Upregulation of the extracellular signal-regulated kinase (ERK) pathway has been shown to contribute to tumour invasion and progression. Since the two predominant ERK isoforms (ERK1 and ERK2) are highly homologous and have indistinguishable kinase activities in vitro, both enzymes were believed to be redundant and interchangeable. To challenge this view, here we show that ERK2 silencing inhibits invasive migration of MDA MB 231 cells, and re-expression of ERK2 (but not ERK1) restores the normal invasive phenotype. A detailed quantitative analysis of cell movement on 3D matrices indicates that ERK2 knockdown impairs cellular motility by decreasing the migration velocity as well as increasing the time that cells remain stationary. We used gene expression arrays to identify rab17 and liprin β2 as genes whose expression was increased by knockdown of ERK2 and restored to normal levels following re expression of ERK2 (but not ERK1). Moreover, we established that both Rab17 and Liprin β2 play inhibitory roles in the invasive behaviour of three independent cancer cell lines, indicating a suppressive role for these proteins in tumour progression. Importantly, knockdown of either Rab17 or Liprin β2 restores invasiveness of ERK2 depleted cells, indicating that ERK2 drives invasion of MDA MB 231 cells by suppressing expression of these genes. Taken together, our data provides evidence that true functional disparities between ERK1 and ERK2 exist with regards to cell migration and identifies Rab17 and Liprin β2 as two novel motility suppressors downstream of ERK2.

The hypothesis of this work was whether IGRT could be safely implemented for clinical use in a busy oncology centre. I aimed to study a number of questions that remain unresolved in the current literature regarding safe and optimised implementation of IGRT techniques. The first study undertaken was the calculation of a local set up margin using two widely recognised margin recipes. This involved the assessment and analysis of multiple images belonging to 100 patients. This allowed progression onto the next project which was assessment of the optimal safe method of delineation of 4DCT. The most efficient method was compared to gold standard. At this point a different aspect of the radiation process was assessed, namely verification. A feasibility study of a simple, efficient form of imaging for use in review of a particular error was performed. This also involved the use of a novel tool which required independent assessment. This progressed into a further study of a larger number of patients using this tool and the images assessed previously to verify a novel form of radiation delivery. Lastly a planning study was performed to quantify the clinical benefit of another delivery system. This involved the delineation and planning of a large number of radical lung patients with standard radiation treatment and the novel radiation treatment and an assessment of the potential clinical benefits. The work presented in this thesis has answered some specific questions in IGRT in lung cancer, and contributed both locally and in the wider lung cancer community to increasing the use of IGRT in lung cancer.

Quantitative pathology as a tool in gynaecological pathology is fairly new. Such techniques allow greater objectivity than histological grading, typing, and residual tumour estimation. This study aims to determine: whether basic morphometry data can predict outcome and chemotherapeutic response, whether newer semi-automated methods of tumour morphometry provide similar results to older methods, and whether advanced image analysis methods can offer further tumour outcome data in ovarian carcinoma. The study was performed on a well-selected group of serous ovarian carcinomas. Tumour outcome, survival and chemotherapeutic response, were investigated in 132 patients treated with the same platinum containing regimes. Traditional clinicopathologic parameters, p53 & Bcl2, mitotic activity index MAn and angiogenesis determinants were initially investigated. Semi-automated analysis, using immunohistochemically based techniques, were applied to estimate volume percentage epithelium (VPE) and nuclear morphometric parameters. Syntactic structure analysis including, minimum spanning tree, and neighbourhood features, was also investigated. Multivariate analysis revealed residual disease status, FIGO stage, MAl, VPE, equivalent nuclear diameter, and angiogenesis parameters to be strong prognosticators for overall and disease free survival. Residual disease status, VPE, nuclear length and angiogenesis parameters were found significant predictors of chemotherapy response. Angiogenesis parameters, as determined by semi-automated image analysis techniques, were found overall to be the strongest prognosticators. Morphometric data can predict outcome and chemotherapeutic response in ovarian serous carcinoma. Semi-automated morphometry techniques provide similar results to older methods, and advanced image analysis can offer further outcome data. The rationale for the application of semi-automated and automated detection is that it may provide an unbiased sampling of a lesion and possibly a more representative estimate of areas that a human expert might label. Such determined, quantitative pathological findings were found to have important value in predicting prognosis in ovarian carcinoma and, if not to supersede, certainly to add to classical prognostic factors.

