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Dystroglycan is central to the dystrophin-associated glycoprotein complex (DGC), which helps provide stability to muscle fibres. The absence of dystrophin in Duchenne muscular dystrophy results in the loss of this complex. The phosphorylation of β-dystroglycan is thought to play an important role in controlling the integrity of the DGC. This phosphorylation event has been shown to promote β-dystroglycan internalisation, possibly as a prerequisite to degradation. The work presented in this thesis aimed to use the zebrafish to investigate new therapeutic approaches to restore dystroglycan to the membrane and assess the extent to which muscle attachments can be strengthened in the dystrophin mutant zebrafish (sapje). As in mammals, the stability of the zebrafish DGC is dependent on dystrophin expression. As such, β-dystroglycan levels in sapje decreased in an age-dependent manner. Loss of dystrophin led to an initial elevation in phosphorylated β-dystroglycan levels. Treatment of sapje fish with proteasome and kinase inhibitors was able to prevent or slow the progression of dystrophy, in a dose-dependent manner. This was associated with a concomitant increase in dystroglycan and decrease in phosphorylated dystroglycan. This work provides insight into the molecular mechanisms that lead to the loss of dystroglycan from the membrane in dystrophic muscles, and may have therapeutic implications in the future.

Recent evidence has shown that adult neurogenesis is sustained in the hypothalamus, a region of the ventral diencephalon that is the central regulator of homeostasis. While studies support a role for adult neurogenesis in energy balance, as yet, the identity of the neural stem/progenitor cell niche remains contested. Tanycyte cells, a unique population to the hypothalamus, present a possible candidate due to their diverse roles, radial-glial like morphology and position adjacent to the 3rd ventricle. Here, I provide in-vivo, in-vitro and ex-vivo data that together support alpha-tanycytes as a neural stem/progenitor cell population. My studies show that the embryonic neural stem/progenitor characteristics of radial glia, including expression profile, a basal process and an apical primary cilium, are maintained in alpha-tanycytes during adulthood. In addition, alphatanycytes are multipotent in-vivo and contribute to the other tanycyte populations, suggesting a lineage relationship of cells within the hypothalamic ventricular zone. A neurosphere assay adds further validity to the idea that there is heterogeneity in progenitor status within tanycyte subpopulations. Furthermore, alpha-tanycytes are responsive to Fgf-signalling in-vivo, a crucial regulator of proliferation and differentiation during embryogenesis, as well as being required for neurosphere formation. In order to further interrogate alpha-tanycytes, I developed and optimised an organotypic slice culture protocol, a technique that has not yet been used to study hypothalamic neural stem/progenitor cell dynamics. This ex-vivo technique provides a number of advantages including efficiency, low-cost, and amenability to manipulation, while maintaining large parts of the niche. Exogenous addition of pharmacological agonists and inhibitors reveals that alpha-tanycytes undergo Fgf-dependent proliferation in response to physiological stimulation, and implicates a role for the hypothalamic niche in the homeostatic control of stress. Together, these studies characterise the component cells of the adult hypothalamic neural stem/progenitor cell niche, providing a framework for future research to further explore the heterogeneity and physiological significance of alpha-tanycytes.

Laminin alpha 1 (LAMA1) is a major component of the earliest basement membranes in the mammalian embryo. Disruption of the murine Lama1 gene result in lethal failure of germ layer differentiation and extraembryonic membrane formation at gastrulation stages, while conditional deletion of Lama1 leads to aberrant organization of retinal neurons and vasculature, and defects in cerebellar glia and granule cell precursors later in development. Similarly, inactivation of lama1 in zebrafish affects lens, retina and anterior notochord development. This diverse range of phenotypes in Lama1-deficient animals reflects the complexity of its expression pattern during embryogenesis, which is largely conserved among vertebrates. Major sites of Lama1 transcription in the mouse embryo are the neural tube, presomitic mesoderm, somites, nephrogenic mesoderm, head mesenchyme and the lens. However, little is known about the signaling mechanisms governing the spatio-temporal control of Lama1 transcription. Previous studies in our lab revealed a requirement for SHH signaling in the transcription of Lama1 in the somites and neural tube of mouse embryos. Therefore, I hypothesized that SHH might directly modulate Lama1 expression via the binding of GLI transcription factors to regulatory regions in the Lama1 locus. In this study, I identified a cis-regulatory element that may be involved in the SHH-dependent control of Lama1 expression in the murine embryo. I began my study with a phylogenetic footprinting approach that uncovered 25 conserved non-coding elements upstream of the murine Lama1 locus, some of which contained GLI binding motifs. Subsequent luciferase reporter-based analysis in cell culture with a subset of the CNEs did not provide convincing evidence for enhancer- and/or silencer-like properties of the elements, except for CNE7. The CNEs were further characterised using an in vivo transgenesis reporter screen in zebrafish, which uncovered a skeletal-muscle specific regulatory region. In parallel, a detailed survey of the existing literature revealed the presence of a non-conserved GLI-occupied region in intron 1 of the murine Lama1 gene. Subsequently, I showed that this element behaves as a tissue-specific enhancer driving reporter expression in the neural tube of mouse and zebrafish embryos. I provided evidence that active Hh signaling is required and sufficient for the activity of this enhancer. Finally, I demonstrated that the GLI binding motifs within the element are essential for its function. Altogether, these results suggest that SHH may directly control Lama1 transcription in the mouse neural tube via an intronic enhancer, and also provide further insight in the relationship between cell signaling and the regulated expression of extracellular matrix components in development and disease.

