Colorectal cancer is thought to originate in the epithelial cells that line the colorectal crypt and, in most cases, is associated with a mutation in Wnt-signalling pathway. These mutations cause cells to alter their proliferative behaviour, make their cytoskeleton less deformable and increase their levels of cell-cell and cell-substrate adhesion. In this thesis we develop three different types of models for the proliferation and movement of epithelial cells in a colorectal crypt. We use these models to investigate how changing the cell adhesion, cytoskeleton and proliferation properties of mutant cells affects their ability to establish a mutant population within the crypt. First we develop a continuum model of two cell populations, normal and mutant, using a spatially-varying source term to model Wnt-dependent proliferation and using Darcy's law to describe cell movement down pressure gradients. We distinguish between mutant cells and normal cells by assuming the former have a spatially independent source term, representing proliferation, and a different viscosity to normal cells, to model changes in their cytoskeleton and levels of adhesion. The model is solved analytically by an asymptotic expansion of the variables and numerically using a collocation method. The results show that the ability of mutant cells to remain in the crypt depends on the position of the initial mutation and their viscosity: the further up the crypt a cell suffers a mutation the more rigid and adhesive the cell must be for a mutation to persist. We then consider a discrete cell-centre model based on the work of Meineke et al. (2001). Cell-cell interaction forces are modelled by springs and are balanced by a viscous drag term. Adaptations to Meineke et al. (2001) include unpinning of stem cells from the bottom of the crypt, dependence of cell-drag on cell size, dependence of cell-cell interaction forces on their area of contact and the inclusion of mutant cells. Using agile software engineering techniques, the software environment, CHASTE, is developed and used to solve the model numerically and to reproduce experimental findings such as crypt homoeostasis and monoclonality. The results again reveal that increasing the drag on the mutant cells increases the likelihood of a mutant population establishing itself within the crypt. The third approach is a discrete cell-vertex model. The model decouples cell-cell adhesion forces from cell deformation forces and movement is determined by a free-energy gradient balanced by a viscous drag term. Numerical simulations show that the model can generate similar results to the cell-centre model, and reveal that increased cell-cell adhesion of the mutant cells increases the likelihood of the mutant population invading the crypt. Finally the three models are compared in terms of their suitability for modelling epithelial tissue.

The purpose of this study was to develop novel optical microscopy techniques in order to investigate colloidal drug particle endocytosis by mammalian cells. A total internal reflection microscope (TIRM) was initially developed for high resolution cellular imaging. TIRM is a non-fluorescent imaging technique based on the principle of ‘scattering’ of the evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass and air. The key design considerations with respect to development of a TIRM instrument are discussed. The technique is also compared and contrasted to the more commonly known non-fluorescent RICM (Reflection Interference Contrast Microscopy) technique using computer simulations. Time-lapse video TIRM is applied to imaging the interaction between A549 and 3T3 cells, and a polylysine coated substrate. Real-time label-free visualisation of 0.5 and 1 m polystyrene particle endocytosis by living cells is then demonstrated. Modifications to the TIRM system to include a dual-colour fluorescent TIRF (Total Internal Reflection Fluorescence) microscope are described in detail. Results are shown which demonstrate the ability of a combined TIRM/TIRF instrument to selectively image the basal cell membrane both label-free and fluorescently. 3T3 fibroblast cells were genetically modified using standard molecular biology protocols to express the fluorescent fusion protein EGFP-Clathrin LCa (enhanced green fluorescent protein clathrin light chain a). Finally, colloidal particle endocytosis by the genetically modified cell was imaged using the TIRM/TIRF microscope. Direct visualisation of the internalisation of 500 nm particles via clathrin coated pits in 3T3 cells was shown for the first time.

Freshwater planarian Schmidtea mediterranea is an emerging model for studying in vivo gene functions and regulation in native cell niches. The obligate asexual strain of this species reproduces by fission, in which succession of soma occurs without passing through the germline. To achieve this somatic immortality the somatic stem cells need to overcome the end replication problem. Therefore it can be hypothesised that somatic telomere maintenance in asexual S. mediterranea must possess a germ-like property, with which age-related erosions can be adequately repaired. In this PhD project, the telomere repeat unit in S. mediterranea was confirmed to be the vertebrate-like TTAGGG. Attrition of whole body telomere length was found in ageing sexual worms and also in asexual worms which had not gone through recent fission events. Opposite telomere length dynamics were observed in regenerated samples of the two strains, with erosion in the sexuals and reset in the asexuals. The telomere maintenance was found to increase during regeneration in both strains, with a higher level of increase in asexual worms. A homolog of the telomerase reverse transcriptase subunit, Smed_Tert, was identified and characterised in this organism. High level of Smed_Tert expression was seen in germ cells in mature sexual worms and adult stem cells in asexual worms. Knockdown of Smed_Tert expression by RNA interference caused progressive telomere erosion, however effects on cell proliferation and viability have not been observed in knockdown samples. Four alternate splice isoforms of Smed_Tert were identified. The enhanced telomerase activity during regeneration correlates with a proportional increase in the full-length isoform and a decrease in isoforms with a truncated TRBD domain, suggesting a dominant negative regulation of telomerase by alternative splicing. Significant increase in the expression of the full-length isoform was seen in regenerating asexual samples but not in sexual strains, which correlates with their telomere length dynamics. It is hoped that the comparative studies between the sexual and asexual strains can improve our understanding of how soma can evolve to become an effective inheritable unit.

