Trypanosoma brucei spp. are protozoan parasites that cause Human African Sleeping Sickness and Nagana, a disease of cattle. The diseases have a very high mortality rate if untreated and the current drug treatments are inadequate due to toxicity and resistance. In order to develop new treatments, potential drug targets can be investigated in a candidate approach by genetic validation. In trypanosomes this can be performed by using RNA interference (RNAi) technology, which is well developed for this organism. One category of potential targets are protein kinases; members of this enzyme family have been shown to be suitable targets for relatively specific and selective, therapeutic inhibition- especially in the field of human oncology. T. brucei possesses 183 eukaryotic protein kinases, atypical protein kinase and pseudokinases that may be suitable for chemical inhibition. This study intended to genetically validated these targets and pursue interesting leads as potential drug targets. In order to perform RNAi studies on large numbers of candidate genes an existing stem-loop RNAi system was adapted to contain Gateway recombination sites. This created a highly efficient system, allowing the rapid generation of RNAi plasmids to target the entire complement of the predicted kinome of this organism. This collection of plasmids was used to generate a library of RNAi cell lines in bloodstream form parasites (BSF) which were screened for growth defects using an alamar blue cell viability assay. This identified 53 genes which were essential or important for proliferation of the BSF parasites. During this screen, a cell line was identified that contained an RNAi construct targeting two NEK kinases (NEK12.1: Tb927.8.7110, NEK12.2: Tb927.4.5310) which displayed a severe growth defect. This was replicated in a mouse model of infection. NEK 12.1 possesses an alanine gatekeeper residue and molecular modelling and virtual screening predicted this would allow the accommodation of bulky protein kinase inhibitors and therefore was investigated in more detail. Expression and purification of NEK12.1 in kinase active and dead forms allowed activity assays to be performed; these could be inhibited with the bulky inhibitor 3-MB-PP1, which also possessed activity against BSF parasites. Experiments to confirm the individual essentiality of NEK 12.1 (and its activity) remain to be performed, therefore NEK 12.1 has been partially genetically and chemically validated in this study. A unique “Orphan” kinase, containing a putative zinc-finger domain and a potential homologue of PDK1 were also investigated after the pTL RNAi screen. In vivo RNAi studies (using a mouse model) correlated well with the in culture RNAi data, demonstrating that these targets are essential in mammalian infections. During the pTL RNAi screen it was noted that the knockdown of one protein kinase, (DFK) led to a change in the morphology of the cells, yet no reduction in the alamar blue ratio was observed. Further investigation showed that after 72 h of RNAi induction 17% of cells expressed EP procyclin. This was associated with detectable changes in the transcriptome (ascertained by RNA -Seq), that were consistent with a BSF-PCF differentiation event. DFK was predicted to contain multiple transmembrane domains and features suggestive of a receptor kinase. Epitope tagging of DFK followed by cell fractionation and immunofluorescence microscopy demonstrated that the protein localised to the cell membrane. Transmission immuno-electron microscopy confirmed the cell membrane localisation and suggested that the protein kinase domain of DFK was intracellular. This study reveals several protein kinases as new drug targets, in turn identifying one as a key regulator of BSF-PCF differentiation. Due to the tractability of T. brucei it also provides a collection of plasmids and cell lines that should further the investigation of other key biological processes in this important pathogen.

In this thesis, I focus on methodological concepts of studying infectious disease transmission between agricultural premises. I used different disease systems as exemplars for spatial and network methods to investigate transmission patterns. Infectious diseases cause tangible economic threat to the farming industry worldwide by damaging livestock populations, reducing farm productivity and causing trade restriction. This implies the importance of veterinary epidemiological studies in control and eradication of pathogens. Recent increase in availability of data and computational power allowed for more opportunities to study mechanisms of pathogenic transmission. Nowadays, the bottleneck is primarily associated with efficient methods that can analyse vast amounts of high-resolution data. Here I address two livestock pathogens that differ in their epidemiology: bacteria Streptococcus agalactiae and foot-and-mouth disease (FMD) virus. Streptococcus agalactiae is a contagious pathogen that causes mastitis in cattle, and thus possesses a substantial economic burden to the dairy industry. Known transmission routes between cattle are restricted to those via milking machines, milkers’ hands and fomites during milking process. Additionally, recent studies suggested potential introductions from other host species: primarily, humans. However, strain typing data showed discrepancies in strain compositions of bacteria isolated from humans and bovines. In this thesis, strain-specific features of between-herd transmission of Streptococcus agalactiae within dairy cattle population in Denmark are investigated. Foot-and-mouth disease (FMD) is a viral infection that affects cloven-hoofed animals and is of big importance mainly because of the trade restrictions against infected regions and countries. Control programmes against FMD usually include vaccination and culling of animals. However, the debate on the optimal control for FMD is still ongoing. In this thesis, I address questions on identification of the routes of infection and on requirements for movement recording systems to be used for efficient contact tracing during an FMD outbreak. This thesis reveals several interesting findings. Firstly, the increased understanding of strain-specific transmission characteristics of Streptococcus agalactiae. One of the observed strains (ST103) showed significant and consistent spatial clustering of its cases among Danish dairy cattle herds in 2009–2011. Secondly, the network analysis of cattle movements and affiliations with veterinary practices showed that veterinary practices were exclusively associated with transmission of ST103 of Streptococcus agalactiae. Contrastingly, movement networks appeared to be important for all the three predominant bacterial strains (ST1, ST23 and ST103). Fourthly, the new extended approach that allows estimation of the whole transmission tree at once was proposed and tested for the Darlington cluster within the 2001 FMD UK epidemic. Finally, in chapter 6, it was shown that mathematical modelling did not suggest any advantages of ensuring smaller delays in the post-silent control of FMD-like pathogens.

