Stem cell research has raised great interest in the scientific community as it has the potential to form multiple cell types and it is believed they hold the key to curing many diseases. However, there is a need for better understanding of how to control these cells and further research investigating methods for controlling and directing these cells is required. Pluripotent stem cells transplanted into immune-deficient mice 'spontaneously’ differentiate and proliferate to form a complex mass of differentiated and undifferentiated cells, teratomas - teratoma assay. Such tumours are generally haphazard in their organisation however; they do contain some structures similar to those observed in the embryo. Teratoma formation is a useful model to explore the developmental potential of stem cells and study aspects of tissue development. Examination of how the anatomical location into which human pluripotent stem cells are grafted influences their growth in situ allows investigation of how these cells are affected by different areas within the body: cells grafted into the liver rapidly produce large tumours containing predominantly immature cells whereas, subcutaneous implants were significantly slower growing and formed tumours composed of differentiated tissues. These different growth patterns indicate how environmental cues within the niche affect stem cell behaviour. One factor which contributes to the maintenance of a niche is the extracellular matrix (ECM). To investigate how endogenous ECM affects teratoma behaviour, co-transplantation is carried out with stem cells and ECM components. The ECM extract Matrigel™ dramatically increased the success rate of teratoma formation and size with no detectable affect on teratoma composition when compared to controls and removal of the growth factors from the co-transplanted ECM extract had no effect on teratoma success rate, growth rate, or composition. To study the effects of the ECM in vitro, components of the ECM are often used topcoat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study a novel technology developed by Oria Protein Technologies for the immobilisation of peptide sequences from ECM proteins is evaluated. By examining: the adherence of cultured PC 12; neurite outgrowth from PC 12 cells; and neuronal differentiation of neural stem/progenitor cells (NSPCs) it is shown that peptides from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with ECM proteins. Collectively, this data demonstrates that peptides from ECM proteins can be immobilised in functional fashion to control cell behaviour. Surfaces with adsorbed proteins and biomimetic surfaces presenting peptide motifs from ECM proteins are used to investigate and explain observations from in vivo teratoma experiments. In vivo, Matrigel™ increases the gene expression of the pluripotent stem cell marker Oct4, increasing the pluripotent cell percentage and thus increases the likelihood of teratoma formation. In vitro, Matrigel™ also increases the gene expression of the proliferative marker Ki67, indicating that large teratomas from by the co-transplantation of stem cells with Matrigel™ could be due to increased cell proliferation.

Cellular migration is dependent upon the efficient formation of focal adhesions at the point where the cytoplasmic components engage the extracellular matrix via the integrin proteins. The leading edge of the migratory cell is also a site where proteins such as actin are synthesised. In order for these events to take place mRNA transcripts must be transported from the nucleus to the leading edge by a protein or protein:protein complexes that are capable of nucleo-cytoplasmic shuttling. For example paxillin is a scaffold protein that is a central component of focal adhesions and is capable of nucleo-cytoplasmic shuttling. Polyadenylate binding protein-1 was found to be an abundant co-immunoprecipitant of paxillin in lamellipodium formations. Using overlapping PABP-1 and paxillin constructs it was found that one of paxillins N-terminal LD domains (LD1) interacted with PABP-1 RRM (RNA recognition motif) 1 and 2. The NMR derived structures of PABP-1 RRMs 1 and 2 in their unbound forms are presented within this report. Previous reports have indicated the presence of a paxillin binding sub-domain within the PABP-1 RRM 1 domain. NMR titration data, using a synthetic paxillin LD1 peptide, revealed a binding interaction site within PABP-1 RRM 2 and not within the PABP-1 RRM 1 domain. Also reported here are the structural details of the PABP-1 RRM 2/paxillin LD1 complex. The binding interaction appears to be in the fast exchange regime with an estimated Kd of ~211μM. Experimental evidence shows the binding to be electrostatically driven and confined to the four stranded antiparallel β-pleated sheet. The interaction site is shared with the polyadenylated tail of nascent mRNA transcripts. NMR titration data indicates a competition for this site to be biased toward mRNA. This linked with other experimental data, presented here, and would suggest a more complicated picture of binding to include multiple sites of contact.