Planar polarity describes the ability of cells to orient themselves in the plane of a tissue. This process is partly mediated by the six core planar polarity proteins: Flamingo, Frizzled, Strabismus, Prickle, Dishevelled and Diego. These proteins localise in an asymmetric, punctate manner at apicolateral cell junctions. Strabismus and Prickle are found on the proximal side of the cell, Frizzled, Dishevelled and Diego on the distal side, and Flamingo on both sides. All six core proteins are required for planar polarity, but the molecular mechanism of the establishment of planar polarity has not been identified. Several lines of evidence suggest that the core proteins form an asymmetric complex across cell membranes. The puncta observed are therefore hypothesised to represent clusters of core complexes with the same polarity. Elucidating the stoichiometry of the putative core complex will provide insights into the core complex and the mechanisms of planar polarity propagation. The simplest possible core complex model that fits the existing data is two Flamingo molecules, with one molecule each of Frizzled, Strabismus, Prickle, Dishevelled and Diego. EGFP-tagged core protein constructs were generated for in vivo expression at endogenous levels, as changes in protein level could potentially affect the stoichiometry. Images of these fusion proteins in the live Drosophila melanogaster pupal wing were gathered by confocal microscopy. A custom method was then developed to detect the puncta in the images and quantify their brightness. The relative stoichiometries of the core proteins in puncta was determined at 28 hours after pupation, when core protein localisation is strongly asymmetric, and also at 20 hours after pupation, when no asymmetry is visible. The stoichiometry of the core proteins in puncta is more complex than the simple initial model. The data additionally reveal an interesting distinction in behaviour between the transmembrane and cytoplasmic core proteins.

In bone tissue engineering, one of the main challenges is to fabricate scaffolds that promote and support osseous reconstruction. The research reported in this thesis considers the use of apatite-wollastonite (A-W) as a bone scaffold. A variety of scaffold fabrication techniques, all based on initially processing powder to create a pre-form for subsequent sintering, have been developed and characterised for their ability to create microporous; and interconnected macroporous scaffolds. A range of powder processing techniques were used: pelleting, dry powder and slurry moulding, and different powder particle size ranges were assessed for the preparation of microporous scaffolds to influence their surface roughness without significantly varying the porosity. To introduce macroporosity within the ceramic scaffolds that would be comparable in terms of scale and organisation to trabecular bone, a variety of methods were employed. Burning off negative templates such as polymeric particles, filaments and fused deposition modelling 3D constructs was investigated, together with a novel method based on thermally induced phase separation (TIPS), freeze-drying and sintering. Selected microporous scaffolds with different surface topography and pore size; and highly interconnected scaffolds with porosity >80% were fabricated with height 2± 0.1 mm and diameter 8- 10 mm depending on the fabrication method and the particle range that was used. The parameters that were evaluated in vitro were the effect of variable topographies on microporous constructs and the influence of high porosity on cell adhesion, proliferation and cell differentiation. It is concluded that the surface area of A-W scaffolds affects their bioactivity, degradation and mechanical properties. Microporous scaffolds with smaller pores allow cell-cell interaction and promote osteogenesis. Further investigation is required to clarify the observed chondrogenesis that occurred when MSCs were cultured on microporous scaffolds with larger pores. Highly porous A-W scaffolds allow cell infiltration, migration and demonstrate signs of osteochondral lineage differentiation.