This thesis examines the development of a novel in vitro biological caries model and its suitability in testing the efficacy of anti-caries approaches. Dental caries remains a public health concern worldwide; with extensive treatment costs and impacts on quality of life. Ineffective removal of all dental plaque from tooth surfaces after brushing, the insufficient delivery of anti-caries therapies; along with continuing shifts towards high frequency, sucrose-rich food consumption, expedites the caries disease process. It is, therefore, important to explore caries risk and development at these sites, particularly when representatively assessing the efficacy of a test agent in preventing caries. This caries model enabled the study of the anti-caries effects of fluoride to assess its efficacy in conditions simulating the modern diet. The current methodology employed the Constant Depth Film Fermenter (CDFF) to investigate the caries disease process in response to fluoride delivered continuously; twice and thrice daily; and at different concentrations. The approach is the first in CDFF research modelling caries inclusive of a biologically relevant microcosm biofilm in addition to enamel demineralisation. Specific members of multispecies biofilm were selectively enumerated using traditional microbiological culture techniques whilst caries was simultaneously quantified with Transverse Microradiography (TMR), Quantitative Light-Induced Fluorescence (QLF), and Non-Contact Surface Profilometry (NCSP). The fluorescence of biofilm illuminated by QLF was also investigated. Results indicated that quantities of total or specific members of the microbial community are not direct indicators of caries risk and turning focus towards the metabolism of oral biofilm bacteria, and how it may be affected, is vital in caries research. TMR and QLF agree when quantifying caries whilst NCSP shows promise in studying surface changes. At 0.05 ppm, fluoride was unable to exert a significant anti-caries effect despite being continuously present during and between sucrose exposures. Laminated lesions confirmed the importance of maintaining elevated levels of fluoride in the oral environment throughout the day. At higher concentrations (1,450 and 228 ppm fluoride) the anti-caries efficacy of fluoride when supplied in frequent applications throughout the day was confirmed. A third application of fluoride did not appear to additively benefit enamel, since all strategies were effective after 10 days regardless of frequency. Nonetheless the increased plaque fluoride reservoir and subtle antimicrobial effects than in twice daily pulsed biofilm, mean the benefit of a third application is likely more discernible in the long term. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDX) elucidated significant calcium fluoride deposits of enamel surfaces beneath biofilm exposed to 1,450 ppm fluoride continuously. In conclusion, the CDFF can produce multispecies biofilm under conditions similar to those of the oral milieu and investigate its cariogenicity in response to diet and experimental anti-caries agents. The model could be examined using an array of techniques to obtain information about aspects of the biofilm, the substratum, and to validate upcoming methods in an orally representative environment. In this regard, the current study contributes not only to enamel caries research but to biofilm research in general by minimising variation.

Ageing is associated with morphologic and functional changes in the brain, and is a major risk factor for cerebral vascular disease and dementia. Exercise is well known to promote cardiovascular health and reduce the age-related cognitive decline, but the mechanisms underlying this protective effect are not fully understood. Cardiovascular diseases, such as atrial fibrillation (AF), can exacerbate the risk of brain disease. This thesis aimed to assess the influence of ageing, exercise and AF on the cerebral blood flow and its regulation. It was observed that a difference of ≈55% in daily physical activity levels in a cohort of healthy old individuals did not influence internal carotid and vertebral artery blood flow. However, a high cardiorespiratory fitness was associated with increased bilateral internal carotid artery blood flow in young and old individuals when compared to their sedentary counterparts, but the influence on cerebral vasodilatory reserve is still unclear. AF patients had lower cerebral vasodilatory reserve, but preserved dynamic cerebral autoregulation, when compared to healthy controls. Future research is needed to elucidate whether cerebral haemodynamics is modified by exercise training in AF patients.