Meningioma subtypes classification is a real world problem from the domain of histological image analysis that requires new methods for its resolution. Computerised histopathology presents a whole new set of problems and introduces new challenges in image classification. High intra-class variation and low inter-class differences in textures is often an issue in histological image analysis problems such as Meningioma subtypes classification. In this thesis, we present an adaptive wavelets based technique that adapts to the variation in the texture of meningioma samples and provides high classification accuracy results. The technique provides a mechanism for attaining an image representation consisting of various spatial frequency resolutions that represent the image and are referred to as subbands. Each subband provides different information pertaining to the texture in the image sample. Our novel method, the Adaptive Discriminant Wavelet Packet Transform (ADWPT), provides a means for selecting the most useful subbands and hence, achieves feature selection. It also provides a mechanism for ranking features based upon the discrimination power of a subband. The more discriminant a subband, the better it is for classification. The results show that high classification accuracies are obtained by selecting subbands with high discrimination power. Moreover, subbands that are more stable i.e. have a higher probability of being selected provide better classification accuracies. Stability and discrimination power have been shown to have a direct relationship with classification accuracy. Hence, ADWPT acquires a subset of subbands that provide a highly discriminant and robust set of features for Meningioma subtype classification. Classification accuracies obtained are greater than 90% for most Meningioma subtypes. Consequently, ADWPT is a robust and adaptive technique which enables it to overcome the issue of high intra-class variation by statistically selecting the most useful subbands for meningioma subtype classification. It overcomes the issue of low inter-class variation by adapting to texture samples and extracting the subbands that are best for differentiating between the various meningioma subtype textures.

The objective of this thesis is to present the development of Raman microscopy for biochemical imaging of living cells. The main aim was to construct a Raman micro-spectrometer with the ability to perform time-course spectral measurements for the non-invasive study of biochemical processes in individual cells. The work can be divided into two parts: first, the development and characterization of the instrument; and second, completion of two experiments that demonstrate the suitability of Raman technique for studies of live cells. Instrumental development includes the design of optics and software for automated measurement. The experiments involve data collection and development of mathematical methods for analysis of the data. Chapter One provides an overview of techniques used in cell biology, with a special focus on Raman spectroscopy. It also highlights the importance of experiments on living cells, especially at the single cell level. Chapter Two explains the theoretical background of Raman spectroscopy. Furthermore, it presents the Raman spectroscopy techniques suitable for cell and biological studies. Chapter Three details the instrumentation and software development. The main parts of the confocal Raman micro-spectrometer, as designed for studying living cells, are: inverted microscope, 785 nm laser and high quality optics, environmental enclosure for maintaining physiological conditions during measurements of cells, and fluorescence wide-field microscopy facility for validation and confirmation of biochemical findings by Raman studies. Chapter Four focuses on the evaluation of the performance of the Raman setup and explains calibration and analysis methods applied to the data. Chapter Five and Six describe experiments performed on living cells. Chapter Five focuses on studies of the immunological synapse formed between primary dendritic and T cells indicating the polarisation of actin. Chapter Six describes time-course experiment performed on cancerous cells in the early phases of the apoptosis process, which enabled detection of the DNA condensation and accumulation of unsaturated lipids. Chapter Seven summarizes the work and gives concluding remarks.

