The blowfly Calliphora is one of the most sophisticated fliers in the animal kingdom. It displays a broad repertoire of visually guided behaviours that can readily be quantified, including gaze and flight stabilization reflexes, male chasing flights, collision avoidance and landing responses. The fly achieves such robust visuo-motor control tasks based on a comparatively simple nervous system that is highly accessible for electrophysiological recordings. The ability to investigate the fly’s performance at the behavioural and electrophysiology levels makes this animal an ideal model system to study closed-loop visual motor control. The aim of this thesis was to develop and characterize the dynamics of a fly-robot interface (FRI) while a fly performs a closed-loop visual stabilization task. A novel experimental setup involving a FRI was developed which allowed for simultaneous measurements of neural activity from the fly and the behavioural performance of the robot. In the setup, the neural activity of an identified visual interneuron, the H1 cell, was recorded and its action potentials were used to control the motion of a mobile robot that was free to rotate along its vertical axis. External visual perturbations were introduced into the closed-loop system through a rotating turn-table with the robot using the neural activity to counter-rotate and to minimize the observed visual motion. The closed-loop control delay of the FRI was 50 ms which is well within the range of visual response delays observed in fly behaviour. With the FRI, the closed-loop dynamics of a static-gain proportional controller were characterized. The results explain significant oscillations in the closed-loop responses as a possible consequence of a high controller gain which were also observed but never fully interpreted in previous behavioural studies. Varying the controller gain also offers competing control benefits to the fly, with different gains maximizing performance for different input frequency ranges and thus different behavioural tasks. Results with the proportional controller indicate the dependence of the FRI frequency response on the angular acceleration of visual motion. An adaptive controller designed to dynamically scale the feedback gain was found to increase the bandwidth of the frequency response when compared with the static-gain proportional controller. The image velocities observed under closed-loop conditions using the proportional and the adaptive controllers were correlated with the spiking activity of the H1-cell. A remarkable qualitative similarity was found between the response dynamics of the cell under closed-loop conditions with those obtained in previous open-loop experiments. Specifically, (i) the peak spike rate decreased when the mean image velocity was increased, (ii) the relationship between spike rate and image velocity was dependant on the standard deviation of the image velocities suggesting adaptive scaling of the cell’s signalling range, and (iii) the cell’s gain decreased linearly with increasing image accelerations. Despite the fact that several sensory modalities - including the motion vision pathway - process information in a non-linear fashion signal integration at stages one to two synapses away from the motor systems and the behavioural output itself have been shown to be linear. Quantifying the closed-loop dynamics of visuo-motor control at both the behavioural and neuronal level, may provide a starting point to discover the neural mechanisms underlying an appropriate combination of complementary non-linear processes which ultimately result in a linear performance of the overall system.

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Parkinson’s disease (PD) is a debilitating neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Human pluripotent stem cells (hPSCs) are capable of generating any cell from the body, including dopaminergic neurons, so are an ideal source of material for a cell replacement therapy for PD. Hypoxia is a cell culture tool used to recapitulate the oxygen tension of the in vivo environment. Atmospheric oxygen is approximately 20 % O2, which is significantly greater than the in vivo environment of hPSCs and dopaminergic neurons. Studies in human foetal neural progenitor cells (NPCs) demonstrate that hypoxia increases the yield of dopaminergic neurons. Therefore, this thesis examines the ability of hypoxia to enhance the yield of dopaminergic neurons from hPSCs and hPSC-derived NPCs. The work of this thesis begins by validating the hypoxic (2 % O2) model and the use of mouse embryonic fibroblasts as a substrate for hPSCs, and then progresses to generate a scalable protocol for the production of dopaminergic neurons from hPSCs. The protocol uses a free floating method to generate a greater number of NPCs per cm2, with the basic protocol producing 16.67 % FOXA2/TH dual positive cells. Methods to increase the yield of the protocol through the use of hypoxia and lentiviral vectors are then assessed. The final chapter assesses how hypoxia might affect canonical Wnt signalling, which is heavily involved in neural development and differentiation. The key findings from this thesis are that an LMX1A-encoding lentivirus causes a reduction in FOXA2 in differentiated neurons, whilst canonical Wnt agonist CHIR99021 causes a decrease in FOXA2 and LMX1A in expanded and differentiated neurospheres, compromising the yield of dopaminergic neurons. However, hypoxia is able to improve the yield of FOXA2/TH dual positive neurons from normoxic mature neurospheres to 23.27 %, which is comparable to recently published monolayer differentiation techniques.

