The thesis describes an experimental investigation of the fluid dynamics within novel single-use bioreactors (SUBs), including stirred, rocked and pneumatically driven mixing systems. Biological studies to ascertain the impact of hydrodynamic conditions within these systems, on the growth and protein productivity of a mammalian cell line, are also presented. Two-dimensional velocity measurements within different SU technology were acquired with the use of a whole flow field laser-based technique, Particle Image Velocimetry (PIV). Fluid dynamic characteristics including velocity, turbulence, turbulent kinetic energy and vorticity were determined from time-resolved and phase-resolved velocity measurements. Commercial bioreactor systems were modified, if needed, in order to perform experiments within bioreactors commonly used for cell culture experiments, in preference to using vessel mimics. The fluid flow characteristics in both the impeller region and bulk fluid of a single-impeller stirred bioreactor were investigated, facilitating an enhanced understanding of the spatial distribution of velocity and turbulence throughout the vessel. PIV was also used to study the flow in a dual-impeller stirred bioreactor, providing a rare examination of the interaction between the flow fields generated by two impellers. The whole flow field velocity and turbulence characteristics measured within a rocked bag and pneumatically driven vessel, allow a unique insight into the flow pattern and turbulence distribution within two novel cell culture systems. Cell viability, size, growth, protein productivity and metabolites concentration were monitored under different cell culture operating conditions. Cell culture experiments, combined with the hydrodynamic information acquired using PIV, offer an insight into the physiological response of the cells to highly disparate flow conditions. This information helped to understand how the hydrodynamics induced by novel commercially used mixing systems, can impact upon a mammalian cell line. Having implications for an augmented capacity for cross-compatibility, in addition to enhanced strategies for scale translation and optimal bioreactor design.

The primary aim of this study is to develop the means to predict mechanisms and rates of material damage and especially the size of particles making up cell suspensions. Computational fluid dynamics (CFD) is employed to define experimental conditions used in ultra scale-down (USD) techniques and relate these to large-scale operations. The validity of this novel scaling methodology as applied to a large-scale membrane filtration unit and disc-stack centrifuge has been investigated. The current state of the art predicts performance of bioprocess operation but not of material damage itself. For the membrane operation, emphasis has been given mainly to flux rate and fouling, and clarification and dewatering are key parameters during centrifugation. Conversely, the effects of hydrodynamic parameters during the operation on the process material, e.g. shear damage, have not been paid much attention. Though CFD has been currently employed to facilitate predictions of the performance of these operations by a number of research groups, the technique alone cannot provide accurate predictions for a given biomaterial. CFD data thus needs to be integrated with experimental data given by other techniques, e.g. scale-down mimics of process equipment, USD device and etc. This study uses CFD and USD techniques to predict this damage. Following preliminary analysis of fluid stresses, the pump has been identified as the key component of the membrane rig responsible for material damage. CFD was then employed to determine the magnitude of maximum energy dissipation rate (EDR) and provide equivalent rotational speeds of the rotating disc device, or USD device, capable of generating the same engineering conditions. The USD experiment was then carried out following recommended speeds given by CFD and experimental data together with proposed mathematical models was used to predict particle characteristics in terms of size reduction. A verification process was finally performed using full-scale experiments. It was found that CFD could provide essentially similar operating conditions for USD experiments mimicking a large-scale operation, especially for operations of a few hours. An initial investigation has also been carried out on the applicability of the CFD-USD technology to predict material damage in the discharge part of the solid-ejecting disc stack centrifuge. The analysis shows that CFD suffered from difficulties in defining details of the problem domain in order to assess flow fields. Moreover, the exposure to gas phase during the discharge and subsequent droplet formation and an unpredictable shape of slurry made it impossible to investigate this problem further using CFD. As a result, it is concluded that this problem should be addressed by other means. Based on findings of this investigation, prediction guidelines are proposed to facilitate the design process and process development by using a small USD device to assess the engineering environment in a large unit operation. More importantly, the concept may theoretically lay the foundation for scaling any unit operation based on key engineering parameters of the system.

