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Regenerative medicine is a rapidly expanding field of science with an exhaustive volume of literature published on the different strategies used to repair diseased or injured tissue. Recently, stem cells have emerged as a promising candidate in this regard owing to their involvement in embryogenesis, homeostatic turnover and normal tissue repair. Despite this potential, stem cell-based therapies have yet to be fully established in a clinical setting owing to complications associated with their limited numbers, immunogenicity, tumour formation and the ethical considerations surrounding their usage. Furthermore, the mechanisms underlying stem cell differentiation are complex and not fully understood, thus expanding stem cell numbers and predictably directing their commitment toward a desired lineage, represent a major challenge for tissue regeneration strategies. In an attempt to circumvent these problems there is currently a rising interest in biomimetic materials that aim to reproduce the physical architecture, chemical composition and plasticity of the in vivo extracellular environment in an in vitro setting. Furthermore, the need to expand stem cells while maintaining the stem cell phenotype has prompted many to look to the stem cell niche for answers. At the centre of most cellular responses to the physical cues embedded within the ECM are integrins. Integrins are mechanosensitive membrane spanning receptors that link the ECM to the cytoskeleton and thus transmit information from outside the cell into the nucleus, affecting gene transcription via a series of intracellular signalling cascades. To that end, many biomimetic systems incorporate integrin- binding ligands such as the tripeptide RGD. In this work glass surfaces functionalised with RGD were used to study changes in mesenchymal stem cell (MSC) responses to increased integrin binding by using an enzymatic ‘switch’ to reveal surface-bound RGD peptides that have been masked by a large chemical cap (Fmoc). The results of this work demonstrated that RGD- functionalised substrates can support MSC growth and influence them to commit to a particular fate. MSCs on surfaces where integrin-ligand binding was blocked developed a fibroblast-like phenotype whereas MSC grown on surfaces that were later enzymatically digested to reveal the underlying RGD ligands developed an osteoblast phenotype similar to RGD controls.

In this thesis light harvesting complexes, the LH2 and core complexes, from several different species of purple photosynthetic bacteria have been analysed both functionally and structurally. Purified monomeric core complexes from Rhodopseudomonas (Rps.) palustris have been used to isolate and identify the putative Protein W. This information was then used to create a Protein W deletion mutant. A low-resolution crystal structure of the monomeric core complex from Allochromatium (Alc.) vinosum is presented which suggests that the LH1 complex is a complete ellipse, unlike the core complex from Rps. palustris. It has previously been shown that some species are able to synthesise LH2 complexes that have different NIR absorption spectra. For example, strains 7050 and 7750 of Rps. acidophila can express both the B800-850 and B800-820 LH2 complexes, whilst strain 10050 only expresses the B800-850 LH2 complex despite evidence to suggest that this strain contains multiple LH2 genes (pucBA genes). It is this homogeneity that has made the LH2 complexes from this strain structurally amenable. Here, genomic DNA from Rps. acidophila strain 10050 has been isolated and sequenced using the next generation sequencing (NGS) technique, Illumina sequencing. So far 8 pucBA gene pairs were identified arranged into 2 distinct operons, one containing B800-850 pucBA genes and pucC, the putative Bchl transporter that is essential for efficient LH2 expression. The second operon contains B800-820 pucBA gene pairs only. Analysis of the protein products of the B800-850 type pucBA gene pairs has shown that none of these proteins match the sequence for the LH2 that is expressed by Rps. acidophila strain 10050. The crystal structure of the LH2 complex from the culture of Rps. acidophila used to isolate the genomic DNA was resolved to 2.05 Å from crystals of the LH2 complex. This structure shows that the protein sequence of the LH2 complex has not changed. Hence, not all the pucBA gene pairs have been identified in the genome sequence data. Currently mate-pair sequencing is being completed to fill in the gaps of sequence data and to complete the genome sequence. LH2 complexes contain carotenoid (Car) and Bchl molecules. In this thesis, the energy transfer mechanisms between Car and Bchl molecules have been investigated using 2-dimensional electronic spectroscopy (2DES). This technique splits the emission and excitation events on 2-dimensions, which can make the less populated ‘dark’ states more visible as overlapping peaks can be separated. Car moleucles are not seen as theoretically efficient in photosynthesis. This is due to short life times of the excited state S2. However, the Car used in photosynthesis have conjugated carbon tails with ≥9 π electrons. According to calculations by Tavan and Schulten, these molecules have the propensity to contain additional excited states that lie below the S2 state that can be involved in energy transfer and increase the efficiency of energy transfer between the Car and Bchl molecules. For the first time an intermediate Car electronic state has been directly observed and shown to be involved in energy transfer between the Car and Bchl molecules.