The research described in this thesis concerns the investigation of technologies for the molecular engineering of the biosensor interface. Two avenues of investigation have been explored: the use of polymer matrices to modify the properties and functions of optical biosensor interfaces and the conjugation of photochromic dyes to protein systems to achieve photomodulation of protein function for biosensor applications. A comparison of the industry standard polymer, carboxymethyl dextran (CMD) was made against carboxymethyl cellulose and mixed systems, including a novel synthetic polymer, carboxylated polynoxylin. While CMD was found to provide the highest surface loading of protein, mixed polymers demonstrated the ability to allow prediction of surface loading, and showed features such as improved resistance to biological degradation. A novel method of depositing interfaces was investigated, allied to a study of liquid handling methods. A system was developed that allowed a printed heterogeneous array to be produced which showed preferential binding of specific analytes to defined areas of the sensor, whilst the other printed arrays retaining a high degree of non-specific interaction. The use of photochromic dyes to modulate protein function was applied to glucose oxidase and horseradish peroxidase. From the initial results, a hypothesis regarding the mechanism of photomodulation and its effect concerning the molecular weight of the conjugated protein was proposed. This was examined by the photomodulation of members of the peroxidase super family, antibodies and Fab fragments. From these results, the hypothesis was proved to be correct but incomplete, and was modified to include the disruption of the hydration shell around the protein caused by photochromic switching. Further research directly related to these experiments, and in novel fields of investigation have been proposed.

The medical device industry requires multi-disciplinary collaboration between researchers and physicians (Freudenthal et al. 2011). In responding to the challenges associated with medical device development, Ogrodnick (2013) proposed a collaborative model in which design teams have a/synchronous access to a repository of information regarding the progress of the development of a medical device. This research investigates the nature of interactions between manufacturers and physicians throughout the process of medical device development to facilitate more effective practice. A literature review explored the history of medical devices; design and development processes; overall approaches to design and case studies involving healthcare practitioners coupled with collaborative processes. From this detailed review, three gaps in knowledge were identified: industrial design practice in medical device development is not well described; the models and frameworks of user involvement were unverified; user involvement was based on formal agency requirements. Empirical studies were undertaken to identify the state of the art in medical device development and opinions of physician users. These studies were conducted to identify the practices, opinions and influences for collaborative medical device design. The industry study consisted of a multiple case study research design and was used to elicit the practices of 18 major medical device manufacturers. Industry participants were selected on the basis of meeting specific criteria in location, size, type of device manufactured and use of industrial design. Interviews with practicing physicians were conducted to gain insights. The responses from the participants were analyzed using NVivo software, card sorting and data visualization to identify routes to more effective engagement in collaboration during medical device design. The findings indicated that there were seven issues ideas priorities for collaborative practice. These included: user integration throughout the development process; the negotiation/ownership of intellectual property; the knowledge of impact to device design; consistent communication between device developers and physician users; timeliness and efficiencies of interactions; the identification/connection of partners; and meeting legal requirements of healthcare laws. These issues were translated into design requirements and six potential tools for cMDD. Using a Pugh matrix, each concept was evaluated against the developed requirements. As a result, the comprehensive computer application concept, which addressed the maximum number of issues, was selected and further developed for the purposes of validation. The navigation and graphic design was completed and a video, which justified the purpose and explained the software, was produced. To evaluate the concept, seven reviewers consisting of both industry representatives and physicians, whom had no previous knowledge of the research project, evaluated the video with positive responses, further potential uses for the software and suggested improvements. This research concludes that within the current regulated process of development there was flexibility in the application of design control during the conceptual phase and that the majority of developers followed a user driven approach to design. Industrial design was responsible for aesthetic design but limited in impact due to a lack of training in clinical science. Physician users are involved however lack knowledge of development process requirements and their impact. Further that there are barriers to collaboration that prevent consistent and valued interactions. Finally, the research resulted in a confirmed app-based tool that would support the promotion of cMDD.

