An investigation into the microwave assisted remediation of organic containing aqueous solutions has been conducted. Tri-n-butyl phosphate (TBP), the nuclear thel reprocessing solvent, and its diluents, are known to be responsible for the formation of interfacial deposits (cruds) in the alkali solvent wash stage of the Purex process for reprocessing spent nuclear fuel. The presence of cruds in the process is managed by regular wash out and the collected cruds are separated from aqueous washings and stored. Chemical oxidation of the cruds is one way of destroying them. This project explored microwave assisted oxidation, using TBP as a simple simulant for the crud. Low concentration TBP streams were circulated through an activated carbon loaded glass reaction vessel. The vessel was then subject to microwave radiation applied in short pulses. Studies were performed to assess the variation in reaction parameters using two oxidants; air and hydrogen peroxide. The analytical techniques used to assess the extent of decomposition were ion chromatography and UV-V is spectroscopy. Results showed TBP was found to decompose to orthophosphate ions in solution and to di-butyl phosphoric acid (HDBP).

Isolation of DNA from skeletonised human remains can be problematic. In addition to DNA degradation, enhanced by high temperature and humidity, there are often potent polymerase chain reaction (PCR) inhibitors present within the samples. It is therefore important to extract the maximum amount of available DNA whilst removing any amplification inhibitors that may be present. Whilst real-time PCR methods are available for quantification and detection of PCR inhibitors the information received is limited as real-time PCR targets amplicons that are much smaller than those typically targeted in forensic analysis. To gain more information on the quality of extracted DNA a new multiplex PCR assay comprising a 4-plex targeting amplicons of 70 base pairs (bp), 194 bp, 305 bp and 384 bp along with two Internal Amplification Contols (IACs) of 90 bp and 410 bp was developed. This multiplex was optimised so that it worked with template amounts ranging between 0.10 ng and 200 ng; partial profiles were obtained with as little as 0.02 ng. The IACs were effective in detecting PCR inhibitors. The multiplex also assessed as a quantification tool. Plotting peak height compared to input DNA of a standard dilution series produced a coefficient of determination (R2) of 0.8308. The multiplex was found to provided reasonable estimates of DNA concentration, when the sample concentration was between 12.5 – 100 ng; relative standard deviations were all below 10% in this range for 30% of tested samples. However, real-time PCR proved to be more precise and was used in the rest of the study for the purposes of quantification. In forensic cases bones and teeth often provide some of the most challenging samples to extract good quality DNA. Using the optimised multiplex to assess the quality of DNA extracts five extraction methods: ChargeSwitch® gDNA Plant Kit, DNA IQTM System Kit, DNeasy® Blood & Tissue Kit, PrepFiler® BTA Forensic DNA Extraction Kit and phenol-chloroform-isoamyl alcohol extaction methods were assessed for their capability for extracting clean DNA from bone samples. Prior to the main experimentation several evaluation studies were carried out to optimise the methods being used. Based on the results, decalcification was not used for any of the extractions as non-decalcified extracts contained higher amounts of DNA. For the phenol-chloroform-isoamyl alcohol extraction it was determined that whilst ethanol precipitation provided higher amounts of DNA, the extracts using Amicon 30kDa filters (Amicon ultra-0.5 centrifugal filter unit with ultracel-30 membrane) were cleaner. Based on poor results with degraded bone samples a pre-process technique was developed; these extractions started with 250 mg of pulverised bone sample which was then concentrated and cleaned up using Amicon 30kDa filters (Amicon ultra-2 ml centrifugal filters for DNA purification and concentration) before carrying out the standard extraction procedures. After optimisation of the extraction methods the comparison study showed that the phenol-chloroform-isoamyl alcohol extraction method produced the highest DNA yields with both fresh and degraded bone samples, followed by DNeasy® Blood & Tissue Kit, ChargeSwitch® gDNA Plant Kit, PrepFiler® BTA Forensic DNA Extraction Kit and DNA IQTM System Kit. However, all produced DNA that could be amplified and did not contain any inhibition. Another application of the multiplex was to assess the effectiveness of different DNA preservation methods by examining the amount and quality of DNA recovered after preservation. Five methods: cell lysis solution (with 1% sodium azide), dehydration / freeze drying, ethanol (96%), freezing and room temperature storage were used to study the effectiveness of preservation methods on fresh and three-month old decomposed pig bone samples which were preserved for 6 weeks, 6 months and 1 year. The results showed that freezing is the best preservation method for both fresh and degraded bone samples for long-term storage followed by ethanol (96%), dehydration / freeze drying and room temperature storage. However, full profiles were obtained from both fresh and degraded bone samples from all methods, except cell lysis solution (with 1% sodium azide). Cell lysis solution (with 1% sodium azide) preservation method tended to be good for short-term storage but with the long-term preservation, less DNA yield was obtained and also the electropherograms showed higher levels of DNA degradation. Finally, using the optimised DNA extraction methods, the multiplex was tested using forensic samples comprising of 30 bone samples from casework in Malaysia and simulated body fluid evidences subjected to environmental insult in the United Arab Emirates. The application illustrated the effectiveness of the multiplex to identify PCR inhibitors and identify DNA degradation, providing supplementary information to real-time PCR.