There is presently increased interest in using germanium (Ge) for both electronic and optical devices on top of silicon (Si) substrates to expand the functionality of Si technology. It has been extremely difficult to form an Ohmic contact to n-Ge due to Fermi level pinning just above the Valence band. A low temperature nickel process has been developed that produces Ohmic contacts to n-Ge with a specific contact resistivity of , which to date is a record. The low contact resistivity is attributed to the low resistivity NiGe phase, which was identified using electron diffraction in a transmission electron microscope. Light emission from Ge light emitting diodes (LEDs) was investigated. Ge is an indirect bandgap semiconductor but the difference in energy between the direct and indirect is small (~136 meV), through a combination of n-type doping and tensile strain, the band structure can be engineered to produce a more direct bandgap material. A silicon nitride (Si3N4) process has been developed that imparts tensile strain into the Ge. The stress in the Si3N4 film can be controlled by the RF power used during the plasma enhanced chemical vapour deposition. LEDs covered with Si3N4 stressors were characterised by Fourier transform infrared spectroscopy. Electroluminescence characterisation (EL) revealed that the peak position of the direct and indirect radiative transitions did not vary with the Si3N4 stressors due to the device geometries being too large. Therefore, nanostructures consisting of pillars smaller than a micron were investigated. Photoluminescence characterisation of 100 nm Ge pillars with Si3N4 stressors show emission at much longer wavelengths compared to bulk Ge (> 2.2 μm). In addition, the EL from Ge quantum wells grown on Si was also investigated. EL characterisation demonstrates two peaks around 1.55 and 1.8 μm, which corresponds to the radiative recombination between the direct and indirect transitions, respectively. This result is the first demonstration of EL above 1.45 μm for Ge quantum wells. Finally, the fabrication of Ge-on-Si single-photon avalanche detectors are presented. A single-photon detection efficiency of 4 % at 1310 nm wavelength was measured at low temperature (100 K). The devices have the lowest reported noise equivalent power for a Ge-on-Si single-photon avalanche detector (1×10-14 WHz-1/2).

Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double-stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared with the seven well-recognized conserved helicase motifs, the role of the Q motif is not well known. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion. To examine the roles of the Q motif in the ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant wild type (WT) and mutant (Q23A) proteins were overexpressed and purified from HEK293T cells. The ChlR1-Q23A mutant abolished the helicase activity of ChlR1, and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but displayed normal ATP binding. The Q motif in FANCJ helicase, a ChlR1 homolog, regulates FANCJ’s dimerization, while our size exclusion chromatography (SEC) indicated that the ChlR1 protein functions as a monomer. A thermal shift assay revealed that ChlR1-Q23A has a similar melting point as ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have similar globular structures, although there are some subtle conformational differences between these two proteins. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not in ATP binding.

We surveyed a number of genome visualisation tools used in biomedical research. We recognised that none of the tools shows all the relevant data geneticists who look for candidate disease genes would like to see. The biological researchers we collaborate with would like to view integrated data from a variety of sources and be able to see both data overviews and details. In response to this need, we developed a new visualisation tool, VisGenome, which allows the users to add their own data or data downloaded from other sources, such as Ensembl. VisGenome visualises single and comparative representations of the rat, the mouse, and the human chromosomes, and can easily be used for other genomes. In the context of VisGenome development we made the following research contributions. We developed a new algorithm (CartoonPlus) which allows the users to see different kinds of data in cartoon scaling depending on a selected basis. Also, two user studies were conducted: an initial quantitative user study and a mixed paradigm user study. The first study showed that neither Ensembl nor VisGenome fulfil all user requirements and can be regarded as user-friendly, as the users make a significant number of mistakes during data navigation. To help users navigate their data easily, we improved existing visualisation techniques in VisGenome and added a new technique CartoonPlus. To verify if this solution was useful, we conducted a second user study. We saw that the users became more familiar with the tool, and found new ways to use the application on its own and in connection with other tools. They frequently used CartoonPlus, which allowed them to see small regions of their data in a way that was not possible before.