Nonsense mediated mRNA decay (NMD) is a translation-coupled process that preferentially destroys mRNAs harboring premature translation termination codons (PTCs). In mammalian cells, NMD is linked to pre-mRNA splicing. Typically, PTCs elicit strong NMD only if positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD. However, recent studies have questioned the importance of splicing and the EJC in NMD and instead they have proposed that, from yeast to mammalian cells, NMD is mostly determined by the distance of the PTC from the 3’ end. In this study, to investigate the link between pre-mRNA splicing and NMD, I used the fission yeast Schizosaccharomyces pombe. Many genes carry introns in fission yeast and unlike in Saccharomyces cerevisiae, the genome encodes for proteins homologous to EJC components. S. pombe is a powerful model organism which is easily genetically manipulated and we envisaged that studying NMD in this organism should facilitate the understanding of the molecular mechanism and in particular the link between NMD and splicing. During my PhD research, I have developed a versatile gene reporter system to study NMD; with it I discovered that splicing strongly enhances NMD in fission yeast and, surprisingly, that the EJC does not appear to be required. Unexpectedly, I found that splicing enhances NMD when the intron is either before or after the PTC, and furthermore, it does so only when the intron is close to the PTC. These observations suggest that the effect of splicing on NMD is direct and not a secondary consequence of splicing enhancing translation. Splicing is not an absolute requirement for NMD; I found that PTCs located early in the coding region could induce NMD even in the absence of a nearby intron. However, against the prediction of current models, I found no strong correlation with the distance of the PTC from the 3’ end. In summary, during my PhD a versatile system has been developed to study NMD in fission yeast and what I have observed challenges current NMD models and provides new mechanistic insights into NMD.

Biofumigation is being increasing used as alternative control method for soil borne pathogens. This method exploits toxic compounds, specifically isothiocyanates (ITCs), which are released during the breakdown of Brassica plant tissues. To date field and glasshouse experimentation assessing the potential for using biofumigation to control agricultural pests and pathogens have produced promising results. Yet large gaps still remain in the specifics of the biofumigation process. It is hoped that further research to analyse how specific toxic compounds produced during Brassica tissue breakdown, specifically ITCs, affect different pathogens. Additionally analysis of the specific isothiocyanates and concentrations produced by Brassica spp. will allow a more pathogen targeted approach to biofumigation to be generated. The importance of assessing the biofumigation process on different scales is also understood, and therefore this study has encompassed work carried out in vitro and using glasshouse experimentation to establish a comprehensive overview of the biofumigation process. Assessing the effects different agricultural treatments have on soil microbial communities has also been recognised and therefore was also be investigated in this study. This study aimed to determine the effects isothiocyanates, produced by Brassica spp., have on three economically important soil borne fungal pathogens, Colletotrichum coccodes, Rhizoctonia solani and Helminthosporium solani. Initial assessment was carried out using in vitro bioassays, allowing assessment of the overall toxicity of each ITC. Results identified that the pathogen response was dependent on both the structure of the ITC and the concentration of ITC present. The most significant pathogen suppression was observed with R. solani when exposed with benzyl or methyl ITC and H. solani when exposed to 2-phenylethyl ITC. To gain understanding of the naturally produced ITCs Gas-Chromatography Mass-Spectrometry analysis was used to analyse specific isothiocyanates produced by a range of different Brassica spp., at different development stages. Results identified Allyl, Benzyl and 2-Phenylthyl ITC as the most commonly produced by the Brassica cultivars used within this study. Overall the Allyl was found within the highest concentrations; however the specific ITCs and concentrations produced were dependent on both the development time and cultivar. Glasshouse experimentation was also carried out to assess both the effects of pure ITCs on R. solani and C. coccodes fungal inoculum within compost and diversity changes within the soil microbial community, in response to isothiocyanate incorporation and the biofumigation process. In order to examine changes in microbial communities‟ analysis was carried out using Terminal Restriction Fragment Length Polymorphism, a DNA fingerprinting method which allows bacterial diversity shift to be traced and statistically analysed. Overall incoproation of pure ITCs did not significantly reduce black scurf or black dot disease symptoms on daughter potato tubers. Additionally after 30 days soil microbial community diversity was not greatly altered by the addition of ITCs. Therefore as it is often suggested that biofumigation is influenced by the soil activity it is thought that this may be due to the addition of Brassica tissue. The increase of organic matter into agricultural soils may influence both biological and chemical processes which may in turn aid pathogen suppression. Overall this study provides a detailed insight into establishing the specific interactions that occur during biofumigation. Results produced findings of ITCs which significantly suppressed the growth of fungal potato pathogens. Development of a novel GC-MS assay revealed previously unknown data of levels and profiles produced by a number of different Brassica plants. Additionally study was also carried out to evaluate the effects of biofumigation of soil microbial communities, which is often ignored within other studies. Overall this study aimed to gain an increased level of knowledge of such processes in order for the methods and the results presented, to be used to establish effective, pathogen targeted biofumigation systems.