The tensin family of cytoskeletal-associated proteins are implicated in fibronectin fibrillogenesis and integrin-mediated cell migration. There are four members of the human tensin family. Tensin1, 2 and 3 are large proteins (~150-200 kDa) with widespread expression in adult tissues that are thought to couple integrins to the actin cytoskeleton, whereas tensin4 (cten) is a smaller protein (77 kDa) with a restricted expression profile that is not thought to interact with actin. To study the role of tensin in fibronectin fibrillogenesis, which is mediated by fibrillar adhesions, I first characterised the isoform specificities of several anti-tensin antibodies. These antibodies were then used to determine the tensin expression profile of human foreskin fibroblasts (HFFs), which were found to express tensin1, 2 and 3. Unfortunately, most of the antibodies did not work sufficiently well for immunofluorescence microscopy so the sub-cellular localisation of the tensins was largely investigated using GFP-tagged proteins. Tensin1, 2 and 3 localised to both focal and fibrillar adhesions, although relative distribution between these two adhesion types was isoform-specific. Since tensin3 preferentially localised to fibrillar adhesions, I used an siRNA approach to investigate its role in fibronectin fibrillogenesis, and found that tensin3 was required for this process. Although tensins play an important role in cell migration, it is unclear whether they act as positive or negative regulators. Therefore, I used over-expression and siRNAmediated knockdown to clarify the role of tensin1, 2 and 3 in both 2D and 3D migration. Interestingly, I found that modulating tensin expression did not affect the 2D migration of HFF, HEK293, or A2780 ovarian cancer cells. However, all three tensins were required for the 3D migration of A2780 cells in fibronectin-containing microenvironments. Over-expressing Rab25, a small GTPase up-regulated in aggressive ovarian cancers, in A2780 cells promotes invasive migration in 3D microenvironments by recycling and retaining integrin at the cell front (Caswell et al., 2007). Tensin was required for the invasive migration and morphology of Rab25-expressing A2780 cells. However, tensin depletion did not affect global α5 integrin recycling or the retention of photoactivated GFP-α5 integrin at the cell front. Interestingly, Rab25 over-expression was also found to promote tensin-dependent fibronectin fibrillogenesis in A2780 cells, suggesting that tensin promotes 3D migration by facilitating fibronectin fibril remodelling.

Senescence is a state of irreversible growth arrest in cells with potentially tumourigenic changes such as irreparable DNA damage. However, senescent cells accumulate with age, altering tissue structure and function and have been observed in atherosclerotic plaques and at sites predisposed to atheroma in man. Recent studies show that long term inhibition of the renin-angiotensin system attenuates the effects of ageing in rodent cardiovascular tissue. Therefore, this thesis investigated whether angiotensin II accelerates senescence of human vascular smooth muscle cells (hVSMC) in vitro and whether the mechanism involves reactive oxygen species, DNA damage, telomere attrition and cell cycle regulatory proteins. Since mitochondria are integral to theories of cellular ageing, the effect of angiotensin II on mitochondrial biogenesis and function were studied. Angiotensin II exposure enhanced superoxide generation via NADPH oxidase in hVSMC, although inhibitors identified the mitochondrial respiratory chain as a contributing source. Angiotensin II induced DNA stand breaks in the Comet assay and accelerated telomere attrition. Senescence associated-β-galactosidase activity was induced by angiotensin II after exposure for 30 days, but also after just 24 hours and after successive short treatments over three days. These effects were attenuated by an angiotensin II type-1 receptor antagonist and antioxidants. Simultaneously, increased expression of p21 and p53 were observed. Angiotensin II induced alterations in mitochondria. A rapid increase in mtDNA content, gene transcript levels involved in initiating mtDNA transcription and replication and ATP levels in hVSMC, suggested mitochondrial biogenesis occurs in response to stress. These data suggest that angiotensin II induces both accelerated replicative senescence (telomere-dependent) and stress-induced premature senescence (telomere-independent) of hVSMC, dependent upon the treatment regime used. These findings explain the anti-ageing effects of life-long angiotensin II blockade in rodents, and may provide a mechanism for accelerated vascular ageing and cardiovascular disease progression in the ageing human population.

Electrical stimulation of the endothelial cell (EC) has been heralded as a potential new approach to the control of directed reorientation and elongation of ECs, these processes being important cell behaviours occurring during angiogenesis. Previous studies by numerous investigators have shown that ECs, like many other cell types, migrate directionally when exposed to an applied electric field (EF). Previous experimental results from our group have shown that VEGF signalling mediates EF-induced EC responses. However, their downstream signalling elements are yet to be identified. The Mitogen-Activated Protein Kinases ERK1/2, JNK, and P38 are involved in a vast array of biological responses and are activated by a variety of stimuli. There have also been numerous reports implicating them in angiogenic processes. I have shown that HUVECs do indeed reorientate and elongate in response to an EF and also that the EF causes activation of the MAPK ERK1/2, but not JNK or P38. ERK1/2 was activated in a biphasic manner, while JNK and P38 remained basally active. Inhibition experiments showed that ERK1/2 and JNK play a role in the EF-induced reorientation and elongation of HUVECs but p38 does not seem to be involved.

