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Transposon mutagenesis is an increasingly used technique in molecular microbiology, antimicrobial development, and bioproduction of compounds. However limited experimental evidence is available for the reproducibility and intrinsic biases of the technique. The reliance on annotated genomes for determination of essentiality is problematic as annotations can change rendering a gene list inaccurate. Genomic features form networks and functional redundancies can be overlooked using the technique, so methods producing double mutations in a similar fashion would provide better resolution of essential processes within a cell. In this thesis, the reproducibility of transposon library construction was assessed by generating mutant libraries. One library is demonstrated to be reproducible when using the BioTradis pipeline, as long as a sufficient density of mutants with insertions inside coding regions is achieved. Transposon insertion frequency was found to be dependent on factors other than gene essentiality, and these should be taken into account during insertion frequency analysis. Some of these insertion biases can be measured and accounted for and insertion counts normalised to reflect this. Also presented is an annotation-independent approach for determining essentiality using change point analysis that can accept normalised insertion data. This enables identification of essential regions that may or may not code for a known product. Finally, the development of Exponential Mutagenesis, the production of mutants containing multiple transposon insertions is demonstrated, producing preliminary data identifying known genetic interactions in metabolic pathways for folate biosynthesis; these are targeted by sulphonamides and trimethoprim, two therapeutic antimicrobials. A high throughput paired mutant approach will lead to a better understanding of the mechanisms and redundancy involved in bacterial survival and antimicrobial resistance. Investigating the full complement of double mutants should also allow us to understand gene interactions in both metabolic and biosynthetic pathways and highlight essential genomic functions rather than gene products.

Extracellular vesicles (EV) are membrane bound carriers released by cells to mediate important aspects of intercellular communication; however, the basic principles of EV-biogenesis, in particular the mechanisms regulating mRNA EV-loading, are poorly characterised. Furthermore, a limited repertoire of tools has thus far made these processes challenging to research. To address these fundamental biological questions, an RNA-sequencing analysis of Drosophila EVs and the cells from which they derive was carried out. A bioinformatic pipeline was subsequently developed with the aim of identifying molecular features within the 3'UTR of mRNAs which may encourage their EV incorporation and long-range transfer. In addition, the role of these EV-enriched features was assessed in vivo, in complex Drosophila tissues, using an adapted version of the Cre-LoxP reporter to visualise long-range cell communication. These analyses revealed a distinct mRNA profile for Drosophila cells versus EVs and identified unique EV-enriched 3'UTR motifs. Motifs were preferentially arranged in distinct combinations and tended to occur in specific RNA secondary structures. These features may modulate their interaction with trans-acting factors, such as RNA binding proteins, with sequence similarity observed between these novel motifs and well-established RNA binding-protein binding sites. Subsequent in vivo analyses highlighted the potential for mRNA 3'UTR modifications to successfully modulate long-range mRNA transfer in complex tissues. EV-enriched motifs within these 3'UTR modifications, however, may only play a contributing role. Future work is needed to assess the effect of specific 3'UTR modifications on mRNA stability, length and GC content, as these factors have the potential to influence long-range mRNA transfer. These investigations provide novel insights into the biological properties facilitating mRNA transfer between Drosophila cells without physical contact. Importantly, these biological features may be evolutionarily conserved, given that similar mRNA properties have been described in mammalian cells. Therefore, these findings could establish the basis to efficiently modulate mRNA transfer and cell function at a distance.

Osteogenesis Imperfecta (OI) is a hereditary disorder effecting approximately 1 in 20,000 births. Symptoms include; low bone mass, recurrent fractures, varying degrees of short stature and deformity. There is currently no disease specific quality of life (QoL) measure for children with OI. This study uses a mixed methods approach to develop a QoL measure for the paediatric OI population. Patient reported outcome measure development is an iterative process, moving back and forth between concept elicitation, questionnaire development, pre-testing and psychometric analysis. In order to encourage a balance between good content validity, alongside promoting a robust, reliable and responsive measure, the methods chosen involved several stages: • Literature review to ensure no suitable QoL measure already existed and to begin eliciting themes. • Interview and focus groups with the target population to uncover relevant concepts, develop a conceptual framework and subsequently validate themes. • Questionnaire development; transforming themes into items, using the children’s’ language to ensure high content validity and acceptability. • Pre-testing the instrument alongside a sample of the OI population, making revisions as required. • Psychometric evaluation to assess validity, reliability and responsiveness of the questionnaire, informing potential item elimination and revision of the measure. Interviews and focus groups with the target population uncovered six main themes when describing QoL in children with OI; being safe and careful, reduced function, pain, fear, independence and isolation. These themes and related sub themes informed the development of the conceptual framework, which alongside the children’s own thematic based quotes, was used to develop the OIQoL. Pre-testing of the OIQoL highlighted logistical issues and understanding, which lead to revisions of the initial version. The final version underwent field testing; concerns around construct validity and internal consistency reliability highlighted the need to undertake further psychometric techniques on a larger cohort prior to item elimination.

Parent involvement in treatment programmes for child anxiety disorders aims to change the parental behaviours and cognitions implicated in the development and maintenance of childhood anxiety disorders. However, very few studies have included parental behaviours and cognitions as outcomes, and the methodological shortcomings of those that have, preclude clear conclusions. This study aimed to provide the first comprehensive examination of change in parental behaviours and cognitions after a guided parent-delivered cognitive-behavioural therapy (CBT) programme compared to a waitlist control. The association between change in parental behaviours and cognitions with child treatment outcome was also considered. Eighty-eight children aged 7 to 12 years old with a diagnosed anxiety disorder were randomised to either an 8-week guided parent-delivered CBT programme (n=41) or waitlist control group (n=47). None of the parents met diagnostic criteria for an anxiety disorder. Observational measures of parental behaviours whilst their child completed an anxiety-provoking task were taken before and after the intervention. Parent expectations were also measured of their child's and own response in the laboratory task, as well as for hypothetical situations that were ambiguous for whether or not they presented a threat. The treatment programme was not associated with greater change in parental behaviours compared to the waitlist control. After the treatment programme there was a change in specific parental cognitions, in that parents perceived themselves and their child to have more control in hypothetical threat ambiguous situations. Change in parental behaviour and cognition was not significantly associated with child treatment outcomes. The results suggest that guided parent-delivered CBT can increase parental self-efficacy in the management of child anxiety. However, the absence of any association of treatment with other parental cognitions or behaviours questions the salience of parental change in the treatment of childhood anxiety disorders.