A continuous culture model system was set up in the laboratory and inoculated with a diverse range of microorganisms, including several bacteria and protozoa, obtained from the local mains water tap supply. This inoculum and was added to the system without any prior culture or other selection process. Biofilms readily developed on glass tiles suspended in the planktonic phase of the system. An avirulent Legionella pneumophila was inoculated into the system and was subsequently isolated from both biofilms and also from the aqueous(planktonic) phase of the chemostat. The attenuation of this strain, determined by its inability to cause disease and death in guinea pigs, remained unaltered despite the long term survival of this strain within the system. Investigations to determine whether the avirulent L. pneumophila was able to infect and proliferate within protozoa were carried out. The results of the present study show that this avirulent L. pneumophila did not proliferate intracellularly and suggest that the association of L. pneumophila with protozoa although probably important in the long term survival of this bacterium especially during periods where adverse conditions prevail, is not essential but opportunistic. In chapter 3 the importance of the presence of these non-legionellae bacteria, which included Flavobacterium sp., Acinetobacter spp. and several species of Pseudomonas, was investigated. The results suggest that the presence of the non-legionellae are relevant to the survival of Legionella especially in environments which favour it's growth, for example water distribution systems. In order that we may gain a further insight into the ecology of microorganisms in their natural environment, it is necessary to visualise them in conditions which allow them to interact in a way which mimics as closely as possible the natural environment. Biofilms were developed on surfaces which could be removed from the model system in their entirety. Direct visualisation techniques, including atomic force microscopy and Hoffman modulation microscopy could then be used which allowed the in vivo examination of biofilms in situ on the surface upon which they had developed. More traditional microscopy methods were also used. Atomic force microscope images of biofilms and individual biofilm bacteria including Legionella were obtained, which clearly showed the presence of exopolymeric substances (EPS). Hoffman modulation contrast microscopy and scanning electron microscopy showed the diverse nature of the biofilms being studied. The results of these investigations suggest that a more complete understanding of the complex nature of biofiims is achieved by the use of a combination of several microscopy techniques. The response of a L. pneumophila serogroup 6 and the avirulent L. pneumophila serogroup 1 to a commercially available biocide, Vantocil IB, was investigated. Both the serogroup 6 and the avirulent serogroup 1 could not be detected following biocide teatment in either the planktonic phase or biofilms. These results suggest that this avirulent L. pneumophila is a suitable model substitute for the virulent L. pneumophila.

