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Investigations of machining optical surfaces into brittle materials using an ultra precision machine tool are presented. The newly developed ultra precision NION machine is evaluated to gain a good appreciation of its operating performance. The machining accuracy capability of this machine is established by careful measurement of its motion accuracy, thermal and dimensional stability and loop stiffness. Corroboration of these measurements are provided by assessment of surfaces which were produced in soft 'easily machined' metal materials. It was found that surfaces smooth to -1 nm Ra could be produced on the NION machine and with a form error of less than 100 nm P-V. The main source of figure error, approximately 80 nm, was found to be caused by the synchronous axial error motion of the workhead spindle. Other elements of the machine, including thermal effects, incurred less than 25 nm of additional figure error. Assessment of the diamond turning process for the producing optical surfaces made in a number of important optical materials, which are ostensibly brittle, were undertaken. Turning tests were carried out to establish the relative difficulty for machining optical surfaces in these materials and to define the most important parameters which affect the attained surface quality. Assessment of the produced surfaces was based on their roughness quality, surface morphology and residual stress condition. It was found that diamond tool edge quality degraded with total cut distance. Tool cut distance was found to be a major influence on achievable material removal rate before micro-fractures became present at the surface. Surface quality and residual stress condition were also greatly influenced by the overall tool cut distance. Diamond grinding trials were also carried out using the NION machine tool. These grinding trials were carried out using a mode of grinding which permits complex shape optical surfaces to be produced. Various grinding technologies were employed to establish the optimum methods. Selected grinding trials were carried out to establish the dominate parameters affecting the optical quality. Assessment of the machined surfaces was in regard of their surface roughness, residual stress and severity of sub-surface micro cracking. It was found that grinding wheel specification was a major influence on surface quality and sub-surface damage. The level of residual stress associated with 'ductile' mode grinding was not found to prohibit its application toward the direct manufacture of optical elements. Selection of grinding parameters which ensured the grain depth of cut, GDOC, parameter did not exceed the materials critical depth, dc, allowed glass surfaces to be ground to 1-2 nm Ra. These ground glass surfaces appeared free of any surface fractures. Sub-surface assessments did however reveal small levels of micro-fractures hidden below the surface. Discussion of both machining processes is provided. Available material removal rates for each process is given when cutting a number of important optical materials. Conclusions regarding the production of both Infrared and visible wavelength optics using the NION machine tool are provided. Recommendations for future work to improve both the understanding of the processes and the effectiveness of applying the processes are suggested.

Mild forms of stress have been found to confer beneficial effects on lifespan of various model organisms. In Caenorhabditis elegans, mitochondrial dysfunction, disruption in the insulin/insulin-like signalling, and reduced protein translation have been documented to increase lifespan. This was proposed to be mediated through the activation of beneficial stress, immune, and detoxification responses. Recently, a modification associated with repression (H3K9 methylation) was found necessary for the mitochondrial stress response and longevity but it is unknown whether chromatin modifications associated with histone 3 lysine 4 (H3K4) methylations play a similar role. Therefore, the overall objective of this study was to investigate the role of H3K4 methylations, mediated by the COMPASS complex (Complex associated with SET1), in regulating stress responses and ageing. To study the effects of H3K4 methylations on long-lived mutants, worms lacking the COMPASS components RBBP-5 and WDR-5 were crossed with different longevity pathway mutants, including mitochondrial mutants (clk-1, isp-1), a protein translation mutant (ife-2), a mTOR pathway mutant (rsks-1) and an insulin-like pathway mutant (daf-2). Herein, we showed that WDR-5 and RBBP-5 contributed to post-embryonic growth and development. The loss of WDR-5 or RBBP-5 further delayed the already slow developing long-lived mutants in an additive manner. It was also demonstrated that WDR-5 or RBBP-5 are required for the mitochondrial unfolded protein response (UPRmt) but not heat or ER stress responses. We showed that RBBP-5 function was important for both natural and induced longevity, whilst WDR-5 was pathway specific. Overall, this research suggests that the mechanism underlying the role of RBBP-5 in longevity involves the coordination and expression of genes associated with lifespan, in particular genes associated with mitochondrial function.