Synaptic vesicle (SV) exocytosis is vital to maintaining neuronal transmission at chemical synapses and defects in this processes has been linked to various psychiatric and neuronal disorders. Further, distinct modes of exocytosis have been implicated in post-synaptic plasticity and these latter processes maybe compromised in many neurodegenerative disorders. Therefore, it is vitally important to elucidate the machinery involved in SV exocytosis and decode the regulatory pathways for distinct modes of SV exocytosis. Herein, synaptosomes, pinched off nerve terminals, prepared from cerebral cortex of adult male Wistar rats were used as a model system to investigate these processes. Especially, A. Ashton had previously demonstrated the existence of KR mode of exocytosis in these synaptosomes and determined that the distinct modes can be regulated by adjusting the activity of various kinases and phosphatases. Synaptosomes were maximally labelled with 100 µM FM2-10 dye such that all the releasable vesicles, from readily releasable pool (RRP) and reserve pool (RP), were loaded with the dye. The exocytosis of the dye was then studied by employing various secretagogues (high K+ {HK}, 4-aminopyridine {4AP} or ionomycin {ION}) in the presence of 5 mM [Ca2+]e; these stimuli only induced a single round of release. This dye release was then directly compared to Glu release from terminals treated identically (more than 80% of these synaptosomes are glutamatergic), and differences between dye and Glu release were studied following various drug treatments. The results show that the inhibition of dynamins can increase the FM2-10 dye release during certain stimulation conditions (4AP5C and ION5C; where 5C represents 5 mM [Ca2+]e) without changing the Glu released indicating that dynamin(s) are required for the closure of the fusion pore (and therefore KR) during the employment of these stimuli. However experiments involving blockade of the ATPase activity of non-muscle myosin-II suggest that myosin-II may also be able to regulate the fusion pore, independent of dynamin-I, when a different stimulus (HK5C) is employed. The three stimuli employed here produced distinct kinetics for changes in [Ca2+]i and suggest that dynamin-I may only be able to regulate KR mode of exocytosis when the Δ[Ca2+]i is relatively lower (overall Δ[Ca2+]i < 140 nM) and that myosin-II replaces dynamin-I in this function when these Ca2+ changes are relatively higher (overall Δ[Ca2+]i < 140 nM). In order to investigate the phosphoregulatory pathways of these two phospho-proteins (dynamins and myosin-II) the activity of various enzymes including protein phosphatase (PP) 2A, PP1, calcineurin and PKC protein kinase C (PKC) was modulated externally. The data indicate that the properties of dynamin-I and myosin-II can be regulated by phosphoregulation induced by PKC and this induces their function in the KR mode of exocytosis. When the Δ[Ca2+]i is lower (overall Δ[Ca2+]i < 140 nM), the relevant PKC remains deactivated and dynamin-I can continue to close the fusion pore of exocytosing vesicles thereby causing KR. On the other hand if the overall Δ[Ca2+]i is greater than 140 nM then relevant PKCs are activated which will then phosphorylate dynamin-I and myosin-II rendering the former inactive and the latter active such that myosin-II can now replace dynamin-I in closing the fusion pore. Western blot analysis revealed that dynamin-I is dephophorylated at Ser 795 residue by PP2A, and that this residue can be phosphorylated by Ca2+ activated PKC as increase in phosphorylation of Ser 795 (by blockade of PP2A) or by supramaximal stimulation of PKC by active phorbol esters leads to a switch in the RRP to a FF mode, it would appear that this site may be important for defining the mode of exocytosis. Activated PKC can also phosphorylate myosin-II but the phosphorylation sites on the myosin-II oligomer that play such a role remain be determined. These significant new findings help establish that SVs can switch between modes of exocytosis and that there are specific proteins implicated in this process. Clearly, further work may reveal the importance of these processes for synaptic plasticity and whether certain psychiatric or neuronal diseases could be explained by perturbation of these distinct modes of exocytosis.

