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Modern microscopy techniques can image beyond the diffraction limit, in three spatial dimensions, and capture sub-cellular resolution videos, providing new biological insight and assisting in drug development. However, such advanced instruments typically require expert engineers and physicists to operate them, limiting their throughput and practicality for answering biological questions. Moreover, analysis of the raw data is complicated and there are significant barriers to publishing and sharing the data with others. This thesis addresses these problems, presenting two tools designed to reduce the level of expertise required to acquire and publish modern microscopy data. The development of a structured illumination microscope (SIM) is described, with a particular emphasis on control and reconstruction software designed to make SIM accessible to biologists who are new to super-resolution microscopy. The microscope's ease-of-use has led to a wide variety of biological investigations, which are presented as case studies to assist readers of this thesis in designing their own SIM experiments. The current practice for publishing 3D data is to show 2D intensity projections or fly-through videos, which present the data only from the author's perspective and do not give readers the opportunity to explore the results themselves. To solve this problem, Chapter 3 introduces my new volumetric rendering program, FPBioimage, which runs in a web browser. By creating a tool that is intuitive and easy to use, FPBioimage enables researchers around the world to immediately view their colleagues' experimental results, even when separated by thousands of miles. Two biological studies are discussed in detail to highlight the ability of these tools to answer the latest questions in cell biology. SIM's combination of high speed and high resolution video capture reveals a pinching phenomenon in the endoplasmic reticulum which was previously unknown, responsible for active flow of luminal proteins. FPBioimage is used to show metal organic frameworks successfully delivering sensitive drugs to cells, establishing a new method of cancer treatment. All software presented in this thesis is freely available, and has been carefully written to be reusable by other researchers. This is evidenced by OMERO, an online microscopy data repository, adopting FPBioimage as their default volumetric renderer. The open-source license under which the software is distributed means that developers can continue to build on the programs, extending the capabilities as new technology becomes available.

Non-alcoholic fatty liver disease (NAFLD) now affects around one in four adults in the human population and parallels the global increase in obesity. Within the spectrum of NAFLD, simple steatosis is associated with insulin resistance and type 2 diabetes while progression to steatohepatitis (NASH) is associated with an increased risk of liver cirrhosis and all-cause mortality. The molecular pathology of NAFLD is incompletely understood, however observational studies in human cohorts suggest the regulation of DNA methylation may play a role. 5-hydroxymethylcytosine (5hmC) is a cytosine modification generated from 5- methylcytosine (5mC) by the Ten eleven translocase isoenzymes (Tets) as part of a demethylation process. The aim of this project was to examine the role of Tet enzyme activity on the pathogenesis and progression of NAFLD. Detailed characterisation of two established murine dietary interventions allowed the selection of a NAFLD mouse model which broadly recapitulated the metabolic, histological and transcriptional features of human disease. Using DNA immunoprecipitation coupled with whole genome next generation sequencing and RNA micro expression arrays I examined the effect of high fat diet feeding (HFD) on hepatic DNA 5hmC levels within annotated gene regions. Whilst the global 5hmC profile was not altered by HFD, there was profound genic enrichment of 5hmC in upregulated mediators of cholesterol synthesis and transport (Lss, Sc4mol, Fdps, Hsd17b7, Cyp17a1, Mvd, Cyp1a2, Dhcr7 and Apoa4) with no enrichment in genes with other pathological functions (drug detoxification, inflammation, cell cycle regulation). Induced peaks of 5hmC enrichment were subsequently abolished following rescue of the NAFLD phenotype by conversion to control diet. Cross species validation was performed in vitro utilising embryonic stem cell derived hepatocytes challenged with a cocktail of high energy substrates. My in vivo findings were broadly replicated with specific 5hmC enrichment in genes synthesising lipotoxic molecules (PLIN2, CIDEC, APOA4, ACADVL, HMGCS2, APOA5, CYP2J2, IGFBP1, PPAP2C, ACSL1, APOC3, ANGPTL4, NRG1) with no enrichment in upregulated genes of alternative function. To determine whether or not the 5hmC enrichment seen is of functional relevance, I studied Tet1-/- C57BL/6J mice. Tet1-/- mice are grossly normal in appearance, however loss of Tet1 conferred a striking resistance to diet induced obesity with reduced body fat mass, improved insulin-sensitivity and near complete absence of NAFLD compared to wild type littermates. Furthermore, the HFD fed Tet1-/- liver transcriptome showed a ‘protective’ profile, with suppression of genes for lipid synthesis, inflammation and fibrosis. Thus, in multiple cross-species models of NAFLD, over nutrition induces genic hydroxymethylation specifically within activated genes driving the synthesis and transport of lipid molecules. Such changes are reversible with resolution of the NAFLD phenotype strengthening functional association. Tet1 deficiency conveys an obesity and NAFLD resistant phenotype. I therefore introduce Tet1 mediated hydroxymethylation as a novel mechanism for NAFLD pathogenesis.

