Mammalian cell cultures are used for production of biopharmaceuticals, e.g. monoclonal antibodies. Only mammalian hybridoma cells contain the pathways for antibody production, but due to their multicellular origin the cells have complex nutrient requirements. Cell growth and antibody production are limited by supply of essential nutrients such as glutamine and accumulation of toxic waste products such as lactate. Many attempts have been made at tackling these challenges, e.g. by optimising growth media to keep metabolite concentrations at optimal levels. These approaches have been hampered by our ability to monitor relevant cell culture parameters such as metabolite concentration dynamics in real time. The aim of this study is to develop a solution to this problem using a synthetic biology approach. Whole-cell bacterial biosensors for important culture parameters, glutamine, leucine, alanine and lactate, were designed, built and characterised. The biosensors were designed from natural metabolite-sensing systems, specifically the Escherichia coli Ntr regulon, Lrp regulon and lldPRD operon and the Bacillus subtilis GlnK-GlnL system. Characterisation of the biosensors in defined medium using known lactate concentrations was followed by validation in mammalian cell culture media and using cell culture samples. A lactate sensor based on the lldPRD operon showed a reliable lactate-response during initial characterisation and was chosen to determine lactate concentrations in cell culture samples in parallel with lactate analysis using a bioprofiler. Generally, the lactate concentrations from the two methods showed a good match. Data points where the results differed showed that there are some sources of error in the usage of the biosensor that could be addressed in future. The results of this study also highlight the many challenges of applying synthetic biology constructs to complex industrial contexts. The biosensors presented in this study are more generally applicable in any experimental context that requires sensing of metabolites.

Invasive aspergillosis is a major cause of morbidity and mortality in acute myeloid leukaemia (AML) (1-3) and Aspergillus fumigatus has been identified as the most common species causing invasive disease in these patients (2,4). The adoptive transfer of natural killer (NK) cells has been described as a promising therapeutic tool for AML (5,6) and it has been suggested as an attractive strategy in the prophylaxis of common infections in these patients. This PhD aimed to characterize the human NK cell response to A. fumigatus in order to assess the potential use of these cells to prevent this fungal infection. The interaction between NK cells and A. fumigatus in vitro resulted in cell adherence to the fungus, polarization of the lytic granules towards A. fumigatus, NK cell degranulation and chemokine release. The co-culture of NK cells and A. fumigatus also resulted in CD56 downregulation on NK cell surface, a process that might represent a novel mechanism of immune evasion for A. fumigatus. The analysis of NK cell antifungal activity demonstrated that NK cells do not contain fungal growth, however the cells have shown to induce fungal DNA release that has been associated to fungal damage (7,8) and might mediate immune cell activation since pathogen-derived DNA has been shown to stimulate the widely expressed TLR9 receptor (9-11). AML-derived NK cells were shown to maintain their ability to polarize the lytic granules towards A. fumigatus and to induce fungal DNA release. In conclusion, my findings suggest that the role of NK cells in the immune response against A. fumigatus infection might be indirect, through the recruitment of immune cells to the site of infection and the induction of immune cell activation mediated by fungal DNA. Therefore, my results suggest that the infusion of NK cells on their own would be insufficient to control A. fumigatus infection in AML patients.

The transparency of the human cornea is vital in maintaining vision. The cells on the surface, corneal epithelium cells, are constantly replaced by differentiated limbal stem cells. Damage to the limbus can cause a deficiency in the limbal stem cell population which can cause cornea opacity and vascularisation consequently leading to blindness. Currently limbal stem cell deficiency can be detected clinically and treated surgically by transplanting stem cells onto the damaged cornea. However, the long term success rate is low 68\% and the results vary from patient to patient. There is an interest to understand the cause for this variability in results and the low long term success rates. The aim of the project was to develop an Ultrahigh resolution OCT (UHROCT) to locate the transplanted corneal epithelium cells. Optical Coherence Tomography system (OCT) is a non-invasive micron resolution imaging technique that can image several millimetres in tissue. The cells to be transplanted are labelled with super paramagnetic iron oxide (SPIO) nanoparticles. After uptake these SPIO embedded cells are displaced with an external magnetic field at a known frequency whilst being imaged by the UHROCT. This technique known as phased resolved magnetomotive OCT, allowed us to locate the oscillating nanoparticle which is embedded in the cell by detecting the location of the modulation frequency. For the Ultrahigh resolution OCT a super luminescent diode (SLD) light with centre wavelength at \num{890e-9}m and a bandwidth of \num{150e-9}m. The system has an axial and lateral resolution of 2.5$\mu$m and 6.20 $\mu$m in air respectively. The system was able to detect as low as 1273 SPIO loaded cells/ $mm^2$ on rabbit cornea. The spectral domain UHROCT constructed was also used to image 3D oral mucosa constructs. The system shows superior contrast in comparison to 1310 nm swept source systems. The UHROCT was able to identify key difference between the normal, dysplastic and malignant construct. For the malignant construct Cal27’s, hematoxylin and eosin staining was used to confirm the formation of a keratinized superficial layer. The keratinized layer was presented as a hyper-reflective thickened layer superficial to a darker region on both OCT platforms. This keratinized layer causes a sharp fall in the signal in the UHROCT making it difficult to visualise the underlying structures of the construct.