Since its first use to solve the structure of sodium chloride in 1915 X-ray crystallography has developed significantly to become the premier technique for obtaining 3D structural information of small molecules and macromolecules alike. As the technique continues to develop and focus its attention on weak diffraction from the likes of micro-crystals and poorly packed crystals of membrane proteins and large protein complexes; as well as ultra-high resolution data and weak anomalous signal from native atoms, data quality is becoming more and more important. Data quality is particularly important in the wake of long wavelength macromolecular crystallography (MX) for phasing using anomalous signal from native sulphur and phosphorous atoms in proteins and DNA. This thesis first investigated the use of a new sample handling technique using a humidity controlled stream to preserve macromolecular crystals while excess surrounding solvent is removed (Chapter 2). Following the successful development of this technique the effects of excess surrounding solvent on data quality was assessed when collecting at standard MX X-ray wavelengths (~ 1 Å) and longer X-ray wavelengths (~ 2 Å). Datasets were collected from large populations of control and test crystals at standard and longer wavelengths to allow robust statistical methods to be applied; a practice not widely adopted in method development studies in X-ray crystallography. This made it possible to assess the small differences in data quality in the presence and absence of excess surrounding solvent. The effects of surrounding solvent at longer wavelengths appear to be protein dependent with some proteins tested showing no significant difference and others a significant decrease in data quality at longer wavelengths (Chapter 3). Originally this project aimed to use the new long wavelength in-vacuum MX beamline, I23, at Diamond Light Source UK to carry out phasing experiments using native sulphurs for structure solution. However, the considerable complexity involved in developing in-vacuum MX meant these experiments could not be carried out during the time frame of this thesis. Chapters 4 and 5 outline the production of a novel cancer protein (cancerous inhibitor of protein phosphatase 2A) and two protein targets from the Achromobacter xylosoxidans (Ax) genome intended for sulphur single wavelength anomalous dispersion phasing experiments on I23. Of these proteins the structure of Ax-α/β hydrolase was solved by conventional methods, the structure of which is discussed in Chapter 5. Of the protein crystals used in long wavelength data quality experiments in Chapter 3 the molecular biology of PA3825-EAL, a biofilm regulating protein essential to the swarming ability of Pseudomonas aeruginosa, was investigated further. The crystal structure of PA3825-EAL was solved in the resting, substrate bound and product bound states to high resolution. Comparison of the crystal structures of monomeric and dimeric PA3825-EAL with the inactive dimeric structure of MucR-EAL suggests dimerisation via helix 8 plays a role in inhibition of EAL domains. Prior to this, dimerisation was thought to be an activating factor in EAL domains. The product bound state of PA3825-EAL showed the presence of a previously unreported third metal binding site which may form an essential component of the reaction mechanism of EAL domains. Inability of MucR-EAL to incorporate this third metal due to dimerisation may explain the lack of activity despite possessing the conserved catalytic residues necessary. The fast detector technology and improvements in automated data processing software that allowed diffraction data for large populations of crystals to be collected in Chapters 2 and 3 have also been applied to development of a serial data collection technique. Of 159 datasets collected from 8 crystals of a copper nitrite reductase from Achromobacter cycloclastes, 45 datasets from a single crystal were analysed to observe the reaction mechanism using high resolution crystal structures. X-ray radiolysis initiated the reaction and high resolution data allowed the conversion of nitrite (NO2) to nitric oxide (NO) to be observed in the crystal. Other aspects of the reaction were investigated from the data series including a conserved water chain connecting the copper sites which may act as a proton wire to donate a proton and produce NO. This technique may have wide applications to the study of the reaction mechanisms of other metallo-proteins.

Tamoxifen is an effective and well-tolerated treatment for early disease and/or pre-menopausal patients with breast cancer (BC); although many women go on to develop resistance. Currently the five-year survival rate following Tamoxifen resistance (TR) is < 20%; hence the mechanisms need to be better understood. Recent research has focussed on specific pathways, however additional mechanisms are involved and we investigated these using cell line models of BC (MCF7) and TR using a variety of proteomic approaches. Differential expression and phosphorylation of proteins between the MCF7 and TR cell lines were detected by antibody arrays; which detected changes in Mitogen Activated Protein Kinases and Receptor Tyrosine Kinases family members, and in apoptosis related proteins. There were 21 novel proteins found to be altered in TR. 262 quantifiable proteins were found using SILAC; 29% over expressed in resistance and 25% down regulated. 5 were subsequently picked for validation by Western blot and 2 of these (IQGAP1 and cortactin) were chosen for further investigation with siRNA and functional assays. IQGAP1 was found to play a role in TR; as decreasing expression of IQGAP1 using SiRNA decreased the proliferation of TR cells and significantly modulated the TR cells ability to invade matrigel.

Circadian rhythms are biochemical, physiological, or behavioural over 24 hours. The avian circadian system is complex, involving numerous oscillators in the brain. I characterised two hypothalamic input mechanism (melatonin receptors and light) and one output mechanism (vasotocin) in the zebra finch. Melatonin receptors were cloned and expression levels investigated in the brain and in peripheral tissues. Receptors were found in all tissues, with some pronounced rhythmic mRNA expression. Tissue-specific differences in temporal distribution, peak expression and amplitude suggests melatonin have varied roles in different tissues and different receptors control/influence these roles. Effect of light in the hypothalamus was investigated by exposing light into the dark phase of an LD cycle and studying the difference in C-FOS expression. C-FOS was found in hypothalamic nuclei associated with photic transduction. C-FOS-IR cells were also found in the two known avian hypothalamic oscillators, the LHN and SCN. Arginine-Vasotocin is a neuropeptide involved in numerous bodily and nervous tissue functions, secreted within the hypothalamus and pituitary gland. Immunofluorescent experiments showed marked differences in expression, as different zeitgeber times and between species. This study has improved our understanding of avian circadian systems, providing new insights into the hypothalamic oscillator of a complex circadian organisation.