The lymphatic system constitutes a vital part of the systemic circulation, maintaining tissue fluid homeostasis; its biological significance is not limited to fluid balance, as it is a part of the immune system, facilitating immune cell trafficking and initiating the immune system response to pathogens. However, the hydrodynamics/biomechanics of the lymphatic system have been relatively understudied when compared to the cardiovascular system. In contrast with the latter, where the heart provides the necessary energy for blood flow, the lymphatic system relies on the active contraction of vessels and one-way valves to generate and sustain flow. The present thesis describes the development of an optical flow diagnostic method for resolving the 2-dimensional flow field at the mid-plane of isolated contracting mesenteric rat lymphatic vessels, in-vitro. The aim was to develop an experimental protocol for accurately estimating flow parameters, and in addition, estimate lymphatic functional parameters, with high spatial and temporal resolution. Previous studies in-situ have relied on lymphocytes as tracers, but their low density however resulted in a reduced spatial resolution. The time-resolved micro-Particle Image Velocimetry technique, a well established method has been employed to study this transient flow. To that end, a bespoke light source was developed, utilising high-power light-emitting diodes, as well as associated control and image processing software. Image analysis was performed with PIV cross-correlation algorithms and was facilitated by custom vessel wall detection algorithms. In terms of flow parameters, flow velocity, flow rate and shear stresses were estimated from the raw images. Additionally, the dynamic pressure was estimated allowing for extraction of pressure volume curves and estimation of work performed during contraction by the lymphatic vessels. It was also possible to obtain an estimation of lymphatic wall stress. The results were extended to lymphatic functional parameter estimation, such as contraction amplitude and fractional pump flow. Statistical analysis was undertaken, in order to reveal possible relationships between the experimental parameters and the derived hydrodynamic/biomechanical results. Analysis of the uncertainty of the measurements showed that method employed provides exceptional accuracy. The results demonstrated the successful application of the micro-PIV technique in lymphatic vessels and yielded interesting observations, either previously unreported in the literature or contradicting previous reports. The wall velocity was shown that it may be comparable to the fluid one; an observations that questions the assumptions of previous attempts to study lymph flow. It was also observed that the flow rate may not increase during contraction depending on the hydrodynamic conditions. Although an incidental finding, vibrations were shown to increasethe contraction frequency of lymphatic vessels.

This thesis focusses on the challenges relating to clinical- and cost-effectiveness analysis in Health Technology Assessment (HTA). It includes methodological developments, both statistical and presentational, in evidence synthesis aiming to address those challenges. In HTA, analysts often face problems with limited availability of data required to inform economic model. This thesis proposes innovative evidence synthesis approaches to address this challenge, illustrated in two examples. Bivariate random-effects meta-analysis (BRMA) and network meta-analysis (NMA) were used to synthesise all available evidence to predict progression-free survival (PFS), in metastatic prostate cancer. This enabled the specification of a three-state Markov model previously limited to two states when PFS was not recorded. In the second example, a scenario in multiple sclerosis is considered where utility data for the trials included in a HTA were not available and external utility data from a single study was used instead. This thesis illustrates how BRMA can be applied to include all available evidence to inform utility estimates for use in a cost-effectiveness analysis. NMA, allowing for a simultaneous and coherent comparison of multiple interventions, is increasingly used in HTA. However, due to the inherent complexity of presenting NMA results, it is important to ease their interpretability. A review of existing methods of presenting NMA results in HTA reports revealed that there is no standardised presentational tool for their reporting. Novel presentational approaches were developed which are presented in this thesis. The original contributions of this thesis are the innovative approaches to incorporate historical data to predict and increase the precision of parameter estimates for cost-effectiveness analysis to better inform health policy decision-making; and three novel graphical tools to aid clear presentation and facilitate interpretation of NMA results. Ultimately, the hope is that the graphical tools developed will be recommended in updated guidance setting the standards for future HTAs.