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In this thesis a nerve-bouton preparation of Purkinje cells has been characterised. Mechanically isolated Purkinje cells are shown to retain active afferent nerve terminals. This provides a simplified system where the effects of manipulating the ion channels in nerve boutons can be studied without the potentially confounding influences of the rest of the presynaptic cell or surrounding tissue. Isolated Purkinje cells were initially identified for whole cell patch-clamp recordings by their distinctive size and shape. Vesicular release of neurotransmitter was evident by spontaneous inward synaptic currents with a characteristic time course. Antagonist application established that isolated Purkinje cells receive a mixture of inhibitory GABAergic and excitatory glutamatergic inputs. Changes in the frequency, amplitude, and burst behaviour of these spontaneously occurring synaptic currents were used to infer properties of the afferent boutons. Because rat Purkinje cells can be distinguished by their lack of postsynaptic NMDA receptors the presynaptic effects of NMDA application could be readily investigated. NMDA caused an increase in the frequency of postsynaptic events. The NMDA-induced increase was found to be sensitive to external magnesium and TTX application. NMDA application was found to increase the frequency of both GABAergic events and glutamatergic events. Physiologically, NMDA receptors in afferent inhibitory terminals are thought to be activated by the retrograde release of glutamate. So experiments were performed to determine if retrograde release of glutamate could also increase the frequency of glutamatergic events, however it was found that this process has a much more pronounced influence on the GABAergic events. Properties of afferent boutons were also probed with a range of potassium channel blockers. The relevant topics covered are pharmacology, synaptic transmission, and the role of NMDA receptors in the cerebellum and the main technique used is whole-cell patch clamp recording.

Why there are two distinct sexes has received little attention compared with that lavished on the value of sexual reproduction. While sex requires two parents, there is no obvious need for these to be of different sexes. Furthermore, self-incompatible gametes seemingly reduce the likelihood of finding a partner. What causes mating types and sexes to predominate in nature remains a conundrum. The uniparental inheritance (UPI) of mitochondria (in which only one sex, usually the female, passes on its mitochondria) is widespread among sexual organisms. Theoretical work suggests that the evolution of two sexes can be understood in the light of mitochondrial inheritance. However, the exact role of UPI is not clearly understood. Part I of this thesis considers the evolution of self-incompatible mating types in relation to this perspective, using probability theory and population genetics. Chapter 2 studies the impact of UPI on interactions between genes in the mitochondria and the nucleus, in an effort to elucidate the role of UPI itself. In Chapter 3, I develop a new, explicit theoretical model that challenges the prominent view that selection for UPI leads to the establishment of self-incompatible mating types and sexes. An alternative hypothesis proposes that mating types evolved as a consequence of selection for asym- metry in gamete attraction and recognition. This idea is based on the assumption that an asymmetry in gamete communication leads to more effective attraction and recognition. In Part II of this thesis, I examine this idea further. In Chapter 4, I perform an extensive literature review of mating type interactions and provide empirical support for the prediction that an asymmetry in signalling is indeed common in nature. The underlying assumptions of this hypothesis are linked to the physical constraints that gametes experi- ence during sex, and the role of polarity in cell-cell interactions. To assess the impact of these constraints rigorously, in Chapter 5 I develop a biophysical model for signaller-detector dynamics based on chemical diffusion, chemotaxis and individual cell movement that can be tested in silico and in vitro. This thesis examines the role and origins of self-incompatible mating types and sexes. The novel theo- retical methods and perspective on the empirical literature presented here place this evolutionary question in a fresh context and encourage further theoretical and empirical work.

This thesis presents a new investigation of image restoration and its application to fluorescence cell microscopy. The first part of the work is to develop advanced image denoising algorithms to restore images from noisy observations by using a novel featurepreserving diffusion approach. I have applied these algorithms to different types of images, including biometric, biological and natural images, and demonstrated their superior performance for noise removal and feature preservation, compared to several state of the art methods. In the second part of my work, I explore a novel, simple and inexpensive super-resolution restoration method for quantitative microscopy in cell biology. In this method, a super-resolution image is restored, through an inverse process, by using multiple diffraction-limited (low) resolution observations, which are acquired from conventional microscopes whilst translating the sample parallel to the image plane, so referred to as translation microscopy (TRAM). A key to this new development is the integration of a robust feature detector, developed in the first part, to the inverse process to restore high resolution images well above the diffraction limit in the presence of strong noise. TRAM is a post-image acquisition computational method and can be implemented with any microscope. Experiments show a nearly 7-fold increase in lateral spatial resolution in noisy biological environments, delivering multi-colour image resolution of ~30 nm.