The potential application for stem cell therapy is vast. Despite a limited understanding of their mechanisms of action, clinical trials assessing stem cells in human stroke are underway. Colony stimulating factors (CSF) such as granulocyte colony stimulating factor (G-CSF), which have been used to mobilise haematopoietic stem cells (HSC), also show promise in treating stroke. Preclinical experiments evaluating the effect of G-CSF in stroke were meta-analysed; G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Further studies assessing dose-response, administration time, length of ischaemia and long-term functional recovery are needed. Tracking iron-labelled cells with MRI may help to establish migratory patterns following transplantation. Our systematic review of iron-labelled stem cells in experimental stroke suggests that compounds already licensed for humans (ferumoxide and protamine) may potentially be used in clinical trials. In a phase IIb single-centre randomised controlled trial (n=60), the safety of G-CSF in recent stroke was assessed (STEMS-2). G-CSF appears safe when administered subacutely and may reduce stroke lesion volume. Phase III trials are required to test efficacy. An updated Cochrane review on CSFs in stroke shows that G-CSF was associated with a non-significant reduction in early impairment but had no effect on functional outcome in 6 small studies. In two trials, erythropoietin therapy was significantly associated with death by the end of the trial. It is too early to know whether G-CSF could improve functional outcome in stroke. In 8 recruits randomised into STEMS-2, mobilised CD34+ HSCs were paramagnetically labelled, re-infused and tracked with serial T2* MRI. Post-stroke HSC labelling appears safe and feasible. There is suggestive evidence in one patient that labelled HSCs migrate to the ischaemic lesion. Our in vitro evaluation of CD34+ HSCs has revealed that uptake of superparamagnetic iron oxide (SPIO) is enhanced but not dependent upon a transfection agent. Iron labelling of CD34+ cells in this manner did not affect cell viability or inhibit growth. This methodology could be applied to clinical trials. We have established the expression of G-CSF protein, its receptor (G-CSFR) and CD34 antigen in post-mortem brain tissue from participants recruited to STEMS-2. Areas of angiogenesis and expression of G-CSFR in acute and chronic infarction suggest potential targets for therapy. There are many preclinical studies reporting the effects of stem cells in treating stroke (with a noticeable lack of neutral or negative articles). Despite the wealth of literature there remain many unanswered questions and patients should not undergo stem cell therapy unless it is as part of a well designed clinical trial.

India is rapidly becoming established as a major player in the stem cell sector. However, concerns have been raised about the use of unproven stem cell therapies and the exploitation of parents for cord blood banking. This study aims to explore the nature of stem cell activities, how key stakeholders generate expectations around them and frame the ethical issues they raise, and why the biomedical governance system is unable to regulate these emerging practices. The study involved a survey, documentary analysis and qualitative interviews with key scientists, clinicians, representatives of firms and policymakers. The thesis observes that, unlike international commentaries which largely focus on embryonic stem cell treatments, in India it is adult and cord blood stem cells which are dominant in research and clinical settings. Expectations are configured on the basis that stem cells have the potential to: solve the problem of organ shortage; help patients with ailments; provide affordable health care; and establish India as a global player. The creation of expectations is ethically problematic given the potential health risks and economic exploitation of both native and international patients. However, the ethically contested activities are justified by clinicians on the basis that the Helsinki Declaration allows to use an experimental therapy; there are many 'desperate patients' demanding these treatments; and adult stem cells are safe. To date, the government of India appears to be unable to prevent these activities. Contrary to suggestions in previous literature and by some informants that new legislation is needed to address the problem, this thesis finds that state-led mechanisms for biomedical governance lack the ability to implement existing oversight measures. This implementation gap is partly because other forms of governance are not strong enough and partly because there are high expectations at state level aimed at establishing India as a global player in the stem cell sector.

