Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be <1% but further analysis by next generation sequencing (NGS) revealed HDR occurred at a level of 2%. This relatively low level of repair was determined to be a consequence of distance from the cut site to the mutation and so rather than designing a new pair of ZFNs, the position of the existing intron 9 ZFNs was exploited and attempts made to correct >80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines.

An investigation of implicit measurement techniques amongst low risk and forensic samples

There are difficulties with utilising self- report and physiological measures of assessment amongst forensic populations. This study investigates implicit based measures amongst sexual offenders, nonsexual offenders and low risk samples. Implicit measurement is a term applied to measurement methods that makes it difficult to influence responses through conscious control. The test battery includes the Implicit Association Test (IAT), Rapid Serial Visual Presentation (RSVP), Viewing Time (VT) and the Structured Clinical interview for disorders. The IAT proposes that people will perform better on a task when they depend on well-practiced cognitive associations. The RSVP task requires participants to identify a single target image that is presented amongst a series of rapidly presented visual images. RSVP operates on the premise that if two target images are presented within 500milliseconds of each other, the possibility that the participant will recognize the second target is significantly reduced when the first target is of salience to the individual. This is the attentional blink phenomenon. VT is based on the principle that people will look longer at images that are of salience. Results showed that on the VT task, child sexual offenders took longer to view images of children than low risk groups. Nude over clothed images induced a greater attentional blink amongst low risk and offending samples on the RSVP task. Sexual offenders took longer than low risk groups on word pairing tasks where sexual words were paired with adult words on the IAT. The SCID highlighted differences between the offending and non offending groups on the sub scales for personality disorders. More erotic stimulus items on the VT and RSVP measures is recommended to better differentiate sexual preference between offending and non offending samples. A pictorial IAT is recommended. Findings provide the basis for further development of implicit measures within the assessment of sexual offenders.

Designing tools that enhance communication and understanding between multiple stakeholders: the case of mobile payment value networks

The pervasive use of mobile technologies has provided new opportunities for organisations to achieve competitive advantage by using a value network of partners to create value for multiple users. The delivery of a mobile payment (m-payment) system is an example of a value network as it requires the collaboration of multiple partners from diverse industries, each bringing their own expertise, motivations and expectations. Consequently, managing partnerships has been identified as a core competence required by organisations to form viable partnerships in an m-payment value network and an important factor in determining the sustainability of an m-payment business model. However, there is evidence that organisations lack this competence which has been witnessed in the m-payment domain where it has been attributed as an influencing factor in a number of failed m-payment initiatives since 2000. In response to this organisational deficiency, this research project leverages the use of design thinking and visualisation tools to enhance communication and understanding between managers who are responsible for managing partnerships within the m-payment domain. By adopting a design science research approach, which is a problem solving paradigm, the research builds and evaluates a visualisation tool in the form of a Partnership Management Canvas. In doing so, this study demonstrates that when organisations encourage their managers to adopt design thinking, as a way to balance their analytical thinking and intuitive thinking, communication and understanding between the partners increases. This can lead to a shared understanding and a shared commitment between the partners. In addition, the research identifies a number of key business model design issues that need to be considered by researchers and practitioners when designing an m-payment business model. As an applied research project, the study makes valuable contributions to the knowledge base and to the practice of management.

The effectiveness of using patient-reported outcome measures as quality improvement tools

Aim: To investigate the value of using PROMs as quality improvement tools. Methods: Two systematic reviews were undertaken. The first reviewed the quantitative literature on the impact of PROMs feedback and the second reviewed the qualitative literature on the use of PROMs in practice. These reviews informed the focus of the primary research. A cluster randomised controlled trial (PROFILE) examined the impact of providing peer benchmarked PROMs feedback to consultant orthopaedic surgeons on improving outcomes for hip replacement surgery. Qualitative interviews with surgeons in the intervention arm of the trial examined the view of and reactions to the feedback. Results: The quantitative review of 17 studies found weak evidence to suggest that providing PROMs feedback to professionals improves patient outcomes. The qualitative review of 16 studies identified the barriers and facilitators to the use of PROMs based on four themes: practical considerations, attitudes towards the data, methodological concerns and the impact of feedback on care. The PROFILE trial included 11 surgeons and 215 patients in the intervention arm, and 10 surgeons and 217 patients in the control arm. The trial found no significant difference in the Oxford Hip Score between the arms (-0.7, 95% CI -1.9-0.5, p=0.2). Interviews with surgeons revealed mixed opinions about the value of the PROMs feedback and the information did not promote explicit changes to their practice. Conclusion: It is important to use PROMs which have been validated for the specific purpose of performance measurement, consult with professionals when developing a PROMs feedback intervention, communicate with professionals about the objectives of the data collection, educate professionals on the properties and interpretation of the data, and support professionals in using the information to improve care. It is also imperative that the burden of data collection and dissemination of the information is minimised.