This thesis describes an investigation into silk spinning, with the objective of producing high performance silk fibres in the laboratory using a novel spinning device based upon observations on natural spinning glands and processes. After an in-depth literature review the work is reported in two sections: natural and artificial spinning. The literature provides fragmented data on different aspects of natural silk production, and artificial spinning has not yet reproduced fibres with the properties of native silk fibres, despite unfounded claims of biomimetic spinning. The first half of the thesis looks at natural silk spinning. The work started with a general study of the morphology of spider and silkworm spinning ducts: First, how the silk fibre develops as the dope flows through the gland; and second the relationship between silk fibre properties and both gland morphology and spinning speed. More detailed studies using histochemical and spectroscopic investigations showed that the silk ducts of the spider Nephila edulis and the silkworm Bombyx mori both contain β-chitin, despite an evolutionarily distant common ancestor. Finally, observations showed that the duct of N. edulis consists of alternating nanoporous discs, and FEA modelling indicated that the duct is optimised for mechanical integrity and permeability. The second half of the thesis describes the development of a spinning device that uses natural silk dope mainly taken from B. mori as feedstock. It begins with a description of the gradual development of the engineering aspects of the spinning device, to meet challenges raised during the spinning investigation. The development of a centrifugal capillary rheometer, for practical quantitative insights into rheological processes is then presented. Finally the spinning investigation is reported: first, the screening of spinning in glass capillaries based upon natural gland dimensions and flow rates, which have been shown to induce fibrillation in silk dope in a rheometer, and also included initiation of instability through heat applied along the capillary; second, the final spinning evaluation, using lessons learned from all the screening trials throughout the project, but also including a key development of a hydrophobic coating on the capillary tip to inhibit droplet formation and massively increase the process stability and ease of fibre production. The main conclusions from this work are that good silk fibre cannot be spun by flow shear stress alone; and, that heat instability induces indiscriminate gelation of the silk, whose disordered molecular structure gives poor silk fibre properties. The body of work behind these conclusions provides fundamental background information and new insights that will contribute to the next stages of development of artificial silk spinning, from obtaining a better understanding of the biology of natural spinning glands to the engineering difficulties of implementing the bioinspired principles.

This dissertation clarifies the concept of evolvability, the increased capacity of some organisms or systems to support evolution, especially the evolution of life-like complexity. I survey the literature, which is spread over the fields of population genetics, developmental biology, artificial life, and microbial and molecular evolution. Finding that researchers have often used the term vaguely and incompatibly I identify five distinct kinds or senses of evolvability. I also identify five key constituent ideas, which I discuss in the context of organismic evolvability, a sense of evolvability with deep roots in the traditional fields of animal development and macroevolution. In these fields research into evolvability has historically been hampered by an insufficiently detailed knowledge of development. Research in molecular evolution has produced a thorough knowledge of the folding of RNA into secondary structure, which can be regarded as a model of development. This has motivated new approaches to evolvability based on representing development via a single genotype-phenotype mapping function. I build on these approaches to invent new mathematical methods to formalise the traditional ideas. I create an exact model illustrating a classic example of evolvability, the capacity for repeated segmentation and simple modularity. I analyse this with two new formal approaches. First is the genospace algebra, a propositional calculus based on graph theory. It is a formal language for describing genotype-phenotype maps. It provides a system for making calculations, proofs, and diagrams about mutational structures in genotype space, and it is flexible enough to allow description at arbitrary degrees of resolution. Second is a pair of concepts, the genetic leverage and the genetic fulcrum. The leverage provides a crude numerical measure of evolvability, and the fulcrum provides a heuristic for identifying the genomic and developmental causes of evolvability. Besides its specific relevance to diversification and development, evolvability is also crucial to the fundamental question of how evolution produces ordinary biological life. Simulation systems that implement only a conventional textbook model of evolution -– systems possessing only variation, inheritance, and selection –- fail to evolve anything resembling the complexity of the biological world. Research into evolvability is our best bet to illuminate the "missing ingredient" for life-like evolution.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. Subplate plays a fundamental role in cortical development. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of corticofugal and thalamic axons. At later stages, these cells are involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. In my thesis, I aimed to further characterize the embryonic murine subplate by establishing a gene expression profile of this population at embryonic day 15.5 (E15.5) using laser capture microdissection combined with microarrays. I found over 250 transcripts with presumed higher expression in the subplate at E15.5. Using quantitative RT-PCR, in situ hybridization and immunohistochemistry, I have confirmed specific expression in the E15.5 subplate for 13 selected genes which have not been previously associated with this compartment. In the reeler mutant, the expression pattern of a majority of these genes was shifted in accordance with the altered position of subplate cells. These genes belong to several functional groups and likely contribute to the maturation and electrophysiological properties of subplate cells and to axonal growth and guidance. The roles of two selected genes - cadherin 10 (Cdh10) and Unc5 homologue c (Unc5c) - were explored in more detail. Preliminary results suggest an involvement of Cdh10 in subplate layer organization while Unc5c could mediate the waiting period of subplate corticothalamic axons in the internal capsule. Finally, I compared the expression of a selection of subplate-specific genes (subplate markers) between mouse and rat and found some surprising species differences. Confirmed subplate markers were used to monitor subplate injury in a rat model of preterm hypoxiaischemia and it appeared that deep cortical layers including subplate showed an increased vulnerability over upper layers. Further characterization of subplate-specific genes will allow us to broaden our understanding of molecular mechanisms underlying subplate properties and functions in normal and pathological development.