During development, polarity is a common feature of many cell types. One example is the polarisation of whole fields of epithelial cells within the plane of the epithelium, a phenomenon called planar cell polarity (PCP). It is widespread in nature and plays important roles in development and physiology. Prominent examples include the epithelial cells of external structures of insects like the fruit fly Drosophila melanogaster, polarised tissue morphogenesis in vertebrates and sensory hair cells in the vertebrate ear. In this work we focus on the wing and the abdomen of Drosophila, where PCP becomes obvious in the alignment of hairs and bristles. The underlying dynamics are not fully understood yet, but two distinct protein networks centred around the transmembrane proteins Frizzled and Dachsous, respectively, have been shown to play essential roles. We will present and analyse five models for different aspects of the process of planar cell polarisation. The first two models assess the nature of PCP in a generic setting, ensuring that the results are valid for whole classes of PCP models. Models three and four are existing more complex models that include detailed assumptions about the underlying protein interactions of the Frizzled system in the Drosophila wing. Model five considers the Dachsous system in the Drosophila abdomen. We describe the features of the different types of mechanisms and determine the conditions under which they can yield polarity. All five models can establish wild-type polarity for a wide range of parameter values. We find, however, that for model one, three and four an inhomogeneous pattern exists for the same parameter values as the polarised state. Therefore, in these cases either specific initial conditions, which are unlikely in nature, or a global bias are necessary to ensure correct polarisation. Furthermore, we present the effects of clonal clusters of cells on the polarity of the surrounding cells in our models and relate them to the phenotypes observed in experiments. Model one and five show the largest discrepance between the numerical and the experimental results. We discuss the biological relevance of these findings and indicate outstanding questions.

The eukaryotic cell cycle primarily consists of five phases, namely a resting state, G0, and four cycling phases G1, S, G2 and M phase, with cells progressing in this order before dividing into two cells back in phase G1. Understanding how a drug affects the cell cycle can give insight into the drug's mechanism of action and may assist research into potential treatment strategies. The pentacyclic acridinium salt RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate) is an attractive agent because it is potentially cell-cycle specific and inhibits the activity of telomerase, an enzyme known for its role in cellular immortalisation in human cancer. The precise mechanism of action of the drug on the cell cycle dynamics, however, remains unclear. We have devised experiments, collected experimental data and formulated a mathematical model describing the cell cycle dynamics of cancer cells and their time- and dose-dependent modulation by RHPS4 to investigate how the compound affects cells in each stage of the cell cycle. In addition to a control case, in which no drug was used, we treated colorectal cancer cells with three different concentrations of the drug and fitted simulations from our models to experimental observations. We have shown that the model is "identifiable", meaning that, at least in principle, the parameter values can be determined from observable quantities. Our fitting procedure also generates information on the sensitivity of parameters in the model. We found that RHPS4 caused a marked concentration-dependent cell death in treated cells, which is well modelled by allowing the rate parameters corresponding to cell death to be sigmoidal functions of time. Since the drug uptake into the nucleus is rapid (saturation within 5 hours), the observed delay effect of 5 days of the compound is unexpected and is a novel finding of our research into this compound. Our results show that, at low concentrations, RHPS4 primarily affects the cells in the G2/M phase, and that the delay decreases at larger doses. We propose that secondary effects lead to the induction of observed cell death and that changes in the molecular structure of the non-coding DNA sequences at chromosome ends, called telomeres, might be a precursor of delayed cell death. We therefore investigated the dynamics of telomere length in different conformational states, that is, t-loops, G-quadruplex structures and those being elongated by telomerase. By formulating differential equation models we studied the effects of various levels of telomerase and RHPS4 concentrations on the distribution of telomere lengths and analysed how these effects evolve over large numbers of cell generations. As well as calculating numerical solutions, we use quasicontinuum methods to approximate the behaviour of the system over time, and predict the shape of the telomere length distribution. We showed that telomere length maintenance is tightly regulated: too high levels of telomerase lead to continuous telomere lengthening, and large concentrations of RHPS4 lead to progressive telomere erosion. Our results suggest different effects of RHPS4 dependent on the drug concentration used: low concentrations reduce telomere length, but do not impair the equilibrium of the system, and high concentrations destabilise the system leading to chromosome degradation and senescence and/or cell death. Moreover, our models predict a positively skewed distribution of telomere lengths at equilibrium, and our model predictions are in good agreement with experimental data.

