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Deafness is a major sensory disorder affecting an enormous number of people worldwide. Loss of the auditory hair cells and their associated spiral ganglion neurons in the cochlea causes the vast majority of hearing loss cases. Therapies currently available are unable to address the root cause of the impairment, and as such are limited in their effectiveness. The field of regenerative medicine is emerging as a viable option for restoring and repairing lost or damaged cells in the inner ear. Human embryonic stem cells have been previously used to generate otic progenitors by employing ligands involved in otic placode induction, FGF 3 and FGF 10. Canonical Wnt signalling is known to play a major role in the expansion of the otic placode. In this work, I have manipulated the canonical Wnt signalling pathway in combination with FGF and explored their effect on otic progenitor differentiation. An initial period of FGF signalling combined with canonical Wnt inhibition was required to promote an ectodermal identity in the differentiating human embryonic stem cells, with a concomitant increase in the expression of otic markers. Following this, a phase of canonical Wnt activation with an attenuated FGF signal further expanded the proportion of otic progenitors, substantially more so than in the standard FGF based protocol. The progenitors derived from this more efficient, developmentally informed protocol also possessed the ability to further differentiate into hair cell-like cells and in particular, auditory neurons. Reporter lines were also generated in order to monitor otic and hair cell differentiation in real time and visualise the effects of manipulating the canonical Wnt signalling pathway with molecular compounds. This work involved the development of an updated in vitro protocol capable of readily and efficiently inducing the differentiation of otic progenitors from human embryonic stem cells. This could be beneficial in the development of potential cell-based therapies for deafness.

Cellular communication and the link between the extracellular and intracellular environments are in part mediated by signalling events from plasma membrane proteins. Their tight regulation by a diverse array of post-translational modifications (PTM) is essential to maintain the cellular integrity. Understanding these modifications would help with understanding cancer cell progression. Dystroglycan is one of the plasma membrane proteins with both structural and signalling properties, and is a core component of the dystrophin associated protein complex (DAPC). The nuclear localisation of dystroglycan and some DAPC members suggests similar roles to those observed in the plasma membrane. Dystroglycan is composed of an alpha and beta subunits that are modified by different PTM. The altered glycosylation of alpha and the phosphorylation of Y890 in beta, are triggering factors for the instability of dystroglycan leading to the disruption of the DAP complex. In cancer studies, it has recently been indicated that in addition to the above-mentioned PTM, other signalling events such as additional phosphorylation sites and ubiquitination, could mediate the rapid turnover of dystroglycan from the plasma membrane and from the nucleus. Here we show that beta-dystroglycan is susceptible to additional phosphorylation, and we were able to demonstrate its multiple ubiquitination. Further experiments revealed that beta-dystroglycan is subject to intramembrane proteolytic events induced by increased cellular density and PDBu treatment. Interestingly, we observed the translocation of beta- dystroglycan to the nucleus due to high cell density growth. By enzymatic inhibition studies we were able to identify gamma-secretase and furin as the enzymes responsible for the shedding of beta-dystroglycan from both the plasma membrane and nuclear envelope. Interestingly, we observed the preferential nuclear translocation and degradation by the proteasome of the cytoplasmic fragment of beta-dystroglycan. An interactome analysis by mass spectrometry techniques revealed that beta-dystroglycan interacts with components of the ubiquitin-proteasome system, the cell-cycle, and the nucleus. This all together suggests that, the regulated intramembrane proteolysis mediated by ubiquitination and phosphorylation PTM of beta-dystroglycan triggers downstream nuclear signalling events. These findings provide more ideas of the mechanisms implicated in the regulation of beta-dystroglycan and importantly, of some nuclear processes wherein beta-dystroglycan is involved. These insights may have further implications in the understanding of the progression of cancer and the development of useful therapies.