A major challenge of the image registration in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is related to the image contrast variations caused by the contrast agent passage. Tracer-kinetic model-driven motion correction is an attractive solution for DCE-MRI, but previous studies only use the 3-parameter modified Tofts model. Firstly, a generalisation based on a 4-parameter 2-compartment tracer-kinetic model is presented. A practical limitation of these models is the need for non-linear least-squares (NLLS) fitting. This is prohibitively slow for image-wide parameter estimations, and is biased by the choice of initial values. To overcome this limitation, a fast linear least-squares (LLS) method to fit the two-compartment exchange and -filtration models (2CFM) to the data is introduced. Simulations of normal and pathological data were used to evaluate calculation time, accuracy and precision of the LLS against the NLLS method. Results show that the LLS method leads to a significant reduction in the calculation times. Secondly, a novel tracer-kinetic model-driven motion correction algorithm is introduced which uses a 4-parameter 2-compartment model to tackle the problem of image registration in 2D renal DCE-MRI. The core architecture of the algorithm can briefly described as follows: the 2CFM is linearly fitted pixel-by-pixel and the model fit is used as target for registration; then a free-form deformation model is used for pairwise co-registration of source and target images at the same time point. Another challenge that has been addressed is the computational complexity of non-rigid registration algorithms by precomputing steps to remove redundant calculations. Results in 5 subjects and simulated phantoms show that the algorithm is computationally efficient and improves alignment of the data. The proposed registration algorithm is then translated to 3D renal dynamic MR data. Translation to 3D is however challenging due to ghosting artefacts caused by within-frame breathing motion. Results in 8 patients show that the algorithm effectively removes between-frame breathing motion despite significant within-frame artefacts. Finally, the effect of motion correction on the clinical utility has been examined. Quantitative evaluation of single-kidney glomerular filtration rate derived from DCE-MRI against reference measurements shows a reduction of the bias, but precision is limited by within-frame artefacts. The suggested registration algorithm with a 4-parameter model is shown to be a computational efficient approach which effectively removes between-frame motion in a series of 2D and 3D renal DCE-MRI data.

Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells and. The work presented in this thesis set out to examine the hypothesis that microenvironment of skeletal muscles can be influenced by immune cell secretions, which affect satellite cell proliferation, and that beneficial immune-muscle interactions in young people are blunted in elderly. The aim of this research was to investigate the role of ageing human lymphocytes on skeletal muscle cell behaviour. For this purpose, lymphocytes were isolated from whole blood of young (aged 18-25 years) and older (aged 78-85 years) healthy volunteers and older healthy volunteers. All the participants were healthy, with no history of muscle disease and not on immunosuppressant or corticosteroids treatment that affect immune function. Lymphocytes were cultured with, or without, anti-CD3/CD28 activators for 4 days to induce release of cytokines, interleukins and growth factors into the media. The secreted proteins were used to prepare conditioned media that were used to culture C2C12 myoblasts. Secretomes were analysed and fifteen secreted Th1/Th2 cytokines and IGF-I were quantified by multiplex immunoassay. The gene expression and protein concentrations of amphiregulin were determined from T-lymphocytes lysates by real-time PCR and ELISA respectively. The levels of CD25 and FoxP3 expression in lymphocytes were examined using flow cytometry. The expression of muscle transcription factors, MyoD and Myogenin were determined by real- time PCR. Activated Mek1/Erk1/2 and Akt/mTOR were measured by multiplex immunoassay. Our results demonstrate for the first time that a decrease in the levels of amphiregulin and CD25 coincides with the increase in FoxP3 with ageing, which may be involved in suppression of lymphocytes. Seven cytokines were differentially secreted by the young- compared with the old-activated lymphocytes. The secretome from young-activated lymphocytes had 30% (P < 0.005) higher IGF-I concentrations compared with old and control treatments. The conditioned media from young -activated lymphocytes increased the rate of proliferation of myoblasts by ~3-fold (P<0.005) and caused an approximate 4-fold (P < 0.005) increase in migration compared with non-activated lymphocyte control media. These responses were characterised the extended proliferation of young -treated myoblasts was also associated with a decrease in MyoD and Myogenin and an increase in mediators of proliferation Mek1/Erk1/2and a decrease in the key proteins for differentiation, Akt/mTOR. In contrast, myoblasts treated with conditioned media from old-activated lymphocytes exhibited a high degree of differentiation.