Sudden cardiac death is one of the leading causes of mortality in the western world. One of the main factors is myocardial ischaemia, when there is a mismatch between blood demand and supply to the heart, which may lead to disturbed cardiac excitation patterns, known as arrhythmias. Ischaemia is a dynamic and complex process, which is characterised by many electrophysiological changes that vary through space and time. Ischaemia-induced arrhythmic mechanisms, and the safety and efficacy of certain therapies are still not fully understood. Most experimental studies are carried out in animal, due to the ethical and practical limitations of human experiments. Therefore, extrapolation of mechanisms from animal to human is challenging, but can be facilitated by in silico models. Since the first cardiac cell model was built over 50 years ago, computer simulations have provided a wealth of information and insight that is not possible to obtain through experiments alone. Therefore, mathematical models and computational simulations provide a powerful and complementary tool for the study of multi-scale problems. The aim of this thesis is to investigate pro-arrhythmic electrophysiological consequences of acute myocardial ischaemia, using a multi-scale computational modelling and simulation framework. Firstly, we present a novel method, combining computational simulations and optical mapping experiments, to characterise ischaemia-induced spatial differences modulating arrhythmic risk in rabbit hearts. Secondly, we use computer models to extend our investigation of acute ischaemia to human, by carrying out a thorough analysis of recent human action potential models under varied ischaemic conditions, to test their applicability to simulate ischaemia. Finally, we combine state-of-the-art knowledge and techniques to build a human whole ventricles model, in which we investigate how anti-arrhythmic drugs modulate arrhythmic mechanisms in the presence of ischaemia.

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.

Colorectal cancer (CRC) arises in part from the dysregulation of cellular proliferation, associated with the canonical Wnt pathway, and differentiation, effected by the Notch signalling network. In this thesis, we develop a mathematical model of ordinary differential equations (ODEs) for the coupled interaction of the Notch and Wnt pathways in cells of the human intestinal epithelium. Our central aim is to understand the role of such crosstalk in the genesis and treatment of CRC. An embedding of this model in cells of a simulated colonic tissue enables computational exploration of the cell fate response to spatially inhomogeneous growth cues in the healthy intestinal epithelium. We also examine an alternative, rule-based model from the literature, which employs a simple binary approach to pathway activity, in which the Notch and Wnt pathways are constitutively on or off. Comparison of the two models demonstrates the substantial advantages of the equation-based paradigm, through its delivery of stable and robust cell fate patterning, and its versatility for exploring the multiscale consequences of a variety of subcellular phenomena. Extension of the ODE-based model to include mutant cells facilitates the study of Notch-mediated therapeutic approaches to CRC. We find a marked synergy between the application of γ-secretase inhibitors and Hath1 stabilisers in the treatment of early-stage intestinal polyps. This combined treatment is an efficient means of inducing mitotic arrest in the cell population of the intestinal epithelium through enforced conversion to a secretory phenotype and is highlighted as a viable route for further theoretical, experimental and clinical study.