Filamin A (FLNA), an actin-binding protein, interacts with a wide range of proteins with great functional diversity. Previous studies have demonstrated that FLNA can regulate transcription factor activity. The mammalian Runx family of transcription factors, Runx1, Runx2 and Runx3 have essential roles in development and disease. In order to regulate transcription the Runx proteins form obligate dimers with a co-regulator, CBFβ. Previous studies have shown that CBFβ interacts with FLNA. In addition, FLNA has been shown to interact directly with Runx2 to supress Runx2-mediated transcription. However, to date the precise nature of the interaction between Runx2 and FLNA has not been determined. The aim of this study was therefore to determine the minimal domains on Runx2 and FLNA that mediate the interaction and subsequently determine the structural basis of the interaction using Nuclear Magnetic Resonance (NMR) spectroscopy. Understanding the structural basis of this interaction may be important in the design of therapeutic reagents to modify Runx function in human disease. A series of Runx2 and FLNA polypeptides were generated and the minimal regions required to interact with each other was determined using GST pulldown assays. It was established that the interaction between Runx2 and FLNA is mediated primarily by Ig domain 23 of FLNA and a short region on Runx2 known as the Nuclear Matrix Targeting Sequence (NMTS). NMR was subsequently used to investigate how FLNA interacts with Runx2, and confirmed that domain 23 of FLNA is responsible for the interaction with the NMTS domain of Runx2. A partial region of the NMTS was resolved using backbone NMR assignment. A two-mode interaction was discovered through chemical shift differences between domain 23 and NMTS. These findings reveal a novel interaction between domain 23 in FLNA and NMTS domain in Runx2. Experiments were also performed on mutant forms of CBFβ found in patients with breast cancer. The impact of these mutations on CBFβ structure and function has not been previously characterised. Nine CBFβ missense mutations were expressed and purified and their ability to interact with the Runt domain of Runx1 was analysed by GST pulldown and electrophoretic mobility shift assays. The mutations affected the formation of the CBFβ/Runt complex either by disrupting the interface or by affecting the stability of the protein.

CBFß, the essential co-regulator of RUNX transcription factors, has a pro-metastatic role in triple negative breast cancer cells. In this context, RUNX1-CBFß and RUNX2-CBFß activate the expression of the EMT master regulator SNAI2, thereby maintaining the mesenchymal state of the cells. However, CBFß is reported to be one of the most frequently mutated genes in ER positive (ER+) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumour suppressor role for CBFß. However, the impact of missense mutations and the loss of CBFß in ER+ breast cancer cells has not previously been studied. Work described in this thesis demonstrates that missense mutations in CBFß accumulate near the Runt-domain binding region. Missense mutations inhibited the ability of CBFß to form CBFß-RUNX-DNA complexes suggesting, that these mutations are indeed loss of function. The loss of CBFß in ER+ breast cancer cells was then modelled by using CRISPR-Cas9-mediated gene-editing to delete CBFß in ER+ MCF7 cells. This led to an increase in cell migration that was dependent on the presence of ERa. Analysis of the potential mechanism revealed that the increase in migration is driven by the co-regulation of Trefoil factor 1 (TFF1) by CBFß and ERa. Subsequent analysis demonstrated that RUNX1-CBFß acts to repress ERa activated expression of TFF1. TFF1 is a motogen that stimulates migration and it was shown that knockdown of TFF1 in CBFß-/- cells inhibits the migratory phenotype. The findings in this thesis therefore reveal a new mechanism by which RUNX1-CBFß and ERa combine to regulate gene expression and a new role for RUNX1-CBFß in the prevention of cell migration by supressing the expression of the motogen TFF1.

In the UK, 12,000 patients die from metastatic breast cancer annually. There is therefore an urgent need to identify the molecules that cause metastasis. Recent work has revealed a role for the RUNX family of transcription factors in the development of metastatic breast cancer. The RUNX proteins form active transcription factor complexes when bound by the heterodimeric partner CBFβ to regulate the expression of metastatic genes. Previous work from our laboratory has demonstrated that knockdown of CBFβ resulted in a decreased invasion capacity of the metastatic breast cancer cell line MDA-MB-231. Three-dimensional culture of MDA-MB-231 cells revealed that loss of CBFβ induces a mesenchymal to epithelial transition (MET). The aim of this project was to determine the role of the RUNX/CBFβ complex in maintaining the mesenchymal phenotype of metastatic breast cancer cells. The data presented show that the phenotype changes were accompanied by changes in EMT marker-gene expression, including Snai2, MMP9, and MMP13. Induction of CBFβ in the CBFβ-knockdown cells remarkably restored both the invasive capacity and the mesenchymal phenotype. Further analysis revealed that maintenance of the mesenchymal phenotype was dependent upon both CBFβ-partner proteins, RUNX1 and RUNX2. Taken together the data presented in this thesis demonstrate that RUNX/CBFβ complexes drive the epithelial to mesenchymal transition (EMT) in breast cancer cells. These findings are likely to be important in the development of potential therapies to inhibit the metastatic spread of breast cancer.

Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.