Adult skeletal muscle regeneration depends on the existence of tissue-specifc stem cells known as satellite cells. Satellite cells are found in a quiescent state in homeostatic conditions but become activated, re-enter the cell cycle, proliferate and differentiate or self-renew in response to muscle injury, exercise or disease. These events are tightly regulated by intrinsic and extrinsic cues, including well-characterised embryonic signalling cascades. The Sonic Hedgehog (Shh) signalling pathway has multiple roles in tissue patterning, cell fate determination, cell survival and proliferation in the embryo. Previous studies have shown that during embryonic myogenesis, Shh signalling controls the specification, migration and proliferation of muscle progenitor cells, as well as muscle patterning by the regulation of genes encoding basement membrane proteins. As the myogenic program carried out by satellite cells recapitulates, to a certain extent, embryonic myogenesis, I hypothesised that Shh signalling controls satellite cell activity in a manner reminiscent to its effect on muscle progenitor cells in the embryo. In this study, through a combination of ex vivo and in vivo approaches, I showed that, although quiescent satellite cells are refractory to Shh signals, activated satellite cells respond to Shh signalling. Shh response persists during the expansion phase and declines as satellite cells enter differentiation. Through the use of pharmacological agonists and antagonists of Shh signalling, as well as of an inducible conditional knockout mouse line of the Smoothened receptor in satellite cells, I demonstrated that Shh signalling contributes to satellite cell proliferation ex vivo and in vivo and to muscle regeneration following injury. Analysis of cell cycle dynamics showed that Shh signalling promotes the entry of satellite cells into the cell cycle and their progression through G1/S phase. Thus, the present study demonstrates that Shh signalling is required for adult skeletal muscle regeneration and provides novel insights into the role of Shh signalling in the control of satellite cell progression through the cell cycle and through myogenesis.

No description available.

No description available.

No description available.

Superbug infection is one of the greatest public health threat with grave implications across all levels of society. Towards a new solution to combat infection by multi-drug resistant bacteria, this thesis presents an engineering framework and genetic tools applied to repurpose commensal bacteria into 'micro-robots' for the treatment of superbug infection. Specifically, a prototype of designer probiotic was developed using the human commensal bacteria Escherichia coli. The engineered commensal was reprogrammed with user-specified functions to sense superbug, produced pathogen-specific killing molecules and released the killing molecules via a lytic mechanism. The engineered commensal was effective in suppressing ~99% of planktonic Pseudomonas and preventing ~ 90% of biofilm formation. To enhance the sensing capabilities of engineered commensal, genetic interfaces comprising orthogonal AND & OR logic devices were developed to mediate the integration and interpretation of binary input signals. Finally, AND, OR and NOT logic gates were networked to generate a myriad of cellular logic operations including half adder and half subtractor. The creation of half adder logic represents a significant advancement of engineering human commensal to be biological equivalent of microprocessor chips in programmable computer with the ability to process input signals into diversified actions. Importantly, this thesis provides exemplary case studies to the attenuation of cellular and genetic context dependent effects through principles elucidated herein, thereby advancing our capability to engineer commensal bacteria.

This study investigates the utility of piezoelectric transduction to assess the structural health of a system through impedance analysis, with application to the field of 'smart' orthopaedic implants. The work is motivated by the high proportion of orthopaedic implant failures that occur due to loosening of the bond between the implant and the corresponding bone surface. The ultimate aim is to prove that piezoelectric sensors embedded within orthopaedic implants have the potential identify implant loosing before it would be shown in imaging techniques. Orthopaedic knee implants were selected as a case study for proof of concept for the proposed health monitoring system. Three distinct experiments were conducted: 1) Small piezoelectric sensors are attached to model tibial trays which are in turn attached with bone cement to sawbone blocks. The measured sensor impedance over a range of input frequencies is measured and analysis of the frequency impedance traces is carried out to determine what changes in the trace are indicative of the bone cement between the sawbone and aluminium curing; 2) Commercially available tibial trays cemented to sawbone tibias are progressively loosened under a fatigue load in a compressive testing rig. Results from three Linear Variable Differential Transducers (LVDTs) measuring the micromotion between the implant and sawbone are compared with frequencyMimpedance traces taken from a piezoelectric sensor attached to the top side of the tibial tray; and 3) Varying amounts of bone cement is used to cover the surface between a sawbone block and model tibial tray. Frequency-impedance readings are taken from a piezoelectric sensor adhered to the top side of the tray. Support vector machines are used to classify between the varying amounts of cement on each test sample. Experimental results and data analysis demonstrate the potential of piezoelectric sensors ability to provide information on the integrity of bone cement bond. Findings include: 1) Piezoelectric sensors can determine at what point bone cement bond between sawbone and an aluminium plate has cured; and 2) It is possible to identify different levels of cement coverage between sawbone and aluminium plate with an accuracy of up to 92 % with piezoelectric sensors. These findings establish the veracity of piezoelectric transduction as a means of identifying orthopaedic implant loosening in vivo. This investigation provides a firm basis for future work bringing the ideal of using piezoelectric sensors as a technique for detecting loose implants in vivo closer to becoming a reality.