As more complex biotherapeutics are produced, the numbers of antibodies exhibiting aggregation phenomenon has increased greatly. It is therefore of growing importance to understand the products and origin of these phenomena and to be able to select candidates that show the greatest stability. The purpose of this work was to assess different methods for determining protein stability and the aspects of stability they measure, analysing the different aggregate species produced to offer a platform solution when dealing with different aggregate phenomenon during process development. The effect of reversible self association (RSA) on the purification of a product was evaluated and shown to only critically effect the operation of viral filtration steps in a typical bioprocess through blockage of filter pores. A custom made, rotating disc, interfacial shear device was evaluated along with thermal, spectroscopic and molecular modelling methods for their ability to determine the relative stabilities of antibodies to aggregation. A capillary interfacial shear device with 10 fold reduced volume was designed and tested, showing comparability of monomer loss in the capillary with the coefficient of monomer decay in the disc device. This surface related damage was further studied by comparison to thermal methods with a range of known modifications to IgG structures, using modelling techniques to indicate aspects of protein structure key to loss of stability. The interfacial shear device provides an orthogonal measurement related to modification of exposed protein residues whereas thermal techniques trend with intramolecular stability. It was also concluded that for full characterisation of an aggregate profile SE-HPLC, Nanosight and Microflow Imaging should be used to enable capture of the entire size range of aggregate species from 10nm to 100μm. The work highlights the future prominence of molecular modelling techniques as part of a fully integrated aggregate mitigating solution to determine aggregation hot spots.

Polysaccharide conjugate vaccines are typically comprised of several different polysaccharides produced with distinct and complex production processes. Clarification and primary recovery operations, such as particle conditioning are integral to purification. Efficient process development of these two unit operations has been constrained by lab-scale models that require large volumes and considerable time to evaluate. A modular approach to develop rapidly purification processes for polysaccharides at the micro-scale would greatly enhance productivity and speed the development of novel conjugate vaccines. To enable high throughput screening, a suite of high throughput analytics was assembled for polysaccharide-containing feedstreams. Three orthogonal generic assays were developed for the high throughput determination of product titre. Assays for clarity and impurity determination were also optimized. The final comprehensive suite of high throughput analytics was qualified for analysis of product titre, product quality, impurity clearance, clarification efficiency, and particle size characterisation. We developed a novel system for high throughput particle conditioning. With this system, 96 individual reaction conditions can be evaluated in parallel, including downstream centrifugal clarification. The scalability of particle conditioning was evaluated between USD reactors with less than 1 mL of volume up to Pilot-scale reactors of 14 L for several biological feedstreams derived from three host species. Product yield, impurity clearance, and product quality were comparable between scales. An engineering characterisation of the reactors was performed and several approaches for scaling particle conditioning processes were evaluated. To support this platform, a custom-built, modular system for depth filtration of eight conditions/filters in parallel was developed using 0.2 cm2 area of media per channel. The reproducibility and scalability of this technology was demonstrated with several filtration media. With this integrated approach to particle conditioning and depth filtration, we have shortened the process development time. The engineering scale up models developed in this thesis will facilitate the creation of more robust processes at pilot and commercial scales of operation.

Cardiovascular disease is a worldwide problem as the number one cause of death and disease. Although improvements in pharmaceutical and surgical interventions have increased patient survival post infarction, approximately half those that recover have a progress to congestive heart failure. Stem cell therapy is a strategy to improve functional recovery after myocardial injury. Many early phase clinical trials have been conducted throughout the world, albeit with variable results. It is postulated that the transplanted cells are not retained within the peri-infarct region and either apoptose or migrate to the spleen. This thesis aims to understand the functional characteristics of bone marrow mononuclear cells (BM-MNC) and Mesenchymal Stem Cells to characterise their adhesion and migration responses that are essential for successful cell retention. In vitro model assays were developed to mimic the physiological environment of the peri-infarct region using hypoxic conditions and fibronectin, an extracellular matrix that is highly upregulated in the infarct region. In vivo rat studies have shown Stromal Derived Factor-1 (SDF-1) stimulated MSCs improved cardiac function post infarction. In this study SDF-1 stimulation had no effect on the in-vitro adhesion and chemotaxis of MSCs and BM-MNCs. Both cell types had significantly decreased adhesion to fibronectin in hypoxic conditions To investigate the importance of Neuropilin-1 (NRP1) MSCs were knockdown for the protein. NRP1 knocked down MSCs had significantly decreased adhesion to fibronectin, chemotaxis to PDGF-AA, and interactions with endothelial cells. Expression of NRP1 was enhanced by basic fibroblast growth factor that enhanced adhesion in hypoxic conditions and increased vascular endothelial growth factor release.