Freeform surfaces find wide application, particularly in optics, from unique single-surface science programmes to mobile phone lenses manufactured in billions. This thesis presents research into the mathematical and algorithmic basis for the generation and measurement of smooth freeform surfaces. Two globally significant cases are reported: 1) research in this thesis created prototype segments for the world’s largest telescope; 2) research in this thesis made surfaces underpinning the redefinition of one of the seven SI base units – the kelvin - and also what will be the newly (and permanently) defined value for the Boltzmann constant. Theresearchdemonstratestwounderlyingphilosophiesofprecisionengineering, the critical roles of determinism and of precision measurement in precise manufacturing. The thesis presents methods, and reports their implementation, for the manufacture of freeform surfaces through a comprehensive strategy for tool path generation using minimum axis-count ultra-precision machine tools. In the context of freeform surface machining, the advantages of deterministic motion performance of three-axis machines are brought to bear through a novel treatment of the mathematics of variable contact point geometry. This is applied to ultra-precision diamond turning and ultra-precision large optics grinding with the Cranfield Box machine. New techniques in freeform surface representation, tool path generation, freeform tool shape representation and error compensation are presented. A comprehensive technique for very high spatial resolution CMM areal scanning of freeform surfaces is presented, with a new treatment of contact error removal, achieving interferometer-equivalent surface representation, with 1,000,000+ points and sub-200 nm rms noise without the use of any low-pass filtering.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family and participates in sensing and integration of a variety of environmental cues. As a central regulator of cell metabolism, mTOR has gained attention due to its role in many human diseases, including cancer and diabetes. Thus, great efforts are being made to understand signalling via the mTOR pathway as it represents an interesting pharmacological target. In higher eukaryotes, mTOR exists in two evolutionary conserved multi-protein complexes, TORC1 and TORC2. These complexes contain core components that define each complex and regulate their activities. mTORC1 signalling has been extensively studied but mTORC2 is still poorly understood. The discovery that mTORC2 is a key regulator of the activities of the AGC family kinases AKT, PKC and SGK1, and is involved in cellular processes such as proliferation and cell survival in response to growth factors, has stimulated the study of its activation and regulation. A core component of mTORC2 is stress-activated protein kinase interacting protein (SIN1), which is essential for the assembly and kinase activity of the complex and has been proposed to act as a scaffold to recruit mTOR substrates to facilitate their phosphorylation. A yeast two-hybrid screen uncovered novel SIN1 binding proteins that may be potential regulators of mTORC2 activity. One of these is subunit D of the Eukaryotic translation initiation factor 3 (eIF3d), a conserved protein involved in protein metabolism and cytoskeletal organization. This interaction has been confirmed by pull-down assays with overexpressed proteins in HEK-293 cells and the C-terminal region of eIF3d has been identified as critical for this interaction. Moreover, it has been confirmed that recombinant and endogenous eIF3d co-precipitate with endogenous SIN1 but not the mTORC2 components RICTOR and mTOR or the mTORC1 component RAPTOR. It is also shown that the interaction of SIN1 and eIF3d is likely to be occurring in the nucleus. In Hela cells, knockdown of eIF3d expression causes a decrease in the phosphorylation of the mTORC2 targets AKT and PKC on their turn motif sites, but also leads to a significant increase in the phosphorylation and activation of the mTORC1 target S6 kinase. This suggests that eIF3d is a regulator of both mTORC1 and mTORC2 signalling. Reduced expression of eIF3d leads to an increase in cell size, decreased cell proliferation and impairment of cell cycle progression, possibly by promotion of mitotic spindle multipolarity. Additionally, eIF3d knockdown leads to a delay in stress granule formation in Hela cells under arsenite stress, indicating that eIF3d might be acting as a negative mTORC1 regulator by contributing to its stress granule mediated inhibition. Thus eIF3d appears to be a regulator of cell size, proliferation and stress responses, in part mediated by modulation of mTOR activity.

In 2012, plasma figuring was proven to be an alternative solution for the fabrication of large scale ultra-precise optical surfaces. Indeed, plasma figuring was successfully demonstrated on a metre class glass surface. The process was exceptionally rapid but residual errors were observed. This thesis addresses this issue by proposing an enhanced tool that provides a highly collimated plasma jet. The enhanced tool is characterized by a targeted material removal footprint in the range 1 to 5 mm FWHM. The energy beam is provided by an Inductively Coupled Plasma (ICP) torch equipped with a De-Laval nozzle. This thesis focuses on characterization and optimisation of the bespoke plasma torch and its plasma jet. Two research investigations were carried out using both numerical and experimental approaches. A novel CFD model was created to analyse and understand the behaviour of high temperature gas in the De-Laval nozzle. The numerical approach, that was based on appropriate profiles of temperature and velocity applied to the nozzle inlet, led to a significant reduction of computational resources. This model enabled to investigate the aerodynamic phenomena observed from the nozzle inlet up to the processed surface. Design rules and the effect of changing nozzle parameters were identified. Sensitivity analysis highlighted that the throat diameter is the most critical parameter. A challenging power dissipation analysis of the plasma torch was carried out. Temperature and flow rate in key components of the torch were measured. Experimental results enabled to calculate the power dissipation values for RF power up to 800 W and for the entire series of designed nozzles. This work enabled to scientifically understand the power dissipation mechanism in the bespoke ICP torches. In addition, by comparing the intensity of the power dissipation values, one nozzle was clearly identified as being more capable to provide a highly efficient plasma jet.

