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The overarching theme of this thesis was to explore if and how sexual reproduction in fungi could be exploited for disease control and strain improvement. Work focussed on two distantly related members of the Pezizomycotina; Pyrenopeziza brassicae, an important pathogen of Brassica napus (oilseed rape) and other Brassica species; and Penicillium roqueforti, the famous species used for commercial production of blue cheeses. First, investigations were undertaken to characterise a putative lipid hormonal compound, "Sex Factor" (SF), involved in controlling the switch between asexual and sexual reproduction in Pyrenopeziza brassicae, the causative agent of light leaf spot in Brassica plant species. SF shows promise as a novel disease control agent because of its reported ability to repress asexual sporulation, an important factor in the spread of disease. Crude SF was extracted from mated cultures of P. brassicae and it was confirmed via bioassays that extracts were able to repress asexual sporulation in this species. By contrast, asexual sporulation of North American "Lineage 2" isolates of P. brassicae was unaffected by SF. Biochemical purification work including the use of LC-MS then identified 16-(β-D-Glucopyranosyloxy)-2,15-dihydroxyhexadecanoic acid as a putative candidate for being an active component of crude SF. Finally, progress was made towards identifying key genes which may be involved with regulation of asexual and sexual development. Overall, this work has provided new insights into the asexual and sexual biology of P. brassicae, and laid the groundwork for developing new methods of disease control against this important fungal pathogen. Secondly, investigations were made to characterise sexual progeny of Penicillium roqueforti. A sexual cycle has only recently been discovered in this species, and therefore it was of academic and of commercial interest to determine whether sex can lead to the production of progeny with novel characteristics relevant to blue cheese production. Four pairs of parental isolates and respective progeny were screened for proteolytic activity and lipolytic activity, and production of certain secondary metabolites. It was found that up to 91 % of progeny had altered proteolytic activities and up to 55 % had altered lipolytic activities, compared to parental isolates. No progeny were found to have novel secondary metabolite production capacity, although significant variation was detected, suggesting novelty may be found with further screening. Thus, it was demonstrated that strains of P. roqueforti with alterations in desirable characteristics can be generated via the sexual cycle. These results may encourage the use of sexual reproduction for strain improvement of this species and other species used in industrial settings. Third, work was undertaken to characterise lipase genes involved in blue cheese maturation given that the genetic basis of the lipolytic system of P. roqueforti, the most important factor in determining the flavour of blue cheese, was largely unknown. Nine putative lipase genes were identified in silico, five of which were found to be more than two-fold upregulated in model blue cheese conditions. Concurrently, a "laboratory strain" of P. roqueforti (ku70::pyrG) was developed which allowed for more efficient genetic manipulation than would be possible using a wild type strain. This laboratory strain was then used to generate lipase knockout strains which led to the identification of two genes, lipD and lipI, which encode for lipases that likely constitute the main extracellular lipolytic activity of P. roqueforti. Finally, the expression of these key genes was determined in parental isolates and sexual progeny of P. roqueforti. Novel expression profiles were found for lipD, indicating that sexual reproduction can lead to variation in lipolytic activity by alteration of gene expression. The development of a laboratory strain more amenable to genetic manipulation will be of interest to researchers working on this species who can now conduct genetic characterisation studies more easily. In addition, identification of lipD and lipI will allow more specific screening for strains which show desired expression levels of or mutations within these genes, and could therefore be of interest to blue cheese producers.

Purpose Investigate changes in retinal structure and function caused by multiple sclerosis (MS) using established and novel modes of image acquisition and analysis, in order to identify candidate biomarkers of disease. Use longitudinal analysis to determine the rate at which changes to the retinal anatomy occur and whether this rate-of-change alters throughout the MS disease course. Method Within the Anne Rowling Regenerative Neurology Clinic (Royal Infirmary of Edinburgh), 72 MS participants (53 female, 19 male, age range 20-79, median age 44) and 80 healthy volunteers (HV) (61 female, 19 male, age range 23-73, median age 38) received spectral domain optical coherence tomography (SD-OCT) and ultra-wide field scanning laser ophthalmoscope (UWF-SLO) scans at baseline. Within the MS group, 16 (9 right eyes and 10 left eyes) had at least one clinically diagnosed episode of optic neuritis (ON). Follow up scans were taken at approximately 3, 6 and 12-month intervals over the course of two years. Visual acuity (VA) as an indicator of retinal function was also measured at each visit using Sloan letter charts at 100% and 2.5% illumination. Through proprietary software, automatic segmentation of SD-OCT images quantified changes to retinal nerve fibre layer (RNFL) thickness and macular volume. Novel image processing algorithms specifically adapted to semi-automatically segment the vasculature in