Brain Machine Interface (BMI) or Brain Computer Interface (BCI) technologies provide the prospect of regenerating or replacing functions lost due to motor disabilities. BCIs connect the brain to a computer which translates the electrical activity of the brain into commands used to control external devices, hence allowing people with motor disability to control their external environment through a non-muscular communication channel. A BCI operates by transforming electrophysiological signals, known as Electroencephalogram (EEG) signals, from the user into device commands under an operating protocol. The protocol initialises and defines the nature of the communication (i.e., discrete or continuous). It also determines the strategy which underpins the generation of the signals used by the system (i.e., what triggers the changes within the EEG signals). While protocols involving brisk and very constrained movements have been widely explored in BCI studies, more natural movements have barely been considered. The choice of protocols that entail non-realistic movements emanates from the generation of well understood neuronal correlates modulated by the execution of such movements, leading to a lack of freedom in the design of BCI protocols. The development of algorithms translating EEG signals into commands that control external devices,a task termed as event detection in the present research, are a central part of any BCI system. However, most of the time, complex methods are used and most event detection in BCI development is devoted to the optimisation of such methods. Furthermore, the methods require extensive training of the user, putting a mental load on the user. The present study aims to investigate simple, but powerful, event detection methods requiring minimal training and the use of a protocol involving natural movements. Scalp EEG data was recorded from nine participants using natural hand movements. In particular, self-paced reaching hand movements were considered. The data was investigated in a pseudo time frequency domain using continuous wavelet coefficients. Methods using wavelet modulus maxima, the Mahalanobis distance, and bootstrapping of the Mahalanobis distance were developed for event detection. The data was analysed over a frequency range from 0.1 Hz to 25 Hz, covering the Slow Cortical Potentials (SCP), Mu and Beta frequency bands. The results showed that the method using wavelet modulus maxima was able to predict reaching hand movements onset in the SCP, Mu and Beta frequency bands about 1 s before movement onsetand yielded an maximum average prediction rate of approximately 80%. The Mahalanolobisdistance and the bootstrap methods were able to predict reaching hand movements initiation inthe SCP band about 1 s before movement onset with a maximum prediction rate of approximately 70%. The study has demonstrated that human voluntary movements can be predicted approximatively 1 s prior to movement onset with a good prediction rate. The study may contribute to the understanding of the planning and the control of human voluntary movements. Furthermore, the present research may contribute in designing advanced assistive devices in general and in particular may contribute in improving BCI systems design. Finally, the results may encourage the use of natural movements during BCI protocols design aiming to predict movement initiation and the monitoring of the mental state of BCI users.

There is a clear clinical need for small diameter blood vessel grafts. Previous studies have shown that decellularised porcine arteries have potential for future development and clinical translation. However, in order to overcome the problems of thrombogenesis and encourage endothelialisation in small diameter applications, it will be necessary to devise innovative approaches. In this study it was hypothesised that a bioactive peptide could be self-assembled within the decellularised tissue to overcome the problems of thrombogenesis and to aid and enhance re-endothelialisation. A method for self-assembling the tape forming peptide, P11-4 within decellularised tissues was developed. The study then went on to explore the P11 series of peptides as materials for tissue engineering by examining biocompatibility and haemocompatibility and demonstrated the use of self-assembled peptide coatings to prevent thrombus formation and enhance re-endothelialisation. The self-assembly of peptide P11-4 within decellularised porcine internal carotid artery was assessed using a range of microscopic and spectroscopic techniques. Fluorescent microscopy was used to show the penetration of the peptide throughout the decellularised conduit. Self-assembly of the peptide was assessed by FTIR spectroscopy. Using CLSM and MPLSM it was shown that the peptide self-assembled around the extracellular matrix of the acellular tissue. Fluorescent microscopy was used in conjunction with a specially designed flow cell to show that the peptide coating remained in the decellularised vessel for over 14 days under model flow conditions. The biocompatibility and haemocompatibility of a library of 43 peptides was assessed to identify ideal candidate peptides for use and to develop design characteristics for the application of self-assembling peptides in biomedical settings. Testing was carried out using cytotoxicity testing, the Chandler loop thrombosis model, a haemolysis assay and a complement inhibition assay. The results showed that large poly-cationic peptides were non-bio or haemo compatible, large neutral peptides enhanced thrombosis formation and that poly-anionic peptides with hydrophobic cores inhibited the complement system. Peptide coatings of P11-4, P11-8 and P11-12 were shown to decrease, and in the case of P11-12 prevent, thrombus formation; showing potential for application in small diameter acellular blood vessels. Peptide P11-4, functionalised with cyclic RGD, was shown to enhance the attachment and retention of ovine endothelial cells on the decellularised vessel, demonstrating the potential of functionalised peptide to enhance re-endothelialisation. In conclusion, this study has demonstrated the potential of self assembling peptide technology for improving the function of acellular porcine arteries in vitro.