Unsustainable use of wildlife is a global conservation challenge. Understanding ecosystem specific patterns of wildlife exploitation is key to addressing this challenge. This thesis explores the nature of wildlife exploitation in and around Ugalla Game Reserve in western Tanzania. The reserve is divided into Ugalla east and Ugalla west tourist hunting blocks. First, I assessed the status of wildlife in the hunting blocks. Overall, estimates of wildlife population parameters suggested that Ugalla west was somewhat more exploited than Ugalla east. Second, I looked at the degree to which the hunting blocks experienced illegal wildlife hunting (poaching) and factors behind this. The spatial distribution of poaching signs and household interviews revealed that poaching was widespread, more so in Ugalla west than Ugalla east. Proximity to the reserve encouraged poaching, although bushmeat consumption increased with distance from the reserve. A wide range of bushmeat species was favoured, but the common species were impala Aepyceros melampus, dik-dik Madoqua kirkii and common duiker Sylvicapra grimmia. Availability of alternative sources of animal protein, agricultural production and income had significant influences on poaching. Different forms of poaching were specialist activities largely independent of each other. To address poaching, the main focus of attention has been on creating wildlife management areas (WMAs) along with allowing legal subsistence hunting by the communities around the reserve. Third, I assessed the impact of legal subsistence hunting on the wildlife species, and showed that it is not well managed and wildlife populations are contracting. This leaves WMAs as a potentially viable option for the conservation of Ugalla. Therefore, lastly, I identified and recounted some options for promoting the sustainability of WMAs. This thesis presents the first detailed assessment of wildlife exploitation in Ugalla, thus contributing to the existing body of knowledge on tackling the bushmeat crisis in Africa.

The work in this thesis has investigated the potential for a unique HMW glucose polymer (Vitargo, Swecarb AB, Sweden; MW of 500-700 g.mol-1) derived from barley starch to augment short-term post exercise muscle glycogen resynthesis above that of an isoenergetic LMW glucose polymer (Maxijul, SHS International, UK; MW of 900 g.mol-1). The HMW glucose polymer has been previously investigated in comparison to a LMW glucose solution with studies reporting a 70% greater muscle glycogen content after 2 hr recovery from glycogen-depleting exercise (Piehl-Aulin et al., 2000) and an enhanced gastric emptying at rest (Leiper et al., 2000). More recently an improved work output (10%) in a maximal exercise test performed 2 hr after exhaustive exercise was demonstrated after ingestion of the HMW glucose polymer compared to an isoenergetic LMW glucose polymer (Stephens et al., 2008). Key observations in this study were a greater rate of rise in blood glucose and serum insulin concentration during recovery with ingestion of the HMW compared to the LMW glucose polymer. Thus it was suggested that the improvement in performance in the secondary exercise bout could potentially be attributed to greater muscle glycogen availability present at the onset of the test. This hypothesis was subsequently tested initially in this thesis with the quantification of muscle glycogen content after cycling to exhaustion and ingestion of the same HMW and LMW glucose polymers. However, despite undertaking an identical exercise protocol, in contrast with the study by Stephens et al (2008), no differences in the rate of rise in blood glucose or serum insulin were observed. Accordingly muscle glycogen resynthesis measured 2 hrs after exhaustive exercise was similar following ingestion of the HMW and LMW glucose polymers (118 vs. 123 mmol.kg-1). Thus exercise performance in a secondary bout was near identical between both polymers (173 vs. 175 kJ). It was concluded that the LMW and HMW glucose polymers elicited similar post exercise muscle glycogen resynthesis however, since the sampling interval in this study using muscle biopsies was large (2 hr), it may have negated to highlight any early differences in muscle glycogen content. Therefore further investigation was undertaken that focused on more subtle sequential fluctuations in muscle glycogen by using ultra-high field 13C MRS following feeding of the same HMW and LMW glucose polymers. Marginal increases in muscle glycogen during 1 hr of recovery from prolonged exercise were reported after ingestion of the HMW and LMW glucose polymers (6 and 4% respectively). Additionally, increases in muscle glycogen after ingestion of both glucose polymers above that of a zero-energy control were not seen after 1 hr of recovery when a greater magnitude of resynthesis would be expected with the former. It was thus postulated that irrespective of the improved sensitivity of ultra-high field 13C MRS, the technique may not be suited to post exercise muscle glycogen resynthesis determination due to the methodological issue of subject positioning inhibiting typical gastric emptying patterns. When considering the implications of these studies it appears that the HMW glucose polymer does not augment post exercise muscle glycogen resynthesis above that of an isoenergetic glucose polymer with a much lower molecular weight. Nonetheless given that the blood glucose and serum insulin profiles over a 2 hr recovery in the first study of this thesis and the study by Stephens et al (2008) were notably different with the same test solutions, it was considered that there may be a disparity with the HMW glucose polymers utilised. Importantly the production of the HMW glucose has altered such that the manufacturing process has deviated from granulation to agglomeration with the native starch evolving from potato to corn and more recently barley. It was suggested that the most recent HMW glucose polymer used presently had deviated away from its initial characteristics leading to the blood glucose and serum insulin responses observed in the first study of this thesis. Indeed by then comparing post exercise ingestion of a previous granulated version of the HMW glucose polymer with a more soluble agglomerated version in the same experimental protocol as the first study, an initial greater rise in serum insulin was observed in the first 55 min of post exercise recovery. Thus alterations in manufacturing from granulation to agglomeration do appear to have affected properties related to postprandial insulin secretion. However this effect on insulin was not seen overall over the 2 hr recovery period and no differences in blood glucose or exercise performance in a secondary bout were observed suggesting other factors such as the native starch may be influential. It can thus be concluded that the difference in postprandial glucose and insulin responses seen between previous work and the present investigation may be due to altered physical characteristics of the HMW glucose polymer. No differences in intrinsic viscosity, rheology or molecular weight were noted between the agglomerated and granulated versions of the HMW glucose polymer thus the alterations in the origin material may account for more influence on digestibility in vivo. Further investigation would be warranted into effects on post exercise muscle glycogen resynthesis and exercise performance provided that the HMW glucose polymer could be returned to its original formulation.