The transmembrane envelope glycoprotein, gp46 of maedi visna virus (MVV) was proposed to mediate membrane fusion between the virus and cell and between infected and uninfected cells on the basis of analogy with other viruses. In order to look at the relationship between fusion and gp46, which is difficult to purify to quantity from virus preparations, recombinant gp46 (rpg46) was made. This first required cloning of the part of the MVV (strain EV1) <I>env</I> gene which encodes gp46. Sequence comparison with other viruses showed that gp46 is a typical transmembrane fusion protein, with a hydrophobic N-terminal fusion peptide. This putative fusion peptide was highly conserved between different MVV isolates although gp46 as a whole was only about 80% conserved. rpg46 was expressed as fusion proteins in yeast and in bacteria, but could only be prepared at low yield and purity principally due to its toxicity to host cells. Immunised animals raised antibodies to rgp46 and sera from MVV-infected sheep specifically reacted with rgp46, whereas serum from uninfected sheep did not. A fusion assay was developed using MVV to determine the effect of serum on MVV-mediated fusion. A significant difference was shown between the activity of sera from MVV-infected and uninfected sheep in the fusion assay: Serum from uninfected sheep generally enhanced fusion at low dilutions of serum (<1:64) and had no effect at higher dilutions, whereas serum from MVV infected animals tended to inhibit fusion at low serum dilutions (<1:16) and enhance at higher dilutions. These results may have important implications in consideration of potential vaccines and also in understanding how the virus can continue to replicate and eventually cause disease in the face of an apparently normal specific immune response.

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Rationale: MRI is a highly specialised imaging modality that uses non-damaging radiation to produce detailed medical images. In most countries, these images are acquired by practitioners who first train as radiographers and then specialise in MRI. Previous research suggests that some MRI practitioners may have insufficient knowledge to practise safely. This research proposes that practitioners should be educated initially and exclusively in MRI via a specialised undergraduate curriculum. This is underpinned by a proposition that the practice of MRI does not require an advancement of previously acquired radiographic knowledge, but instead reflects a difference in knowledge. Methodology: As there are educational and professional implications to this research, a mixed-methodology approach is chosen. A convergent nested design with a dominant educational quantitative strand, supported by professional qualitative data, is used. Quantitative data are collected via an objective structured clinical examination (OSCE) to explore whether there is any difference in MRI knowledge between graduate and experiential practitioners. Graduate practitioners (n=25) learn MRI only via a specialised undergraduate degree. Experiential practitioners (n=23) learn only experientially postqualification as a radiographer. Qualitative data exploring the professional implications of direct entry into MRI are collected via semi-structured interviews with key stakeholders (n=8). These are professionals who have either experience of graduate and experiential practitioners or an influence on policy in this area. The data from both strands are merged and analysed using a connections matrix. Findings: Statistical analysis of the quantitative data shows that graduate practitioners score more highly in the OSCE at a significance of p < 0.05, especially in topics related to general principles of MRI, image contrast and image acquisition. The qualitative data support direct entry but raise concerns about limited scope of practice and registration. Contribution to knowledge: This is the first study that considers whether the radiographic specialism is best learned initially and exclusively at undergraduate level and whether it is necessary to qualify as a radiographer to practise MRI. It is also the first study that uses a mixed-methodology approach to explore the feasibility of early specialisation in radiography.