal and chemical stability. These experimental results corroborated with computational predictions, which provide promise for an alternative method to rapidly engineer chemical stability. Additional work analysing the effects of crystal packing on protein structure is also discussed. It was found that the crystalline environment caused small changes to side chain orientation, mostly at crystallographic interfaces and flexible surface loops, with only minor changes to the protein backbone.

BACKGROUND.A resuscitation event involves a team of emergency personnel who attempt to ensure that the patient receives the best possible chance of survival. The team comprises of both doctors and nurses who work in a variety of resuscitation roles and hold a number of responsibilities during the event. There is a growing movement among practitioners to involve family members during resuscitation events. Family presence during resuscitation (FPDR) in both adult and paediatric resuscitations was formally endorsed in the year 2000 by leading Emergency Associations and Resuscitation Councils (American Heart Association, 2000; Association, 2000) who were responsible for releasing practice guidelines. The debate continues with little known about FPDR implementation and practice in emergency departments. This study aimed to investigate the implementation and practice of FPDR with the objective of identifying the benefits, barriers and enablers, evaluating the role of the family support person, and assessing the level of education and training in rural and metropolitan emergency departments.METHOD.A mixed methods approach with a sequential explanatory design was utilised for this research study. The study was predominately quantitative (QUAN) followed by an interpretative qualitative component (qual). Phase One involved a ten page questionnaire which was distributed to 18 emergency departments in the state of Victoria, Australia. Phase Two of the study incorporated a two week period of observation in a single rural and single metropolitan emergency department. A combination of field notes, resuscitation event template and semi-structured interviews were used.RESULTS.A total of 347 questionnaires were included in the final data set with a 27% response rate representing emergency personnel from rural and metropolitan emergency departments. Descriptive and inferential statistics were used to describe theFamily Presence During Resuscitation (FPDR): Searching for Evidence 6of Implementation and Practice in the Emergency Departmentpopulation followed by a factor analysis of the 26 statements on FPDR. A total of 65 doctors and 282 nurses completed the questionnaire, with a mean age of 37.2 years and a mean of 7.8 years working emergency care. A total of 61% (n=173) of the nurses and 65% (n=42) of the doctors had a post graduate qualification and approximately 50% of the doctors and 80% of the nurses had completed adult and paediatric advanced life support training. The staff believed that FPDR occurred 0-20% of the time with adult resuscitations and 80-100% of the time for paediatric resuscitations. The doctors (77%, n=50) and nurses (79%, n=222) believed that family had a right to be present during resuscitation events and that it helped with the grieving process (54% of doctors and 62% of nurses). The staff greatly agreed that a designated support person was essential when allowing family to be present (89% of doctors and 92% of nurses). It was considered important that family members be given the opportunity to say good bye (83% of doctors and 93% of nurses). The perceived benefits, barriers and enablers for the implementation and practice of FPDR were discussed. Following a content analysis of the open ended responses the acronym ER-DRIP was developed which helped to define the essential information that family required during a resuscitation event. The acronym stands for E-emergency personnel, R-reassurance, D-diagnosis, R-regular updates, P-prognosis.During the observations in Phase Two of the study a total of 29 interviews were conducted together with observation of six rural and 18 metropolitan resuscitations. The interviews were audiotaped and later transcribed for analysis. A content analysis was conducted and six major themes emerged including; the importance of the care coordinator, balance of power, delivering bad news, life experience generates confidence, allocating roles and family centre care in action.CONCLUSION.Although not a new research topic, FPDR continues to invoke debate among emergency personnel. This research study aimed to develop an understanding of staff training and education, and to explore the perceived benefits, barriers and enablers to FPDR implementation and practice. In addition comparisons were made between adult and paediatric FPDR practice across both rural and metropolitan emergency departments. Further research is warranted in order to develop an understanding of the impact FPDR has on the patient and the family members. It is theFamily Presence During Resuscitation (FPDR): Searching for Evidence 7of Implementation and Practice in the Emergency Departmentrecommendation of this study that a comprehensive training program be developed which incorporates the acronym ER-DRIP to ensure emergency personnel develop the necessary skills and communication techniques required to feel confident with family presence during adult and paediatric resuscitations.