Melanocytes within benign human nevi are the paradigm for tumor suppressive senescent cells in a pre-malignant neoplasm. These cells typically contain mutations in either the BRAF or NRAS oncogene and express markers of senescence, including p16INK4A 1-4. However, a nevus can contain 10s to 100s of thousands of clonal melanocytes and approximately 25% of melanomas are thought to arise in association with a pre-existing nevus 5-9. Neither observation is indicative of fail-safe senescence-associated proliferation arrest and tumor suppression. I set out to better understand the status of nevus melanocytes. Proliferation-promoting Wnt target genes, such as Cyclin D1 and c-Myc, were repressed in oncogene-induced senescent melanocytes in vitro, and repression of Wnt signaling in these cells induced a senescent-like state. In contrast, Cyclin D1 and c-Myc were expressed in many melanocytes of human benign nevi. Specifically, activated Wnt signaling in nevi correlated inversely with nevus maturation, an established dermatopathological parameter linked to clinical benignancy 10,11. Single cell analyses of lone interfollicular epidermal melanocytes and nevus melanocytes in tissue showed that expression of proliferation-promoting Wnt targets correlates with prior proliferative expansion of p16INK4A-expressing nevus melanocytes. In a mouse model, activation of Wnt signaling delayed, but did not bypass, senescence of oncogene-expressing melanocytes, leading to massive accumulation of proliferation-arrested, p16INK4A-positive, non-malignant melanocytes. I conclude that clonal hyperproliferation of oncogene-expressing melanocytes to form a nevus is facilitated by transient delay of senescence due to activated Wnt signaling. The observation that activation of Wnt signaling correlates inversely with nevus maturation, an indicator of lower malignant potential, supports the notion that persistent destabilization of senescence by Wnt signaling contributes to the malignant potential of nevi.

Ribosomes are responsible for the synthesis of all cellular proteins. It was initially believed that translating nascent chains would not interact with the ribosome exit tunnel, however, a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest. Escherichia coli (E.coli) secretion monitor (SecM) is one such stalling peptide. SecM monitors the SecYEG translocon export activity through its own translocation to the periplasm and upregulates translation of SecA, an ATPase involved in the SecYEG translocation machinery, when translocation is reduced. How stalling peptides interact with the ribosome exit tunnel is not fully understood, however, a key feature required is an essential amino acid arrest motif at their C-terminus, and additionally some peptides, including SecM, undergo compaction of the nascent chain within the exit tunnel upon stalling. In this study analysis of SecM peptides with both alanine and conservative mutations of key arrest motif residues were investigated. This identified three conservative mutants that can retain a degree of stalling; and this level of stalling is further increased when coupled with mutation of a non-essential arrest motif residue P153A. Further analysis of these mutants by pegylation assays indicates that this increase in stalling ability is due to the ability of the P153A mutation to reintroduce compaction of the nascent chain within the exit tunnel, possibly due to the improved flexibility of the nascent chain provided by the removal of the restrictive proline residue. These methods highlight the significance of the interactions between the nascent chain and the exit tunnel, which contribute to translation arrest. This study also examines the ability of stalling peptides to undergo translation arrest in ribosomes of alternative domains, investigating in particular the ability of E.coli SecM and TnaC and fungal Neurospora crassa (N.crassa) AAP to arrest in eukaryotic Wheat Germ and prokaryotic E.coli ribosomes. This study concludes that stalling peptides only induce translation arrest in ribosomes of the same domain. In addition, it also revealed the ability of inducible stalling peptides to undergo translational pausing, prior to the commitment to full translation arrest, a process that does not appear to occur in intrinsic stalling peptides.