Rydberg atoms are often proposed as the basis of quantum computing and quantum information protocols. One of the central reasons for this is that they provide a strong and long-ranged interaction that can be coherently switched on and off. This thesis details two techniques which use the exaggerated properties of Rydberg atoms to manipulate both the quantum state of the atom itself and that of the external light field. The first proposal initially focuses on the creation of many-body quantum states from two-level atoms trapped in a two-dimensional lattice. This approach uses the van der Waals interaction present between alkali metal atoms in highly excited Rydberg s-states. The approximate solution of the corresponding Hamiltonian is detailed in the regime where the laser driving is the largest energy scale of the system. The states which are most likely achieved using an oscillating laser detuning are then determined. These states are then taken as the basis for the creation of deterministic single-photons, whose properties are shown to rely on the interplay between interatomic spacing and the geometry of the lattice. The second technique described uses the coupling between a Rydberg atom and a moving electron to manipulate the atomic state. In this system, the atom is initially excited to a Rydberg s-state and trapped at a finite distance from an electron waveguide. Two analytical methods are used to show that the final state of the atom depends strongly on the direction and modulus of the electron momentum. A complementary numerical simulation shows that the atoms may be left in a polarised state, suggesting the possibility of using this setup to ‘switch on’ permanent electric dipoles in the atoms. This investigation leads naturally to a system where multiple interacting atoms are trapped close to the waveguide, allowing various many-body states to be accessed.