<p>The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. <i>Helicobacter pylori</i> might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of <i>H. pylori</i> on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat.</p><p>A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (<20μm) and discontinuous in the small intestine.</p><p>Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from <i>H. pylori</i> (HPE). In animals with a chronic <i>H. pylori</i> infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. </p><p>HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.</p>

The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. Helicobacter pylori might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of H. pylori on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat. A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (&lt;20μm) and discontinuous in the small intestine. Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from H. pylori (HPE). In animals with a chronic H. pylori infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.

Background: Research in heart disease can be aided by modelling myocardial hemodynamics with knowledge of coronary pressure and vascular resistance measured from the geometry and morphometry of coronary vasculature. This study presents methods to automatically reconstruct accurate detailed coronary vascular anatomical models from high-resolution three-dimensional optical fluorescence cryomicrotomography image volumes for simulating blood flow in coronary arterial trees. Methods: Images of fluorescent cast and bead particles perfused into the same heart comprise the vasculature and microsphere datasets, employed in a novel combined approach to measure vasculature and simulate a flow model on the extracted coronary vascular tree for estimating regional myocardial perfusion. The microspheres are used in two capacities - as fiducial biomarker point sources for measuring the image formation in order to accurately measure the vasculature dataset and as flowing particles for measuring regional myocardial perfusion through the reconstructed vasculature. A new model-based template-matching method of vascular radius estimation is proposed that incorporates a model of the optical fluorescent image formation measured from the microspheres and a template of the vessels’ tubular geometry. Results: The new method reduced the error in vessel radius estimation from 42.9% to 0.6% in a 170 micrometer vessel as compared to the Full-Width Half Maximum method. Whole-organ porcine coronary vascular trees, automatically reconstructed with the proposed method, contained on the order of 92,000+ vessel segments in the range 0.03 – 1.9 mm radius. Discrepancy between the microsphere perfusion measurements and regional flow estimated with a 1-D steady state linear static blood flow simulation on the reconstructed vasculature was modelled with daughter-to-parent area ratio and branching angle as the parameters. Correcting the flow simulation by incorporating this model of disproportionate distribution of microspheres reduced the error from 24% to 7.4% in the estimation of fractional microsphere distribution in oblique branches with angles of 100°-120°.

In this thesis I apply new sequencing technologies and analytical methods derived from genomics and computer science to the neuroanatomy of gene expression. The first project explores characteristics of gene expression across adult neocortical layers in a representative mammal – the mouse. Amongst the thousands of genes and transcripts differentially expressed across layers, I found common functional characteristics of genes that define certain layers, candidate cases of isoform switching, and over a thousand apparent long intergenic non-coding RNA transcripts. The second project compares patterns of gene expression in the structurally diverged adult derivatives of the pallium in mice and chickens. Overall, gene expression levels were moderately correlated between the two species. While expression patterns of ‘marker’ genes were only poorly conserved in these regions, there nevertheless was significant conservation of cross-species marker genes for homologous structures, cell types and functionally analogous regions. Many aspects of these data from both projects can now be easily browsed and searched from custom-built web interfaces. In addition to generating unprecedented genome-wide resources for the neuroscience community to explore the functional and structural dimensions of gene expression amongst different pallial regions in mammals and birds, this work also provides new insights into the widespread evolutionary shuffling of adult marker gene expression.