The work in this thesis is split into two parts. The introduction and following two chapters pertain to the investigation of gene regulation using Chip-seq data and linear modelling. The final chapter pertains to the prediction of hot-spot residues in protein-protein interactions. The rapid escalation in the speed and quality of DNA sequencing has lead to a wealth of data for the location of transcription factor binding and histone modifications across the genomes. Using Chromatin ImmunoPrecipitation followed by sequencing (ChIP-seq) data, we have generated a new binding metric based on the enrichment of the read-counts for each gene. Eight datasets from mouse macrophage cells (two histone modifications, five transcription factors, DNase I hypersensitivity) were used to model the binding of RNA polymerase II. It was found that a linear model just using the DNase I hypersensitivity and histone modification data was better than any of the models containing the transcription factor data. Investigation of the outlying genes for the model revealed no pattern in their Gene Ontology terms or macrophagespecific genes. Human embryonic stem cell data (23 transcription factor and 24 histone modification datasets) were used in combination with LASSO regression to model the binding of RNA polymerase II. The resultant models contrasted with the results from the mouse macrophage linear models in that using the histone modifications data in combination with the transcription factor data lead to the best models. A much more complicated picture of the regulation of RNA polymerase II binding was produced using the LASSO models. Protein-protein interactions are essential for every function within a cell and being able to predict them has large consequences for drug discovery and understanding the vast proteininteraction networks that occur within cells. Predicting protein-protein interactions is difficult due to the large number of possible conformations; predicting hot-spot residues can greatly reduce this. InterBasePro was compared with experimental data and subsequently adaptation was done to assess its usefulness for predicting hot-spot residues. An alternative approach was also made into classifying hot-spot residues based on atomic contacts.

Light and ultrasound are both non-ionizing radiations, ideal for biomedical applications. Recent studies on combining ultrasound and light for biomedical imaging show new promises in improving imaging quality and/or providing complementary imaging contrast. Among a variety of the imaging modalities that simultaneously use ultrasound and light, this work focuses on optical detection of tissue responses to acoustic radiation force (ARF). The applications include optical shear wave elastography and ultrasound modulated optical tomography. The first half of the thesis provides a systematic study on tracking shear waves in optical turbid media using CCD-based laser speckle contrast analysis. The theory, simulation and experiment are developed and cross-validated. The simulation quantitatively relates CCD speckle contrast signal with shear waves, providing useful information to understand the underlying physics. In addition, multiple shear waves are tracked using laser speckle contrast detection. Results show that two counter-propagating shear waves produce a modulation pattern in the optical signal, and the modulation pattern was suggested by simulation as a result of the dual shear wave interference. Shear wave speed measurements in phantoms suggest that the dual shear wave approach is more accurate than the single shear wave approach as that the standard deviation of the speed measurement is reduced by a factor of at least 2. The the dual shear wave approach also provides a reduced boundary effect. Both factors suggest that the dual shear wave approach should improve the accuracy of elasticity measurements. In the second half of the thesis, instead of detecting ARF response in the late phase, the study is motivated by detecting ARF response in the early stage for enhancement of ultrasound modulation of light. A pilot study on incorporating perfluorocarbon-based phase change contrast agent with ultrasound modulated optical tomography is explored. To understand the phase transition process, a quantitative measurement of acoustic nanodroplet vapourisation is developed. A preliminary result also showed that a single ultrasound burst can simultaneously vaporise the nanodroplets and sonify the converted microbubbles to provide additional ultrasound modulation of light. This additional light modulation was shown to increase the laser speckle contrast signal detected on a CCD camera.

Important and novel electric birefringence measurements of immediate significance in the biomedical field are reported on a family of materials which form the major constituents of the cartilage connective tissue. Outstanding amongst the reported results is the confirmation of the Hardingham-Muir model for proteoglycan-hyaluronic acid aggregation, and the relative ease with which the technique was both able to substantiate the model, and, for the first time, to observe the gradual formation of the aggregate. The complex heteropolysaccharide, proteoglycan, has also been characterised by a wide ranging series of electric birefringence measurements. Other conformational changes studied by the technique on other cartilage materials and reported herein are the effects of pH changes on hyaluronic acid conformation, the enzymatic degradation of hyaluronic acid and the thermal denaturation of collagen. The work also considers aspects of the problems of application of theoretical models to flexible polymers and demonstrates what can be achieved on existing crude theories with the nitrocellulose in acetone system. Some exploratory measurements in the interfacial area between solute and solvent are reported for the bacterium E. coli and an aqueous suspension of PTFE particles. A diverse range of materials was studied with particular reference to the values of particle rotary diffusion constants derived from dispersion of birefringence with frequency and analysis of birefringence relaxation time decay rates following the application of a pulsed DC field. Comparisons are drawn between these values and explanations of the discrepancies observed are considered. Last, but not least, the design of an advanced and high sensitivity apparatus for the measurement of electric birefringence is reported. Unusually, the system adopts a vertical mode and the novel Kerr Cell design incorporated dispenses. with end windows. The use of this new compact design is suggested as the basis for commercial applications.