Non-coding RNA (nc-RNA) plays an important role in biological processes. To understand a non-coding RNA function, we constructed twelve molecular bar-coded deletion mutants in Saccharomyces cerevisiae including five snRNAs, three RNAs of unknown function (RUFs), TLC1, SCR1, NME1 and RPR1. Nine of the twelve genes were found to be essential. RUF20 was particularly interesting as it was essential and overlaps the 3' untranslated region (UTR) of SEC4, a GTPase essential for vesicle-mediated exocytic secretion and autophagy. Shorter RUF20 deletions and SEC4 plasmid complementation in RUF20 knock-out strains concluded that RUF20 essentiality was derived from overlap with the SEC4 3' UTR. The SEC4 3' UTR is required for localisation of SEC4 mRNA to bud tips and the cell membrane. To investigate the molecular mechanisms of how RUF20 regulates SEC4 3' UTR formation or SEC4 function, RUF20 expression was turned off by using the TetO7 promoter system. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were performed to determine mRNA abundance for the targeted genes (RUF20 and SEC4). It was found that SEC4 mRNA expression was decreased when RUF20 mRNA expression was suppressed. SEC4 3' UTR formation was checked by RT-PCR primer walking, which indicated that SEC4 3' UTR processing was affected and not formed when RUF20 expression was inhibited. To investigate the localisation of RUF20 and SEC4 in the presence/absence of RUF20 RNA, fluorescence in situ hybridisation (FISH) was performed and it was found that RUF20 RNA displayed a similar pattern of localisation with SEC4 mRNA and there was a mislocalisation of SEC4 mRNA if RUF20 RNA was not expressed. We have identified a novel role for a non-coding RNA suggesting that RUF20 is required for SEC4 mRNA expression and influences the 3' end formation and SEC4 mRNA localisation.

Embryonic stem (ES) cells are cells able to give rise to any somatic cell types. A lot is known about how ES cell pluripotency is maintained, but only a little is known about how differentiation is promoted. In particular, cell fate decisions are regulated by interactions within transcriptional and signalling networks. Indeed, recently it has been discovered that pluripotency of mouse ES cells can be maintained using inhibitors of the Extracellular-signal Regulated Kinase (Erk) and Glycogen Synthase Kinase 3 (Gsk3) pathways (called "2i condition"). Erk and Gsk3 pathways have been previously associated with differentiation and developmental processes and with the regulation of gene expression. These observations suggest that Erk and Gsk3 pathways play a key role in the decision-making process of ES cells. The aim of this study is to understand how activation of the Erk and Gsk3 pathways (upon 2i removal) contributes to cell fate decisions. A genome-wide RNAi screen was recently performed in our laboratory and identified factors that facilitate pluripotency maintenance or that are involved in promoting the exit of ES cells from pluripotency after activation of the Erk and Gsk3 pathways. This PhD project aims to understand the mechanisms by which some of the identified transcription regulators contribute to cell fate decisions, with particular focus on how MAPK and Gsk3 pathway activation might give rise to the transcription and epigenetic profiles which are required for ES cells to exit from the pluripotent state. In this study the behaviour of Erk and Gsk3 during early differentiation was analysed and several transcription regulators that potentially act downstream of these pathways were identified, among which the transcription factors Jun and Elk1. Both Jun and Elk1 are well-known Erk targets. Jun expression is upregulated within days upon 2i removal and the activatory phosphorylation of Elk1 is induced within minutes upon 2i withdrawal. The study highlighted a role played by Jun in mouse ES cells undergoing differentiation in regulating the expression of adhesion-associated genes. Furthermore, the analysis of Elk1 binding sites (using Chromatin Immunoprecipitation followed by next generation sequencing) revealed a redundant role of Elk1 and Elk4 in inducing the expression of a novel long non-coding RNA, Ite1 (Inducible Transcript in ESCs), which is upregulated after days upon 2i removal in ES cells that are exiting the pluripotent state.

The histone variant H2A.Z is an evolutionarily conserved variant which is an essential chromatin component for many organisms. H2A.Z plays a pivotal role in a diverse array of chromatin-based processes such as gene transcription and chromosome segregation. In yeast, H2A.Z is acetylated at four N-terminal lysine residues (K3, K8, K10 and K14). Previous studies have shown that these acetylation sites are critical for H2A.Z function. My research aim was to examine how these four acetylatable lysines act to regulate the function of H2A.Z. Genome mapping of the acetylated K8, K10, K14 isoforms revealed that these acetyl marks are co-localised across the budding yeast genome, indicating that acetylation is a common feature of H2A.Z. Examinations of individual acetylation sites using mutational and phenotypical analyses did not reveal any distinct phenotypes between individual lysine residues. These findings indicated that individual acetylation sites are functionally redundant. Intriguingly, H2A.Z is mis-regulated when all four lysine were mutated to arginine (H2A.Z K3, 8, 10, 14 R) by showing sensitivity to a variety of agents. The global distribution profiles of H2A.Z, however, were unaffected by N-terminal lysine mutations. In fact, unacetylatable H2A.Z alleles perturbed H2A.Z chromatin abundance. Biochemical evidence showed that the altered chromatin level was severely defective when combined unacetylatable allele with mutations of SWR-C components. Together, the data presented here suggested that the N-terminal acetylation of H2A.Z regulates its genome abundance independent of its deposition pathway by SWR-C complex.