Molecular hydrogen (H2) is seen as an ideal replacement for fossil fuels; however the current methods of its synthesis are unsustainable and environmentally damaging. Research is therefore needed to devise new, more suitable modes of H2 derivation. Biohydrogen – i.e. H2 derived from cellular metabolism – is a particularly promising future fuel, owing to it being a truly renewable means to generate H2 that would contribute to neither pollutant nor greenhouse gas emissions. The predominant enzymes responsible for microbial H2 evolution are called hydrogenases. The model organism Escherichia coli produces H2 during mixed acid fermentation by the action of it native hydrogenases. However, the natural level of H2 produced is too low to meet current industrial demand for H2 or any future uses as a fuel. The context of the work presented in this Thesis was therefore towards the augmentation of E. coli H2 yield. This was addressed through a number of specific aims that employed synthetic biology techniques. The first aim was to engineer E. coli to heterologously express genes that lead to the biosynthesis of a foreign NADH-consuming [NiFe]-hydrogenase, native to Cupriavidus necator. It was shown that this hydrogenase was assembled in E. coli and it exhibited activity both in vitro and in vivo. Serendipitously, it was found that the native E. coli maturase system, critical for biosynthesis of the [NiFe] cofactor, could assemble a functional enzyme. The converse was also found: that the C. necator maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. A second approach to augment E. coli H2 yield utilised in this Thesis was the generation of synthetic chimeric metalloenzymes that might allow for new substrates to be used. One such chimera, consisting of a fusion between E. coli Hyd-3 and a ferredoxin from Thermotoga maritima, was characterised and shown to exhibit in vivo H2 evolution when co-produced with a heterologous pyruvate: ferredoxin oxidoreductase. This allowed pyruvate to be employed as a new electron donor for H2 production. In addition, the further characterisation of a chimeric complex combining subunits of E. coli Hyd-2 and Salmonella enterica thiosulfate reductase was undertaken. This allowed H2 production to be driven by respiratory electron donors such as glycerol 3-phosphate. An additional aim of this work was to develop an intracellular H2 biosensor in E. coli, with a view to obtaining a screening method that could be used to scan mutant libraries for increased H2 production. The strategy was to utilise the H2-sensing regulatory [NiFe]-hydrogenase (RH) from C. necator in E. coli to control reporter gene transcription. Synthetic operons were designed for optimum expression of RH-encoding genes, and the heterologous biosynthesis of the RH apparatus was established. Various reporter gene constructs were also generated. However, the system was not found to be functional, and further experiments needed to address this are discussed.

No description available.

This thesis investigates the development of sensing devices based on molecularly imprinted polymers for the detection of clinically relevant analytes. Three analytes were considered, metronidazole, creatinine and propofol. A molecularly imprinted polymer (MIP) was computationally designed for metronidazole and tested using SPE techniques. This polymer was then grafted onto a transducer surface using an immobilised initiator. Amperometric and impedance detection of metronidazole were investigated. The capacitive detection of creatinine was reproduced from the literature (Panasyuk- Delaney et al., 2002) as this approach could be applied to other MIPs to form a universal platform for sensor development. However, the sensors produced using this methodology were difficult to reproduce and attempts to improve them were unsuccessful. A model for capacitive electrodes was developed to explain the obtained results. To address the key challenges found in the aforementioned work, a dual polymerisable monomer was used as a conductive anchor for the amperometric and impedance detection of propofol. The developed amperometric sensors demonstrated very high sensitivity (limit of detection was below 5 µM), although the electrodes lacked in selectivity. In conclusion, this thesis illustrates some of the key areas which need to be considered in the development of MIP-based devices and investigates some innovative solutions to these problems.