Presently, clinical management of sexually transmitted infection (STI) is performed empirically, in the absence of microbiological diagnosis and consequentially the use of suboptimal antimicrobial therapy. Current diagnostic methodologies cannot facilitate the polymicrobial detection required to identify the potential multiple organisms that contribute to STI's. In order to meet this requirement a PCR coupled micro array capable of detecting the presence or absence of 23 organisms relevant to STI was developed and evaluated. This method was applied to explore the differential organism prevalence in 129 first void urine specimens in patients of varying symptomology, revealing the absence of Lactobacilli may be associated with urethritis. Findings of this study also documented the inadequacy of Gram stained urethral smear (GSUS) for identification and stratification of urethritis cases. Performance of rapid automated urine flow cytometric (AUFC) of urinary white cell count (UWCC) as a potential replacement for GSUS was validated in a separate study of male clinic attendees (n=436). UWCC testing demonstrated significantly enhanced specificity when compared to GSUS; universal application of this method would have significantly improved identification of urethritis cases at the point of care. The degree of urethral inflammation implied by UWCC analysis exhibited a stronger association with pathogen load of Mycoplasma genitalium when compared to Chlamydia trachomatis. A notably high prevalence of azithromycin resistant M. genitalium infection was observed amongst study participants; subsequent phylogenetic analysis implied this finding was applicable beyond this patient population. The findings of this thesis contribute new approaches to tackling the inadequacies of current empirical approaches to STI management. Modification of treatment guidelines in light of these findings and adoption of UWCC testing may improve treatment efficacy and enhance detection of nongonococcal urethritis and related syndromes. Results observed also provide an insight into the pathogenic mechanisms of different STI organisms in relation to host inflammatory responses.

The current pace of today's information technologies might lead to the casual observer to believe that this is all new. However the reality is that, as with most things, this is really a long evolution of processes based on tried, tested and re-adapted techniques. This thesis represents 12 years of predominantly technology driven research and covers a whole range of characterising, evaluating and fabricating devices based on liquid crystalline systems. Firstly polymer liquid crystals are discussed with respect to the fabrication of a flexible substrate display based on standard printing techniques and this is shown to have improved display viewing properties over a standard polymer dispersed liquid crystal (PDLC) device. Following on form this work is presented that involves the production of regular grid arrays in isotropic polymers that are used as control structures in nematic liquid crystal systems. This progresses onto a now patented device that allows the production of robust ferroelectric devices based on PDLC technology. Whilst the development of production techniques is important for the advancement of devices it would not be possible to keep up the pace without continued research into the basic liquid crystalline systems. The final chapter reviews work currently under supervision of the author based on flexoelectric effects in symmetric bimesogens. These materials possess responses times of the order of ~ 100ms with an effective optic axis switching angle that is linear with the applied field and can be in well in excess of 90°.