Cleft lip and cleft palate are among the most common congenital birth defects affecting the craniofacial region. Development of the head and face requires tight molecular control of time- and site-specific events. The importance of these regulatory processes is seen when even a small error in the molecular hierarchy has devastating effects on normal development leading to clefting phenotypes. Recently, a number of isolated cleft palate cases have been identified as a result of mutations in SATB2. This thesis reveals that SATB2 expression is conserved across vertebrate lineages. SATB2 is expressed throughout secondary palate development and most abundant in the anterior region of the developing mouse palatal shelves. On fusion of the palatal shelves, Satb2 expression is observed in the ossification centres of the palate. Newborn Satb2-/- mice exhibit cleft palate, fewer disorganised palatal rugae, expansion of the soft palate and hypoplasia of the bones forming the hard palate. Correlating with Satb2 expression during palatal shelf growth, Satb2-/- embryos exhibit altered morphology of the anterior palatal shelves as a result of aberrant cell proliferation. Moreover, the anterior region of the palate lost the potential to fuse, which is underlined by the regional absence of Tgfb3 in the medial edge epithelia. Temporo-spatial expression analysis of the palatal shelves of Satb2-/- embryos revealed marked reduction of Shh in the palatal rugae and significant up-regulation of Fgf10 in the anterior palate mesenchyme, which correlates with the abnormal palatal rugae. Moreover, genes implicated in Bmp signalling including Bmp4, Msx1 and Pax9, which control anterior palate growth, were up-regulated correlating with increased proliferation in the anterior Satb2-/- palatal shelf mesenchyme. Microarray analysis of E13.5 Satb2-/- palatal shelves revealed down-regulation of a group of genes promoting bone development and up-regulation of inhibitors of bone development, which correlates with hypoplasia of the hard palate. Moreover, microarray analysis revealed down-regulation of En1, Cadm3, Sema3e and Shox2, which are specific for the anterior region of the palatal shelves, in Satb2-/- embryos. Interestingly, Tbx22, which is expressed specifically in the posterior region of palatal shelves, was up-regulated resulting in ectopic extension into the anterior region of Satb2-/- palatal shelves. As a result, the well-defined Shox2 and Tbx22 expression border was disrupted, suggesting that the anterior palate mesenchyme had lost its molecular identity. Bioinformatics analysis revealed Satb2-binding motifs around Shox2 and Tbx22, suggesting direct regulation. In addition, Shox2 and Tbx22 are under indirect Bmp signalling. As the region upstream from the Satb2 promoter contains Smad-binding sites, it can be hypothesised that Satb2 receives Bmp signals in order to regulate Shox2 and Tbx22. Motif analysis also revealed potential Satb2-binding sites around Bmp4, Msx1 and Pax9, suggesting that Satb2 may be involved in a feedback loop, thereby fine-tuning the Bmp signalling during secondary palate development.

Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells. Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.

Achievable cutting distance of a diamond tool during turning is finite and is a limiting factor in the size of component that can be turned. This limit is particularly problematic when attempting to turn brittle materials, such as those used in infra-red optics. Natural diamond tools have been used for this application. However natural diamond introduces problems: the gems can contain possible contamination with a range of impurities and strong residual stresses from formation. Cutting distance is therefore inconsistent when using natural diamond. Industry is keen to increase possible cutting distance and to increase the consistency of cutting distance. One possible solution is synthetic diamond materials. New CVD single crystal synthetic diamonds possess high purity and consistent growth conditions and therefore have the potential to be a superior tool-material that provides longer achievable cutting distance and extremely consistent cutting behaviour. This new material is compared against natural and HPHT synthetic diamonds in machining tests against silicon workpieces in a selection of tool-orientations. Aluminium workpieces are machined with MCC and natural diamond tools to assess the performance of the new material against this commonly diamond turned material. While analysing the results from these cutting trials the failure modes of diamond tools were examined closely, resulting in discovering the existence of two separate failure modes and the development of a new wear-model. Natural diamond tools were carefully tested using a range of techniques hoping to find a root cause of the wide variability seen. FTIR offered a strong clue as to the defect within natural diamond tools that leads to occasional high cutting life.