The formation of conjugates between the electrophilic reactive metabolites of drugs and nucleophilic protein sites is known to be associated with toxicological risk. At present there is no low cost and high throughput means of reliably detecting the presence of drug-protein adducts in vitro or in vivo. The development of a reliable high throughput methodology would facilitate the study of underlying mechanisms of toxicity and prove useful in early screening of potential drug molecules. Assays using liver microsomes and trapping agents such as glutathione are used to produce and detect a wide range of drug reactive metabolites which are then characterised by mass spectrometry. The glutathione trapping is effective for metabolite identifications but, the modification of proteins by means of electrophilic attack on nucleophilic centres often occurs in an enzyme independent manner and is unlikely to be analogous to the glutathione model. In order to create a more suitable model system, three short polypeptides were designed and synthesised. These peptides were incubated with clozapine and human liver microsomes. The resulting metabolite-peptide conjugates were analysed by nanoLC-MS. Results indicated that a characteristic conjugate specific ion at 359.1 Da could be detected for each of the peptides. This data was used to create a precursor ion scan specific for the presence of this characteristic ion. Protein separation techniques including SCX, Offgel IEF and 1d-gel electrophoresis, in conjunction with LC-MS (with the precursor 359 scan), were applied to microsome prep samples in order to identify modified proteins. Using these approaches some 1700 protein identifications were made, more than 1000 of these were unique hits. The precursor ion scan was found to have poor selectivity identifying roughly 1/3 as many proteins as the information dependant acquisition approach. No drug-protein adducts were identified. Further to this a novel application of saturation DIGE was applied in order to enrich for the presence of protein adducts. The DiGE approach was used to identify some 15 proteins with apparent change in abundance (fluorescence intensity) between clozapine treated and untreated samples. Spots were excised from the 2d gel digested and analysed by reversed phase liquid chromatography mass spectrometry. The IDA scans identified some 147 unique protein hits, the precursor ion scans identified 18. Again no drug-protein adducts were found. Biotinylated desmethyl clozapine was metabolised in the human liver microsome assay. Western blotting was carried out on a 2d gel run from an assay sample. The Western membrane was probed using an HRP-Streptavidin probe. Imaging of the membrane revealed the presence of several biotin bearing proteins, many of which were not present in the negative control sample. A print out of the image was used as a map for the excision of modified proteins from a duplicate gel. Digestion and LCMS analysis of the samples revealed the presence of several proteins but no protein-adducts were found.

Microfluidic technologies and devices now provide powerful tools for many biological studies to gain knowledge and insight into cell behaviour because of their potential to control the local in vitro environment. This thesis aims to develop microfluidic devices for the single cell proliferation and migration studies that are fundamental in determining cell and tissue behaviour. There are two designs of microfluidic devices that have been used in this project. The first one is hydrodynamic single cell trap device having a bagatelle- like structure. The bagatelle-like devices were used to trap modified MCF7 cells expressing both mcherry-tubulin and GFP-actin and also to study the influences of the oestrogen hormone on MCF7 cells. It was found that the MCF7 cell proliferation could not be seen in the bagatelle-like devices either in the presence or absence of oestrogen. It was hypothesised that this might be due to cell stresses arising from being in a constrained area (trap) and subjected to strong fluid flow forces. The second, novel, device consists of three segregated layers and is termed a microhole device. It was specifically designed, fabricated, characterised and utilised in cancer cell proliferation and migration studies in this thesis. The microhole devices were designed to address the limitations of the bagatelle-like device. In each microhole device, the lower layer comprises of a network of submerged channels linked to an upper layer through cavity-like holes. The networks of submerged channels provide a route through which cells can migrate. The middle layer consists of an array of circular holes used to organise single cells into the cavities beneath. The top layer is a PDMS chamber for cell loading and culture medium perfusion. It was found that the recirculatory flow patterns inside the devices facilitate cell trapping, while also serving to separate high velocity flow in the top chamber from the middle and the bottom layer thereby protecting the cells from shear stress. MDA-MB-231 cells were used in this study. It was found that they can undergo cell cycling normally in the microhole devices, and migrate along an SDF-1α solution gradient produced inside the device, towards high SDF-1α concentration. To explore whether the cells were sensitive to SDF-1α on the surface to which they adhered (as opposed to solution gradients), the microhole devices were modified to have SDF-1α immobilised on selected interior surfaces. Despite each stage of the immobilisation process being verified using the appropriate fluorescence assays, relatively low levels of SDF-1α were detected in the completed devices. This may be due to fabrication processes that might deteriorate the immobilised SDF-1α functionality. It was found that unlike the situation when SDF-1α is in solution form, the MDA-MB-231 cells showed no migratory preference toward the immobilised SDF-1α. Taken together, the microhole devices developed in this thesis provide suitable environments for study cell migration toward stimuli under perfusion conditions. The geometry and the flow characteristics inside the array facilitate cell trapping and serve to protect cells from shear stress caused by high fluid flow. Further applications of the multilayer microhole devices can be found through modifying the different layers to accommodate different geometries for different cell types as well as more complex stimulation conditions, or in other application areas associated with droplet microfluidics and synthetic biology.