The intracellular transport of organelles and vesicles is thought to utilise both microtubules and actin filaments, which mediate long and short-range transport, respectively. Melanosomes, synthesised in melanocytes, are a convenient model organelle to study intracellular transport, since they are visible using brightfield microscopy. They are believed to be transported from the perinuclear area to the actin cortex along microtubules, and then captured by the myosin-Va/melanophilin/Rab27a complex which traffics them along actin filaments to the plasma membrane. In contrast, data presented here demonstrate that anterograde melanosome transport relies only upon the actin cytoskeleton. Myosin-Va null melanocytes were used to test the importance of microtubules and actin on long-range organelle transport. In these cells, melanosomes cluster around the perinuclear area, but disperse into peripheral dendrites upon reintroduction of the myosin-Va gene. When this assay was repeated in the absence of microtubules, melanosomes still dispersed indicating that microtubule-based motors are not necessary for long-range anterograde trafficking. However, depolymerising F-actin, or freezing actin dynamics with latrunculin A or jasplakinolide inhibited the dispersion of pigment granules in myosin-Va null cells melanocytes and induced a clustered phenotype in WT melanocytes. This effect was abolished if microtubules were absent, suggesting that microtubules are only required for retrograde transport whilst dynamic actin is essential for anterograde melanosome transport. Moreover, when Kif5B was forcibly recruited to the melanosome membrane via an inducible dimerisation system, the melanosomes dispersed abnormally. An siRNA knockdown screen of over 120 actin binding proteins identified several proteins including formin-1, Arpc1b, cofilin-1, gamma-actin and spire1/2, which appear to be necessary for maintaining peripherally dispersed melanosomes. This evidence further underlines the importance of the actin cytoskeleton, rather than the microtubule network as previously thought, for the anterograde trafficking of melanosomes.

Tissue engineering aims to regenerate, repair or replace organs or tissues which have become defective due to trauma, disease or age related degeneration. This engineering may take place within the patient's body or tissue can be regenerated in a bioreactor for later implantation into the patient. Regeneration of soft tissue is one of the most demanding applications of tissue engineering. Producing proper nutrient supply, uniform cell distribution and high cell density are the important challenges. Many experimental models exist for tissue growth in a bioreactor. It is important to put experiments into a theoretical framework. Mathematical modelling in terms of physical and biochemical mechanisms is the best tool to understand experimental results. In this work a mathematical model of convective and diffusive transport of nutrients and cell growth in a perfusion bioreactor is developed. A cell-seeded porous scaffold is placed in a perfusion bioreactor and fluid delivers the nutrients to the cells for their growth. The model describes the key features of the tissue engineering processes which includes the interaction between the cell growth,variation of material porosity, flow of fluid through the material and delivery of nutrients to the cells. The fluid flow through the porous scaffold is modelled by Darcy's law, and the delivery of nutrients to the cells is modelled by the advection-diffusion equation. A non-linear reaction diffusion system is used to model the cell growth. The cell diffusion depends on the cell density and growth of cells is modelled by logistic growth. The effect of shear stress on nutrient consumption and cell growth is also included in the model. COMSOL (a commercial finite element solver) is used to numerically solve the model. The results show that the distribution of cells and total cell number in the scaffold depends on the initial cell density and porosity. We suggest various seeding strategies and scaffold designs to improve the cell growth rate and total cell yield.

The seaweed fly, Coelopa frigida, lives in piles of rotting seaweed deposited on beaches by tides and winds. In all populations studied it is polymorphic for two gene arrangements on Chromosome I. A polymorphism at the alcohol dehydrogenase locus is strongly associated with this inversion and can be used to estimate karyotype frequencies. An extensive series of samples from natural populations has revealed a seasonal cycle in inversion frequencies but otherwise frequencies are remarkably constant both geographically and temporally. There is a consistent excess of heterokaryotypes in these samples. Three selective forces influencing inversion frequencies have been investigated. 1) An association between karyotype and development time, previously observed in the laboratory, has been demonstrated in conditions case to those in natural populations. 2) Viability differences between karyotypes have been examined. In natural populations there is some evidence that the excess of heterokaryotypes increases with larval density. In the laboratory heterokaryotypes are shown to have higher viability than either homokaryotype but the strong density dependence reported previously has not been observed. Viability differences are concentrated in the first two days of larval life and are probably related to the rate of supply of nutrients. 3) An association is demonstrated between karyotype and adult size - especially in males. Adult size is shown to correlate with longevity and fecundity of both sexes. Several experiments indicate that large males enjoy greater mating success than small males. The relationship between larval density, development time and adult size is described. The possibility that the effect of the inversion varies between populations or between alcohol dehydrogenase genotypes has also been investigated. A simulation has been used to study how these selective forces interact with one another, and with the changeable environment in which the flies live, and to examine the extent to which they can account for the observed karyotype frequencies.