In this thesis, the research and development of a novel rapid figuring procedure for large ultra-precise optics by Reactive Atom Plasma technology is reported. The hypothesis proved in this research is that a metre scale surface with a form accuracy of ~1 μm PV can be figure corrected to 20 – 30 nm RMS in ten hours. This reduces the processing time by a factor ten with respect to state-of-the-art techniques like Ion Beam Figuring. The need for large scale ultra-precise optics has seen enormous growth in the last decade due to large scale international research programmes. A bottleneck in production is seen in the final figure correction stage. State-of-the-art processes capable of compliance with requisites of form accuracy of one part in 108 (CNC polishing, Magneto-Rheological Finishing and Ion Beam Figuring) have failed to meet the time and cost frame targets of the new optics market. Reactive Atom Plasma (RAP) is a means of plasma chemical etching that makes use of a Radio Frequency Inductively Coupled Plasma (ICP) torch operating at atmospheric pressure. It constitutes an ideal figuring alternative, combining the advantages of a non-contact tool with very high material removal rates and nanometre level repeatability. Despite the rapid figuring potential of this process, research preceding the work presented in this manuscript had made little progress towards design and implementation of a procedure for metre-class optics. The experimental work performed in this PhD project was conducted on Helios 1200, a unique large-scale RAP figuring facility at Cranfield University. Characterisation experiments were carried out on ULE and fused silica surfaces to determine optimum process parameters. Here, the influence of power, surface distance, tool speed and surface temperature was investigated. Subsequently, raster-scanning tests were performed to build an understanding on spaced multiple passes ... [cont.].

Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip and cleft palate (CLP), lip pits, skin-folds, syndactyly and oral adhesions. VWS and PPS are caused by mutations in the transcription factor Interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved DNA-binding domain (DBD) and a less conserved protein-binding domain. Mutation analysis reveals a general genotype-phenotype correlation between the distribution of mutations and the manifestation of either VWS or PPS. However, the molecular mechanisms by which these mutations affect IRF6 function are still unknown. Therefore the aim of this project was to characterize the molecular function and regulation of IRF6 and subsequently determine the effects of VWS and PPS mutations on IRF6 function. DNA binding site-selection showed that the IRF6-DBD exhibited optimal binding site selectivity for the consensus sequence AACCGAAACC/T. This sequence was used to investigate the effects of VWS and PPS mutations in the DBD. Almost all mutations abrogated DNA-binding. It was subsequently demonstrated that a VP16AD-IRF6-DBD fusion protein was able to activate transcription from a promoter containing the derived binding site. However, wildtype IRF6 was shown to be autoinhibited and compartmentalized in the cytoplasm. To determine what signals are involved in the nuclear translocation of IRF6, internal signals that facilitate nucleocytoplasmic trafficking were mapped. The cytoplasmic localization of IRF6 suggested that the identified nuclear export signal (NES) 159IQDTFPFLNI168 is constitutively active and dominant over the nuclear localization signal 5PRRVRLK11. Disruption of the NES leads to nuclear retention; however nuclear localization alone is insufficient for functional activation of IRF6. Therefore, the role of phosphorylation in the regulation of IRF6 was assessed. Five novel phosphorylation sites were identified in IRF6 at residues S47, S131, S153, S424 and T425. Phosphorylation at S424/S425 was necessary for combinatorial phosphorylation at the serine/threonine rich region to induce nuclear retention, whereas, phosphorylation in the DBD at S47 abrogates DNA-binding. The understanding of IRF6 molecular function and mechanisms of regulation provides further insight into the pathways involved in the pathogenesis of VWS and PPS.

Filamin A is a well-characterised cytoskeletal protein which regulates cell shape and migration by cross-linking with actin. Filamin A mutations cause a number of human developmental disorders, many of which exhibit skeletal dysplasia. However, the molecular mechanisms by which Filamin A affects skeletal development are unknown. The transcription factor Runx2 is a master regulator of osteoblast and chondrocyte differentiation. Data presented in this thesis show that Filamin A forms a complex with Runx2 in osteoblastic cell lines. Moreover, it is demonstrated that Filamin A is present in the nucleus in several cell lines, including those of osteoblastic origin. The data presented show that the Filamin A/Runx2 complex suppresses the expression of the gene encoding the matrix-degrading enzyme, matrix metalloproteinase-13 (MMP-13), which is an important osteoblastic differentiation marker. ChIP assays were employed to demonstrate that endogenously expressed Filamin A associates with the promoter of the MMP-13 gene. In addition, Filamin A is not only located in the nucleus but also in the nucleolus, an important nuclear compartment involved in ribosomal RNA (rRNA) transcription. Ribosomal DNA promoter-driven reporter assays, Filamin A-knockdown experiments and exogenous Filamin A transfections demonstrated that Filamin A and Runx2 can repress ribosomal gene expression activity. Importantly, Filamin A is recruited to the human ribosomal DNA promoter, suggesting its direct involvement in the regulation of rRNA transcription. These findings reveal a novel role of Filamin A in the direct regulation of ribosomal gene expression. Finally, by using microarray technology, changes in gene expression were identified when Filamin A was downregulated. Some of the differentially expressed genes were known orchestrators of bone development. The data presented in this thesis strengthen the link between Filamin A and bone development and provide a molecular rationale for how Filamin A, acting as a regulator of gene expression, might influence osteoblastic differentiation.