The decommissioning of nuclear power plants around the world will produce a major waste stream of irradiated graphite. Graphite has been used extensively as a reactor moderator and reflector material that becomes irradiated and contaminated over time. In the coming years ~250,000 tonnes of irradiated graphite will require management making this a significant waste management issue worldwide. Irradiated graphite is categorised as Intermediate Level Waste mostly due to its content of Carbon-14 (C-14) which is a long-lived radioisotope which could be released into the biosphere. In addition the Low Level Waste (LLW) repository at Drigg has very strict guidelines regarding C-14 authorisation and there is currently no deep geological repository available in the UK. Varying amounts of carbonaceous deposits have been identified on irradiated graphite samples removed from reactor cores. If these deposits are rich in C-14, treatment of the waste graphite by oxidation could reduce the C-14 inventory of the remaining graphite. This is the primary focus of this research. In order to investigate a technique that would decontaminate graphite from the carbonaceous deposits it was necessary to produce a range of carbonaceous deposits on virgin graphite material to act as a simulant for the deposits present on reactor graphite. Two deposition techniques were investigated: microwave plasma assisted chemical vapour deposition and a combination of solution deposition and charring. C-13 precursors were used as they facilitate the study of the selective removal of the deposit by mass spectrometry and spectroscopy. Using C-13 analogues instead of C-14 prevents the need to work in active laboratories and allows higher concentrations of deposit to be used which is beneficial when developing a technique for selective removal. A thermal treatment which utilised the application of a vacuum was investigated to determine whether the carbonaceous deposits could be selectively removed with minimal oxidation to the underlying graphite. As carbon deposits were more amorphous than crystalline graphite it was thought that they would oxidise quicker at lower temperatures than graphite. Virgin graphite and samples with deposits were characterised before and after thermal treatment using Scanning Electron Microscopy, Raman Spectroscopy, Thermal Gravimetric Analysis and Mass Spectrometry. An additional area of investigation was conducted using thermogravimetric studies of the oxidation of irradiated graphite which was carried out at the National Nuclear Laboratory’s Preston Lab. This would determine the distribution of C-14 in the carbonaceous deposits and underlying irradiated graphite which could be a key factor in the determination of possible treatments and eventual storage/disposal routes of the waste graphite.

The term glioma encompasses brain tumours arising from the glial cells. Malignant glioma are characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue, hence diagnosis and treatment is difficult, and patient survival is poor. Aptamers are small molecular ligands composed of short oligonucleotides that bind to a target with high specificity and affinity. They are produced in vitro through a method called systematic evolution of ligands by exponential enrichment (SELEX). The aim of the study was to examine the binding selectivity of DNA aptamers on commercial glial cell lines and primary glioma tissues. RNA aptamers and their DNA homologues (SA44, SA43, SA56) were selected for study which showed strong binding affinity to the target U87MG cells as measured by flow cytometry. SA44 and SA43 showed higher uptake and cytoplasmic localisation in U87MG and 1321N1 glioma cell lines compared to non-cancerous SVGP12 cells and non-glioma MCF-7 and T24 cells as measured by confocal microscopy. The data was confirmed quantitatively by flow cytometry analysis, which showed that the aptamers were able to actively internalise in U87MG and 1321N1 tumorigenic cells compared to the non-cancerous and non-glioma cell types. Histochemistry staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was found to be significantly higher for only SA43 aptamer (p < 0.05) in glioma tissues (grade I, II, III and IV) compared to the non-cancerous and tissues. Aptamer SA43 also showed cell type selectivity within the tissue. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, show promise for histological diagnosis of glioma and targeted delivery. In the future, targeting tumour cells and tissues through the use of SA43 aptamer will help develop molecular imaging, targeted delivery by reduction of the non-specific toxicity of chemotherapy and selectively directing anti-cancer drugs to tumour cells.

This thesis aimed to evaluate the relevancy of different in vitro cell and culture models for osteoblastic-linage cells. Cell lines provide a convenient and accessible alternative to primary human osteoblast cells. However, the direct comparison of these cells demonstrated limited similarity of cell lines to the primary human osteoblasts indicating that their use should be limited to appropriate and specific research questions. To investigate the paracrine regulation of osteogenic development, the immature human osteoblasts and human bone-derived mesenchymal stem cells (MSCs) were co-cultured in monolayer or high density culture. Results from this part of the study suggested the presence of an active signalling pathway between MSCs and osteoblasts. What is more, the effect of cell-cell crosstalk depended on the type of culture system. Co-culture in a 3D micromass system stimulated the osteogenic differentiation of progenitor cells, while in monolayer this was not seen. While the stimulation of MSCs with inflammatory and chemotactic factors successfully regulated the cell gene expression and secretion profile, no effect of the secrotome on the osteogenic differentiation of unstimulated cells in monolayer was demonstrated. Altogether, these results indicated the importance of cell-to-matrix interconnectivity. Therefore, the last part of this thesis focused on the assessment of osteogenic differentiation in 2D and 3D cell culture models, which are physiologically relevant. The progression in osteogenesis depends on the applied 3D culture model. While in both, micromass and type I collagen-hydroxyapatite gel, the differentiation is enhanced compared to monolayer, the regulation of this process is triggered in a different manner in these 3D culture models. Together these findings demonstrate how diverse outcomes can be obtained by the application of different models in in vitro research. Ultimately, the 3D in vitro models provide a better choice for a more in vivo-related osteogenic differentiation and its regulation.