Controlling lineage specific differentiation of stem cells is crucial for functional tissue engineering, and current methods have drawbacks and limitations. Histone deacetlyase (HDAC) proteins are key to cellular epigenetics, and a number of studies have shown that their inhibitors (HDACis) can control stem cell fate. The aim of this study was to investigate the effect of a novel HDAC2 and HDAC3 selective inhibitor, MI192, on human adipose derived stem cells (ADSCs), exploring the potential of utilising MI192 in controlling the osteogenic differentiation of ADSCs, with the long term goal of improving the efficacy of bone tissue engineering. MI192 was synthesised in house and characterised with NMR analysis. The effect of MI192 was explored on commercially (Life Technologies) available ADSCs. It was found that 1 µM to 100 µM MI192 was cytotoxic and reduced ADSC viability, with a dose response observed. Using propidium iodide staining, it was demonstrated that MI192 halted the cell cycle in the G2M phase. FACs analysis showed that MI192 altered the stem cell marker expression profile of ADSCs; increasing expression of some markers (CD34 and CD146) and decreasing some (CD29, CD44, CD73, CD105, CD166). When utilised in a pre-treatment strategy in 2D models, MI192 improved the osteogenic differentiation of ADSCs, strongly increasing production of alkaline phosphatase protein, with 2 days of 30 µM pre-treatment being the optimal concentration and treatment length. MI192 pre-treatment also increased ADSC mineralisation (calcium accumulation stained with Alizarin Red and mineral nodule formation stained with Von Kossa) and increased osteogenic gene expression (BMP2, RUNX2, COL1 and ALP). The adipogenic differentiation of the ADSCs was inhibited by MI192 pre-treatment, with reduced lipid droplet accumulation (stained with Oil Red O) and adipogenic gene (PPARγ and ADIPOQ) expression seen. Increased osteogenic differentiation was further demonstrated in 3D models, using Am silk scaffolds. Cell attachment was assessed with live cell labelling, and collagen production, mineralisation and protein production analysed with histology and immunohistochemistry. Finally, TaqMan® Gene Expression Array Cards were utilised to investigate how two days of MI192 treatment altered the expression of 96 different genes. Some key Wnt related genes, as well as other key osteogenic genes such as BMP2 were up-regulated, providing some mechanistic explanation for the increase in osteogenic differentiation potential seen with MI192 pre-treatment. This thesis demonstrates the potential of utilising MI192 to improve bone tissue engineering strategies.