The Reactive Atom Plasma® (RAP) process is a plasma chemical etching process. RAP was developed at RAPT Industries as a process for removing subsurface damage from silicon carbide optics. The process is being investigated at Cranfield University as a novel method for the fine surface correction of large optics, with the aim of shortening the manufacturing period of the next generation of large telescopes. RAP offers material removal rates that are up to 10 times higher than those of ion beam figuring, the current state-of-the-art technique and the convenience in that it can be operated at atmospheric pressure. Cont/d.

A dynamically optimal design is essential for a motion system to perform high speed operation without compromising its accuracy, settling time and vibration specification. Good design practice which accounts for dynamic characteristics in the modelling of a motion system warrants higher performance precision machines and cuts down redevelopment effort to ‘patch’ inherent shortcoming of the machine dynamics. This research aimed to accurately describe the non-linear dynamics of a non-mechanical contact aerostatic guideway system in order to achieve an accurate FEA model of the design stage. The single axis aerostatic guideway is comprised of several machine in¬terfaces that impact the dynamic behaviour of the guideway. Modelling each air bear¬ing pad by a single stiffness element is not adequate to predict the guideway modal behaviour accurately. The aerostatic guideway has been broken down into several key machine interface elements. In-depth investigation of the air film and the air bearing mounting mechanism was carried out. A dedicated air film test rig was designed and built to acquire insight of the air film dynamic characteristics. It is observed that the mounting mechanism of the air bearing constitutes to a signifi-cant dynamic effect to the entire air bearing setup. Based on the findings of the mount-ing mechanism’s stiffness properties, a method was developed to estimate ‘true’ air gap heights which cannot be easily assessed and measured directly in most aerostatic guideway carriages. The estimation method enables a more rigorous FE model of the aerostatic guideway system. The comprehensive dynamic analysis methodology pro-posed in this research greatly increases the confidence and accuracy of the aerostatic guideway’s FE model.

Large scale nuclear fusion and astronomy scientific programmes have increased the demand for large freeform mirrors and lenses. Thousands of one metre class, high quality aspherical optical components are required within the next five to ten years. Current manufacturing process chains production time need to be reduced from hundred hours to ten hours. As part of a new process chain for making large optics, an efficient low damage precision grinding process has been proposed. This grinding process aims to shorten the subsequent manufacturing operations. The BoX R grinding machine, built by Cranfield University, provides a rapid and economic solution for grinding large off-axis aspherical and free-form optical components. This thesis reports the development of a precision grinding process for rapid manufacturing of large optics using this grinding mode. Grinding process targets were; form accuracy of 1 m over 1 metre, surface roughness 150 nm (Ra) and subsurface damage below 5 m. Process time target aims to remove 1 mm thickness of material over a metre in ten hours. Grinding experiments were conducted on a 5 axes Edgetek high speed grinding machine and BoX R grinding machine. The surface characteristics obtained on optical materials (ULE, SiC and Zerodur) are investigated. Grinding machine influence on surface roughness, surface profile, subsurface damage, grinding forces and grinding power are discussed. This precision grinding process was validated on large spherical parts, 400 mm ULE and SiC parts and a 1 m Zerodur hexagonal part. A process time of ten hours was achieved using maximum removal rate of 187.5 mm 3 /s on ULE and Zerodur and 112.5 mm 3 /s on SiC. The subsurface damage distribution is shown to be "process" related and "machine dynamics" related. The research proves that a stiffer grinding machine, BoX, induces low subsurface damage depth in glass and glass ceramic.

Machine tools are made up of many different parts connected together. In order to understand and create more accurate thermal and deformation numerical models the heat transfer characteristics of machine tool interfaces need to be understood. Previously heat transfer across interfaces has not been incorporated in finite element models in thermal and deformation analysis of machine tools. To a varying degree the issue of interfaces will have an effect on the overall performance of machine tools, and clearly if not considered and resolved at the strategic design stage they may be difficult and expensive to correct, once machine tools are operational. An inability to adequately understand and model the interfaces in machine tools is therefore a major limitation to current knowledge. In this research programme heat transfer and corresponding thermal deformation of metal blocks in contact having pre-defined interface geometries, were experimentally and numerically investigated. Factors such as the contact pressure, the contact conformance, and the interface material were investigated. It was found that the most influential factors were contact pressure and contact conformance. Interface material was also found to have a bigger influence if the contact area was non-conforming. When interface material was thicker than the surface roughness, the heat flow across the interface was reduced as fewer contact asperities came into contact. In order to optimise modelling it was necessary to optimise the convection coefficient - this was found to have an effect on overall heat transfer across the interface. It was also found that the overall contact area is important and not the spread of contact regions for the same overall contact. This was investigated using two different contact geometry arrangements. The numerical prediction of heat transfer across an interface is accurate if the contact is conforming. However, if the contact is non-conforming an accurate prediction is not possible. This is due to the fact that direct modelling of non-conformance is not achievable. It was found that the most influential factor on thermal deformation is contact pressure, on the other hand factors such as interface material and interface geometry showed very little effect.