Electrochemical biosensors have been made to detect the metabolic markers, pH, O2 and glucose, and nitric oxide (NO), the signalling molecule involved in angiogenesis. A novel three-dimensional (3-D) graphene/ionic liquid (IL) nanocomposite demonstrates highly sensitive detection of NO in phosphate buffered saline (PBS). An arginine-glycine-aspartic acid (RGD) peptide-functionalised biomimetic graphene film has been used as both a cell culture and sensing matrix to detect NO released by human umbilical vein endothelial cells (HUVECs) in real-time under acetylcholine (Ach) stimulation. The amount of NO released is dose-dependent and inhibited by NG-nitro-L-arginine methyl ester (L-NAME). A poly(ethyleneimine) (PEI)-coated anodically electrodeposited iridium oxide film (AEIROF) exhibiting super-Nernstian response to pH functions as miniature pH sensor for detecting acute changes in extracellular pH due to the interaction of porcine aortic ECs (PAECs) with fibronectin and thrombin. Thrombin causes dissolution of fibronectin, extracellular acidification of PAECs and a change in cell morphology from stretched to round cells. O2 and glucose biosensors based on a novel electropolymerised redox polymer are developed and prepared by one-step electropolymerisation of methylene blue (MB+) and pyrrole for the O2 biosensor, with the addition of glucose oxidase (GOD) for the glucose biosensor. The O2 biosensor demonstrates superior sensitivity towards dissolved O2 at atmospheric (atm) O2 concentrations and below and is insensitive to pH. The glucose biosensor exhibits direct electron transfer (DET) and is insensitive to pH from pH 6 to 8 in N2-purged PBS and from pH 4 to 8 in atm O2 PBS.

The aim of this thesis is to determine shoulder function, during specific functional tasks and relate this to key parameters associated with pathology. The long-term aim is that the results of this work will influence rehabilitating the shoulder after surgery, improving clinical assessment or preventing specific upper limb injuries. For this, initial studies are required to design and build a simulator to quantify upper limb function during a set of functional tasks, and to quantify upper limb anthropometrics and combine these with a musculoskeletal model. The first part of the thesis focuses on anthropometry. Three different methods are studied for defining anthropometrics. First, a modified regression equation data set is introduced; this can be used for calculating body segment parameters considering a subject’s body mass, height, race, gender and age. The new regressions are compared to cadaveric data from the literature and found to improve the moment of inertia calculations. Two different geometrical modelling approaches are also introduced. This found that the geometrical solid shape representing the body segments can lead to noticeable differences in the body segment parameter results. Finally a laser scanner device is developed and applied to measuring these parameters. A mannequin’s upper arm is scanned and its volume found from the 3D image is compared to the actual one giving an average difference of 3.1%.; in addition, a standard-sized object was scanned allowing the validation of the scanning method for calculating body segment parameters. Finally, these different approaches are analysed and applied to a large set of subjects. This then provides key information for the second part of the thesis. The second part then focuses on the muscle forces in functional activities. Six functional daily activities are used in this study. In addition, a driving simulator is designed in order to quantify kinematics, kinetics and external forces during steering at different conditions and postures. At the same time, a computational musculoskeletal model of upper limb is used for measuring the muscle forces during the six functional tasks. The methodology used and the results of muscle forces in functional activities are presented and analysed; from the literature it is found that the failure strengths for repairs of supraspinatus are close to the muscle forces predicted in this study (224 ± 148 N). Finally, the results of this study could help to improve ergonomics for cars, such as driving wheel and car-sits, and inform return to activity recommendations after upper limb surgery to specific muscles. Keywords: Musculoskeletal biomechanics, upper limb, musculoskeletal modelling, anthropometry, body segment parameters, geometrical modelling, regression analysis, 3D laser surface scanner, functional activities, activities of daily living, driving simulator for steering, shoulder functionality on driving, joint forces, joint stability, muscle forces.