Coherent Antistokes Raman Scattering (CARS) microscopy has emerged in the last decade as a powerful multiphoton microscopy technique to rapidly image lipid droplets (LDs) label-free with intrinsic three-dimensional spatial resolution in cells. In this thesis I investigate and compare the ability of hyperspectral CARS and dual-frequency/differential CARS (D-CARS) to enable the chemical specificity required to distinguish lipids of different chemical composition. In hyperspectral CARS a series of spatially-resolved images are acquired over a frequency range thus proving high chemical specificity. In D-CARS two vibrational frequencies are simultaneously excited and probed, and the resulting sum and difference CARS intensities are detected by a fast and efficient single photomultiplier. This results in a higher image speed than hyperspectral CARS and in an improved image contrast against the nonresonant CARS background with a straightforward data analysis. D-CARS and hyperspectral CARS techniques were applied to LDs in model and cellular systems. In model systems made by agarose gel, droplets of pure lipids with different degree of unsaturation (number of carbon-carbon double bonds in the fatty acyl chain) were used as test sample to compare Raman spectra with CARS spectra, and measure D-CARS images at specific chemically-selective wavenumbers. Building from this knowledge, cytosolic droplets induced by loading fatty acids to the culture media of human adipose-derived stem cells (ADSCs) were distinguished in composition both in fixed cells and in living cells during differentiation into adipocytes. Furthermore, the application of a in-house developed Hyperspectral Image Analysis (HIA) software on hyperspectral data provided spatial distributions and absolute concentrations for the chemical components of the investigated specimens. In particular quantitative information was extracted about the concentration of pure neutral lipid components within cytosolic LDs, and changes over time were inferred in living ADSCs according to the type of pure fatty acid added to the culture media.

Background: Craniofacial morphology has been reported to be highly heritable, but little is known about which genetic variants influence normal facial variation in the general population. Aim: To identify facial variation and explore phenotype-genotype associations in a 15-year-old population (2514 females and 2233 males). Subjects and Methods: The subjects involved in this study were recruited from the Avon Longitudinal Study of Parents and Children (ALSPAC). Three-dimensional (3D) facial images were obtained for each subject using two high-resolution Konica Minolta laser scanners. Twenty-one reproducible facial soft tissue landmarks and one constructed mid-endocanthion point (men) were identified and their coordinates were recorded. The 3D facial images were registered using Procrustes analysis (with and without scaling). Principal Component Analysis (PCA) was then employed to identify independent groups ‘principal components, PCs’ of correlated landmark coordinates that represent key facial features contributing to normal facial variation. A novel surface-based method of facial averaging was employed to visualize facial variation. Facial parameters (distances, angles, and ratios) were also generated using facial landmarks. Sex prediction based on facial parameters was explored using discriminant function analysis. A discovery-phase genome-wide association analysis (GWAS) was carried out for 2,185 ALSPAC subjects and replication was undertaken in a further 1,622 ALSPAC individuals. Results: 14 (unscaled) and 17 (scaled) PCs were identified explaining 82% of the total variance in facial form and shape. 250 facial parameters were derived (90 distances, 118 angles, 42 ratios). 24 facial parameters were found to provide sex prediction efficiency of over 70%, 23 of these parameters are distances that describe variation in face height, nose width, and prominence of various facial structures. 54 distances associated with previous reported high heritability and the 14 (unscaled) PCs were included in the discovery-phase GWAS. Four genetic associations with the distances were identified in the discovery analysis, and one of these, the association between the common ‘intronic’ SNP (rs7559271) in PAX3 gene on chromosome (2) and the nasion to mid-endocanthion 3D distance (n-men) was replicated strongly (p = 4 x 10-7). PAX3 gene encodes a transcription factor that plays crucial role in fetal development including craniofacial bones. PAX3 contains two DNA-binding domains, a paired-box domain and a homeodomain. The protein made from PAX3 gene directs the activity of other genes that signal neural crest cells to form specialized tissues such as craniofacial bones. PAX3 different mutations may lead to non-functional PAX3 polypeptides and destroy the ability of the PAX3 proteins to bind to DNA and regulate the activity of other genes to form bones and other specific tissues. Conclusions: The variation in facial form and shape can be accurately quantified and visualized as a multidimensional statistical continuum with respect to the principal components. The derived PCs may be useful to identify and classify faces according to a scale of normality. A strong genetic association was identified between the common SNP (rs7559271) in PAX3 gene on chromosome (2) and the nasion to mid-endocanthion 3D distance (n-men). Variation in this distance leads to nasal bridge prominence.