Driven by a requirement to extend the lifespan of self-aligning lined spherical bearings, this research investigates the use of Elid (electrolytic in-process dressing) as a method of improving ball surface finish. Elid is a continuous and self-regulating electrochemical dressing process that modifies the surface of a grinding, lapping, or superfinishing wheel. It provides improved grit protrusion, impedes wheel loading / glazing and promotes effective cutting. The characteristics of the newly-developed Elid superfinishing process are in many ways fundamentally different to conventional superfinishing. The main difference is that the use of super-abrasives prevents the wheel from self-sharpening; the normal mechanism by which dulled conventional abrasives are removed and a wheel’s surface refreshed. Because the wheel’s performance and condition is continually maintained inprocess by the Elid system, metal resin bonded (MRB) wheels containing very small super-abrasives can be used. It is the utilization of these fine abrasives (30 to 0.12 μm) that enables surface roughness values below 5 nm Ra to be consistently produced on the spherical surface of corrosion-resistant steel balls. This research provides an in-depth understanding of the Elid spherical superfinishing process; investigating the most effective use of the Elid system, wheel dressing requirements and process performance. Optimisation is provided in terms of evaluating the critical operating parameters, the most effective superfinishing cycle and the implications to the complete ball production chain. A range of techniques are used to evaluate processing performance and ball output quality. These include in-process monitoring of Elid and wheel spindle power levels, analysis of wheel condition, rates of ball surface generation and material removal, ball finish and form. Although predominantly concentrated on corrosion-resistant steel, testing is also conducted on titanium and various ball coatings. In investigating various ways of using the Elid system, this work considers electrodischarge truing, pre-process dressing, Elid 1, Elid 2, Elid 3, and Elid combined with electrolytically assisted superfinishing. The initial process solution of Elid 3 (electrodeless) superfinishing provides the capability of working on all standard size balls, however the dressing system lacks stability. The development of a fixturing system that has a small separate electrode enables Elid 1 (conventional) to be used on the majority of ball sizes. Elid 1 allows more aggressive and consistent dressing, a faster rate of ball material removal and thus a substantially reduced processing time. Results with a #12,000 wheel show that surface quality is vastly improved through the use of Elid whilst maintaining current production standards of form accuracy. Surface finishes of 2nm Ra are achieved, which is an order of magnitude better than balls currently produced using barrelling / polishing. Processing times are equivalent or faster when using Elid 1. Alternatively, consistently sub 10 nm Ra finishes can be reached with a #2,000 wheel using Elid 2 (interval dressing). Generally MRB-CBN wheels provide a more effective carbide cutting action than conventional superfinishing wheels. Controlling wheel condition and achieving full and even ball to wheel conformity are the two most significant contributory factors to the success of Elid spherical superfinishing. Insufficient control of these factors results in poor output quality. Monitoring of wheel spindle and Elid power usage provides useful information in assessing the condition of the wheel and identifying potential problems. High spindle power correlates with fast material removal and is a result of high loads and a free cutting action. Elid processing can be employed for improving surface finish after the conventional honing stage, or after cylindrical grinding for improving both ball form and finish.

The main aim of this work was the machining by hard turning of surfaces with optical surface quality. A numerical target had been set as a surface roughness Ra = 10nm. It has been shown that achieving roughness of that magnitude by hard turning is possible. Individual work pieces exhibited the desired surface properties for short lengths at a time, but it proved to be very difficult to achieve these surfaces consistently and over longer cuts. The factors influencing the surface roughness were identified as tool defects and machine vibration in addition to the standard cutting parameters and choice of cutting tool. A model of surface generation in hard turning has been developed and good correlation between simulated and experimentally determined surface roughnesses was achieved. By introducing a material partition equation which determines the proportional contribution of material removal mechanisms in the undeformed chip a comprehensive method for assessing the contributing factors in material removal was developed. While it has been shown that surfaces in hard turning are almost exclusively generated by chip removal and plastic deformation the developed model is versatile enough to include elastic deformation of the work piece. With the help of the model of surface generation in hard turning it has been possible to attribute magnitudes of the influencing factors with respect to the cutting parameters such as feed rate and tool corner radius, and the main disturbances - tool defects and machine vibration. From this conclusions were drawn on the requirements for machine tools and cutting tools, which will need to be realised to make ultra-precision hard turning of surfaces of optical quality a feasible manufacturing process.