UWF-SLO images (using multi-scale filtering and supervised classification) allowed for measurement of vessel diameter. Data from the 3 participant groups (MS (no ON), MS (ON) and HV) was first tested statistically for significant differences. Linear regression modelling with the inclusion of disease, sex, length-of-disease and age as predictors was then used to estimate the true effects of disease on the retinal anatomy without confounders. Results Baseline data showed that individuals with MS (no ON) have a reduced RNFL thickness in all peripapillary regions and a reduced macular volume when compared with HVs. The reduction in RNFL thickness was seen globally in both eyes (OS: -10μm, OD: -11μm, p<0.001) but was greatest in the inferior temporal region (OS: -15μm, OD: -13μm, p<0.01). In the MS (ON) group the reduction in RNFL thickness was even greater, again being largest in the inferior temporal region (OS: -29μm, OD: -24μm). Linear regression models confirmed the effect of disease on RNFL thickness and macular volume with significant coefficients in all regions but the inferior nasal. Follow-up analysis showed that the MS (no ON) group had a larger annual rate-of-thinning of the RNFL than HVs in the majority of regions, the largest difference being in the inferior nasal (OS: -1.32μm/year, OD: -1.06μm/year, p<0.05) and superior nasal (OS: - 1.11μm/year, OD: -1.13μm/year, p<0.05) regions. Retinal arteries were shown to have a significantly reduced diameter in only the inferior nasal quadrant, where the greatest reduction in vessel width was found (OS: -0.005mm, OD: - 0.007mm, p<0.05). For mean venous width, the inferior nasal quadrant of the right eye also showed a statistically significant difference between the two groups (OS: -0.005mm, OD: - 0.005mm). Linear regression models were more frequently significant for arterial rather than venous data, despite both vessel types having significant disease coefficients in the inferior nasal quadrant. Follow-up data was inconclusive on the effect that disease has on the annual rate-of-change of vessel thinning. Median baseline high contrast VA (OS: -6, OD: -3, p< 0.05) and low contrast VA (OS: -6, OD: -6, p<0.01) scores were significantly reduced in the MS (no ON) group when compared to HVs. In this study, MS seemed responsible for a 1 line drop in both high contrast and low contrast VA scores in the MS (no ON) group. A significant difference between the HV group and MS (ON) group was only found in the median low contrast VA score of the left eye (OS: -21, OD: -4, p< 0.05). As with vessel analysis, follow-up data showed no significant rates-of-change in VA score. Conclusions At baseline, the RNFL and blood vessels of the retina were significantly thinner in the MS (no ON) group when compared to HVs in particular regions. Consistent with previous reports in the literature, both RNFL and macular volume showed a significant decrease in individuals with MS (no ON). However, this is the first study to show that both arteries and veins in the retinal periphery also have a reduced width in the inferior nasal quadrant. Such changes in the retinal anatomy correlate with a drop in visual function as measured by VA. An increased annual rate of RNFL thinning was also seen in participants with MS (no ON) indicating that anatomical changes occur over time rather than acutely, however this rate-of-change is largest early in the disease course. A similar pattern was observed with the rate at which macular volume decreases in individuals with MS (no ON). Whether changes to the retinal blood vessels occur over time was not determinable within this study, due to the effect size being too small for the number of participants recruited. Future studies should focus on replicating findings pertaining to retinal blood vessels and determining when in the MS disease course these changes occur.

Organophosphates are a known danger to human health with an array of biochemical targets and complex toxic effects. Phenyl saligenin phosphate (PSP) is an organophosphate and related phenoxy substituted organophosphates irreversibly bind to a variety of important enzymes causing the condition known as organophosphate induced delayed neuropathy (OPIDN). These compounds also potentially cause cardiac muscle damage but little is known about how this class of compounds effect cardiomyocytes. The identification of proteins targeted by these toxins is of interest. This study investigates the synthesis and application of novel fluorescently labelled organophosphates to investigate organophosphate interaction with H9c2 cardiomyoblasts. The synthesis of probes with BODIPY, pyrene and rhodamine fluorescent component is explored with success in preparation of pyrene and rhodamine probes. The fluorescent behaviour of three rhodamine labelled phenyl saligenin phosphates probes with alkyl and PEG linkers is examined and the cytotoxicity of the probes assessed by monitoring MTT reduction, and LDH release from exposed H9c2 cardiomyoblasts. All three analogues showed cytotoxicity (4 h exposure; 100 μM). These probes were found to bind to proteins within mitochondria and spots from 2D-gel electrophoresis experiments, visualised at 532 nm, which are undergoing MS analysis. This project also describes progress towards a photoreactive-fluorescent probe to identify the site of action of the potassium channel opener, diazoxide, which activates cardioprotective pathways. The drug is believed to target mitochondrial KATP channels, but the protein structure of these channels has yet to be elucidated. The fluorescence labelling of whole cardiac cells with a dansyl fluorescent -benzophnone-photoreactoive diazoxide probe has been demonstrated and the progress toward a rhodamine fluorescent analogue is presented.