In recent years, there has been an increased emphasis to treat patients with parenteral nutrition (PN) at home in an attempt to reduce costs and improve clinical outcomes. This increased interest in home parenteral nutrition (HPN) has stimulated researchers to investigate potential sources of instability. One of the more unstable groups in PN is micronutrients, which can be divided into two groups: vitamins and trace elements. This thesis investigates the effects of artificial light sources (cool white, warm white and UVA) on the physicoP chemical stability of vitamins. Vitamins were chemically analysed using a novel stability indicating HPLC assay that could quantify five waterPsoluble and three fatPsoluble vitamins simultaneously in one run. Samples were physically analysed by visual analysis, microscope analysis, laser diffraction, pH and osmolality. Initial experiments investigated the physicoPchemical stability of vitamins exposed to artificial light sources over a period of 24 hours. In cool and warm white light there was approximately a 20% loss of riboflavin and 10% loss of retinol. In UVA light there was approximately a 20% loss of retinol. All other analysed vitamins were stable over the time period to these artificial light sources. Further experiments investigated these conditions following 6 days of storage between 2P8 C. These experiments revealed similar results in the three types of artificial light source. v The protective effects of lipid emulsions on retinol were then investigated in containers and administration sets. Samples containing lipid emulsions in syringes and administration sets had a statistically significant increase in retinol stability. Nevertheless, degradation in excess of 10% still occurred in these groups. The protective mechanism of lipid emulsions was primarily though to be a result of light obscuration. However, soybean oil (SBO), a clear liquid, provided unexpected obscuration of UVA light suggesting it may reflect or absorb damaging rays thereby improving retinol stability.

Breast cancer is the most common cancer in the UK, but despite recent encouraging increases in survival rates, is still the second most common cause of cancer death in women in the UK. To try to reduce systemic toxicity during treatment of cancer patients, a plethora of targeted therapies are in various stages of development. PARP inhibitors have been shown to be particularly effective in BRCA-deficient cells, making them a contender as a personalised therapy. One of the challenges for targeted therapies is that of resistance, which limits the extent of benefit to the patient. The work described in this study continues previous work within our laboratory, investigating the PARP inhibitor olaparib in a conditional mouse model of BRCA2-mutated human breast cancer. The data presented here establish a correlation between histological tumour type and response to olaparib therapy, with poor responders classified exclusively as mesenchymal-like metaplastic spindle cell carcinomas (MSCC). This suggests that further patient stratification is required when deciding on whether this therapy may be suitable, and may explain why not all patients with BRCAmutated breast cancer have benefitted from olaparib therapy in current clinical trials. Investigation of olaparib resistance in this study indicated that several currently proposed mechanisms of resistance were not pertinent to the Brca2/p53 model, hence novel mechanisms were sought. Histopathological analysis of resistant tumours showed that the majority were MSCCs, representing a significant change in the proportion compared to an untreated cohort. Other resistant tumour types had epithelial morphology, but showed an increase in expression in some mesenchymal-like genes compared to untreated cohorts, suggesting that mesenchymal features may be important in causing resistance to olaparib. A similar tumour model, incorporating the additional deletion of E-cadherin, was used to investigate whether lack of this protein in tumours affected response and resistance to olaparib therapy. Loss of Cdh1 led to an increase in invasive ductal carcinomas of no special type (IDC-NST) and the absence of MSCCs, suggesting that genetic loss of expression does not drive the formation of mesenchymal-like tumours. Correlating with this, loss of E-cadherin did not drive epithelial-to-mesenchymal transition in these tumours and had no effect on response to olaparib therapy or resistance to the inhibitor. Taken together, the data presented in this thesis suggest that MSCCs have an intrinsic resistance to olaparib therapy, and tumours which initially respond to olaparib therapy harness or acquire certain mesenchymal characteristics in order to develop resistance during treatment.