Glaucoma is the second most common cause of blindness worldwide and is often associated with an increased intraocular pressure (IOP). IOP is determined by the dynamics of aqueous humour, the liquid filling the anterior segment of the eye. In primary-open angle glaucoma, the elevated IOP is caused by a decreased outflow facility of aqueous humour through the conventional pathway (decreased conventional facility, C). Existing glaucoma therapies aim to lower IOP, but remain inefficient because they fail to target C. As a result, there is growing interest in using the mouse to unravel the mechanisms controlling C. The mouse is a particularly powerful model because it can be routinely manipulated genetically, thereby giving insight into molecules and genes involved in determining C. However, it is still not clear whether mice are suitable surrogates for studying human C. To fill this gap, we aim to demonstrate that the mouse is a suitable model for human IOP regulation, and to then use this model to investigate key processes in IOP regulation. To achieve this aim we used an existing perfusion system to measure C in enucleated mouse eyes. First, we improved the perfusion system by including hydration and temperature control to better mimic in vivo perfusions. Secondly, selected receptor-mediated drugs were found to have similar effects on C in mice as they did in past human studies. Finally, we show, amongst other studies, anti-metabolic agents decreased C, suggesting aqueous humour outflow is metabolic dependent. We conclude that the mouse is a valid model for studying human conventional facility, yielding novel insight into the mechanisms controlling conventional facility. Importantly, this will help the design of novel efficient anti-glaucoma treatments. Notably, this project will have brought fundamental insight into mouse eye perfusions, thereby consolidating the technique for future studies.

This thesis addresses changes in the supraspinatus muscle and tendon architecture, the relationship to fat infiltration and the effect of tear propagation using magnetic resonance imaging (MRI) and functional biomechanics testing using human tissues. The first hypothesis tests the relationship between the anterior and posterior portions of the supraspinatus and the central tendon when normal with no tear (NT), and pathological full thickness tears (FTT) groups. The changes in the pennation angles and central tendon associated with a FTT and the magnitude of the tear size were all statistically significant. The central tendon was found to lie anterior to the long axis of the supraspinous fossa as it passed laterally towards its insertion in the NT group. This relationship was reversed in the FTT group with the tendon lying more posteriorly or in the long axis. The second study hypothesis was that the degree of fatty infiltration of the supraspinatus is positively correlated to the maximal degree of central tendon retraction (CTR) from its insertion seen on the same MRI. The results found this relationship to be statistically significant. The aims of the cadaveric study were to establish the influence on abduction moments of full thickness tears with specific reference to tears to the central tendon. A new method of testing the biomechanics of in-vitro rotator cuff tears was developed through specimen-specific loading protocols through the use of a musculoskeletal dynamics model. A pair-wise comparison of the sections then revealed that the sectioning of the central tendon, regardless of whether the tear starts anteriorly or posteriorly, does the most significant damage to the moment producing capacity of supraspinatus. The overall contribution of this thesis is a clear understanding of the functional biomechanics of the central tendon of the supraspinatus in rotator cuff tears.

The thesis presents targeted sensor-based devices and methods for the training and assessment of upper extremity. These systems are all passive (non-actuated) thus intrinsically safe for (semi) independent use. An isometric assessment system is first presented, which uses a handle fixed on a force/torque sensor to investigate the force signal parameters and their relation to functional disability scales. The results from multiple sclerosis and healthy populations establish relation of isometric control and strength measures, its dependence on direction and how they are related to functional scales. The dissertation then introduces the novel platform MIMATE, Multimodal Interactive Motor Assessment and Training Environment, which is a wireless embedded platform for designing systems for training and assessing sensorimotor behaviour. MIMATE's potential for designing clinically useful neurorehabilitation systems was demonstrated in a rehabilitation technology course. Based on MIMATE, intelligent objects (IObjects) are presented, which can measure position and force during training and assessing of manipulation tasks relevant to activities of daily living. A preliminary study with an IObject exhibits potential metrics and techniques that can be used to assess motor performance during fine manipulation tasks. The IObjects are part of the SITAR system, which is a novel sensor-based platform based on a force sensitive touchscreen and IObjects. It is used for training and assessment of sensorimotor deficits by focusing on meaningful functional tasks. Pilot assessment study with SITAR indicated a significant difference in performance of stroke and healthy populations during different sensorimotor tasks. Finally the thesis presents LOBSTER, a low cost, portable, bimanual self-trainer for exercising hand opening/closing, wrist flexion/extension or pronation/supination. The major novelty of the system relies on exploiting the movement of the unaffected limb to train the affected limb, making it safe for independent use. Study with LOBSTER will determine its usability for home based use.