Peroxisome Proliferators (PPs) are a class of chemicals that cause a programme of augmentative liver growth, however, the mechanism which regulates the induction of hepatic DNA synthesis as a result of exposure to peroxisome proliferators is currently uncharacterized. This study sets out to characterise the induction of DNA synthesis in mouse by peroxisome proliferators, as a prerequisite for investigating and identifying the genes that are responsible for induction of DNA synthesis to control liver growth. Administration of BrdU in drinking water can reduce mouse body weight; an optimized protocol was devised, which does not lead to body weight loss, and which enables reliable measurement of DNA synthesis. Male 129S4/SvJae mice were treated with a single dose of ciprofibrate (100-400 mg kg-1) or methylclofenapate (25 mg kg-1) for two days. Although liver to body weight ratios increased significantly at all doses, no induction in DNA synthesis was observed within 2 days. Subsequent time course studies with ciprofibrate (100 mg kg-1day-1) or methylclofenapate (25 mg kg-1day-1) showed that liver-to-body weight ratio was significantly increased in treated groups by day 2, but that the induction of DNA synthesis was increased significantly only after three days of treatment, for both compounds. No induction of hepatic DNA synthesis was observed in PPARa null mice after treatment with ciprofibrate (100mg kg-1day-1) for 2 or 6 days, showing that the effect required the PPARa. A dose-response study with 0,1,3,10,30,100 or 200 mg kg-1 day-1 ciprofibrate for 3 days, or with 0,10,30,100 mg kg-1 day-1 ciprofibrate for 4 days revealed that liver to body weight ratios were significantly increased in 129S4/SvJae mice treated with 10mg kg-1day-1 and greater ciprofibrate at 3 and 4 days, whereas hepatic labelling index was significantly increased at 100 mg kg-1 day-1 ciprofibrate at 3 days after dosing, with progressive increases at doses of 30 and 100 mg kg-1 day-1 ciprofibrate at 4 days after dosing. In order to explain the early time course of induction of DNA synthesis reported by Styles [113] [164] in Alderley Park mice, a time course study was performed between 1-4 days in Alderley park mice using methylclofenapate (25mg kg-1day-1). The study showed that liver growth was induced by day 2, but DNA synthesis was significantly induced only after 3 days of dosing. To evaluate species differences, the time-course of induction of DNA synthesis was examined in F-344 rats treated with ciprofibrate (50mg kg-1day-1) for 1-4 days. The liver-to-body weight ratio was significantly increased in all time points, but DNA synthesis was significantly increased after 2 days of dosing. These findings demonstrate that there was a delay in induction of DNA synthesis by peroxisome proliferators in mouse by at least 48 hours. This delay in response is not due to strain differences. Moreover, induction of DNA synthesis in rat was earlier than those in mouse, which makes rats a feasible experimental model to study the immediate early genes/ proteins induced by peroxisome proliferators to induce liver growth.

Optical interferometry offers a powerful tool for the study of the mechanical motion of micro- and nano-electromechanical systems (MEMS and NEMS). By examining the modulation of reflected light the displacement can be measured with sub-nanometre precision. Recent work with fibre interferometers carried out by other groups has studied the motion of nanomechanical systems down to temperatures as low as 1 K. Dissipation measurements in the last few years of a number of devices fabricated from high-stress amorphous silicon nitride have shown a marked increase in quality factors when compared to similar low-stress devices. The high quality factors and small masses of these devices have attracted a great deal of interest within the nanomechanical and optomechanical communities. Measurements of dissipation in nanomechanical resonators carried out in Nottingham to date have used the magnetomotive effect to detect nanomechanical motion. This has required that a layer of metal be applied to the high-stress silicon nitride, modifying the mechanical properties. In this thesis we present an overview of the design and construction of an optical detection system designed to study MEMS and NEMS devices from room temperature to liquid helium temperatures. Optical detection is able to measure the displacement of purely dielectric structures and as such is an ideal method with which to measure dissipation in these high-Q silicon nitride resonators, complementing the other nanomechanical measurement techniques available within Nottingham. Using this system, measurements have been made on a number of micro- and nano-electromechanical systems fabricated using processes developed during this work. Confocal images of these devices obtained using the fibre interferometer show a spatial resolution of 0.75 um, a value close to the diffraction limit of the system. Micromechanical quartz tuning forks have been measured to confirm the frequency response of the interferometer, with a value for the piezo-electro-mechanical coupling constant of 2.18 +/- 0.06 uC/m obtained that is in very good agreement with the values published in the literature. Nanomechanical measurements of 200 um square high-stress silicon nitride membranes have revealed thermoelastic damping to be the limiting dissipation mechanism for these resonators at room temperature. Using elastic theory it is possible to quantify the fQ floor predicted by thermoelastic damping seeing good agreement with experimental data. At lower temperatures inter-membrane coupling was observed, with acoustic vibrations from neighbouring membranes coupling into and being amplified by the membrane under observation. Discrepancies in quality factor between the observed and unobserved membranes are most likely due to optomechanical damping of the observed membrane by the laser. This inter-membrane coupling offers a powerful technique for the indirect observation of the flexural modes of nearby membranes without optically damping the response.