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Tissue engineering has emerged as a practical approach to tackle with the prosthetic industry limitations. Its methods merge aspects from developmental biology, engineering, material sciences and medicine, with the aim to produce fully-functional bone tissue ex vivo, to further replacement or regeneration of real bone injuries and/ or defects. Traditional bone tissue engineering cell culture technique, includes the seeding of SCs in three-dimensional matrices, cultured in fed-batch rotating bioreactors, working jointly with biological cues to produce biomimics. Nonetheless, fed-batch bioprocessing has found some difficulties. Namely, mass transport limitations in the seeded scaffolds and accumulation of cellular waste, yielding poor nutrition and oxygenation of the cells. Thus producing heterogeneous distributed cell/ bone constructs. Perfusion of media has been proven to improve mass transport in the culture system, along with removing cellular waste. To overcome heterogeneity, finding the adequate cells and proper cues to drive osteogenic differentiation, are as important as the bioprocess to host the culture. hDPSCs are a promising source of stem cells for the production of bioactive biomaterial for skeletal tissue reconstruction. They lodge immunosuppressive and regenerative functions, high proliferation rates and ease in access. Their source and the subtle nature of the extraction procedure, harbour less moral concerns and variability than ESCs and most of the MSCs. In this study, the characterisation of hDPSCs as MSCs under the minimal criteria set by The International Society for Cellular Therapy was performed. Further, the osteogenic differentiation of hDPSCs encapsulated in alginate/ gelatin hydrogel subjected to suspended culture in a novel perfusion-RWV bioreactor was studied, and compared with traditional fed-batch and static culture methodologies. Finally, the effect of osteogenic cues as physiological BMP2 and simvastatin were studied to enhance the designed bioprocess. The characterisation results demonstrated that passage 4 donor hDPSCs were ideal to perform the 3D osteogenic differentiation. These cells, allowing enough production of cells while maintaining the multipotent phenotype of the parental cells under several conditions, including highly dense long-term culture. These cells were able to undergo osteogenesis in 2D and 3D. The novel high throughput perfusion-RWV bioreactor bioprocessing, demonstrated to be successful in the mitigation of nutrient and oxygen transport limitations, external to three-dimensional cell/alginate constructs, performing above fed-batch RWV bioreactor and static culture, and able to produce more homogeneous, denser and functional bone constructs, rich in mature osteoblasts and mineralising osteocytes. Both BMP2 and simvastatin, demonstrated to enhance the quality of bone constructs produced by the perfusion-RWV unit, yielding more homogeneous constructs, with higher alkaline phosphatase activity, mineralisation and showing a more mature gene pattern. Interestingly. BMP2 produced constructs rich in mature osteoblast, while simvastatin, constructs rich in osteocytes. In particular, this experiment proved effective in producing osteogenic differentiation with minimal use of BMP2, offering a potential mean to avoid dosage dependant safety risks of BMP2. In conclusion, this thesis reports the development of a novel bioprocess to produce homogeneous bone tissue constructs able to support the transfer of the 'on the bench' research, to the clinical facility.

The field of tissue engineering has the potential to improve the quality of life of individuals through combining the knowledge of engineering and life sciences in creating engineered biological substitutes that repair, support and enhance tissue function. Inkjet printing is a versatile tool that can be used for a broad range of applications. Ubiquitous in households, offices and industry, there has been growing interest in the use of inkjet printing for biological applications. Inkjet printing allows the user to deposit nano-picolitre volume of inks of low viscosity with high precision and high repeatability. Within this thesis, inkjet printing was used to explore its applications in the life sciences, with jetting behaviour and scaffold design optimised. The creation of cell-friendly scaffolds was investigated. Gelatin scaffolds, crosslinked with inkjet printed glutaraldehyde were fabricated. Fibroblasts were seeded onto these fabricated scaffolds and shown to proliferate without hindrance, allowing a method to create sub-millimetre cell-friendly fibres for tissue engineering applications. The ability for inkjet printing to create scaffolds to control cell alignment was investigated. Cell orientation can be controlled through inkjet printing paraffin wax to restrict cell proliferation on a substrate. Paraffin wax is not harmful or toxic to cells, and cells were able to grow within the negative spaces between the wax patterns, to create aligned cell culture as cells proliferated. An advantage with the wax scaffolds was that the wax scaffold was readily removable with a scalpel that allowed further analysis of cell behaviour when proliferating into an unrestricted space. A proportion of cells was also detached upon wax removal, proportional to cell density within the wax scaffold and wax channel width. After wax removal, cell cultures quickly lost their ordered appearance within 3 days as they proliferated randomly across the substrate. The creation of in vitro vasculature models through the use of a combination of inkjet-printed wax, PDMS moulding and wax-loss method to create medical phantoms for the study of rheological behaviour was studied. The scalloping behaviour of the printed wax vessel was reduced in the final phantom created, as there would be a thin lining of wax that covers the interior of the PDMS mould after wax removal, making the vessel smoother. Cell printing of neuronally relevant cells were investigated. NG108-15 and porcine Schwann cells (along with fibroblasts to act as a control experiment) were inkjet printed, studying cell viability during and after inkjet printing. It was concluded that cells were not significantly damaged during inkjet printing over a wide range of voltages (50 V-230 V), and no correlation was seen to show an increase in cell death with increasing voltages. Inkjet printed NG108-15 cells showed they produced longer neurites compared to control samples after 7 days. Further to results, it was confirmed that cell printing is limited to a duration of less than 40 minutes due to cell aggregation within the reservoir of the printing system, causing a steady significant decrease in cell numbers during printing.