The native corneal structure is highly organised and unified in architecture with structural and functional integration which mediates its transparency and mechanical strength. Two of the most demanding challenges in corneal tissue engineering are the replication of the native corneal stromal architecture and the preservation of stromal cell phenotype which prevents scar-like tissue formation. A concerted effort in this thesis has been devoted to the generation of a functional human corneal stromal model by the manipulation of chemical, topographical and cellular cues. To achieve this, previously built non-destructive, online, real-time monitoring techniques, micro-indentation and optical coherence tomography (OCT), which allow for the mechanical and contraction properties of corneal equivalents to be monitored, have been improved. These macroscopic parameters have been cross-validated by histological, imunohistochemical, morphological and genetic expression analysis.

With the progression of multivariate statistics, the creation of population specific equations is on the rise. Multivariate analysis generally revolves around metric methods or geometric morphometrics, not on morphoscopic features. A total of eight samples were analysed spanning from prehistoric American to modern day South African and ranged between pygmy populations from the Andaman Islands to medieval British populations. With a sample size of more than 1100 individuals, each os coxa was scored using eight morphoscopic features most commonly used by physical anthropologists and osteoarchaeologists. Trait frequencies were compiled and compared between each of the eight samples. Then, the samples were placed into two groups: a known age and sex group (Christ Church Spitalfields, South African White, South African Black, and South African Coloured), and an unknown archaeological group (Poulton, St. Owens, Chumash, and Andaman). When comparing trait frequencies, slight differences between the samples could be seen. Ordinal Logistic Regressions (OLR) were applied onto each of the four samples from the known age and sex group to create population specific sexing equations (cross-validated). Results from these four equations ranged from 90.24% (South African Black population specific equation) to 96.38% (Christ Church, Spitalfields population specific equation). Population specifity was tested by applying all of the equations onto each sample in this group. In an attempt to reduce this, two new equations were created by combining samples together resulting in a South African specific equation (92.54% accuracy) and a "Summary Sex" equation (92.98% accuracy). After applying each of the six new OLR equations onto the four archaeological samples, high percentage accuracies (ranging from 92.59% to 100.00%) were found when comparing them to the previous records. The only sample that did not produce as high of an accuracy was the Chumash sample with 82.35%. In the attempt to analyse fragmented remains, three avenues were taken. Firstly, all missing values were replaced by the median score. Secondly, the original six OLR equations were 'sectioned' to make three smaller sets of equations. Lastly, to mirror the sectioned equations, three new sets of OLR equations were generated. This study shows that when using morphoscopic traits for sex estimation, applying multivariate techniques can be used to obtain a high accuracy even when dealing with fragmented samples.

In this thesis, a phase sensitive interferometer is successfully implemented to perform birefringent object surface-profile measurement, based on a polarisation adjustment approach. Using monochromatic light, a novel polarization interferometric method is developed, incorporating the birefringence technique and a waveplate. In our experiments, a birefringent wedge is designed for generating carrier fringes in the polariscope. Retardance is calculated from phase shifting using a phase matching technique. The accuracy of the method has been demonstrated to have an error of less than 0.02 radians. The accuracy and resolution quantitative analysis presented in this thesis can be used to determine accurately the phase-shifting interferometry for high-precision surface profile and bio-structure, such as fibre and collagen measurements with low cost. FFT technique and phase-stepping methods are described to determine birefringence within the cornea. The distribution of human corneal lamellar collagen is determined through a microscopic technique using the combination of a circular polariser and a quarter-wave retarder. A quantitative measure of corneal birefringence is achieved by phase unwrapping. The experimental findings of elliptic and hyperbolic populations of collagen fibrils may explain the optical phenomena of central corneal retardation with biaxial-like behaviour in more peripheral areas. A low cost, simple, and direct approach has been developed to make the required microscopic measurement. The traditional transmission system is improved by applying a reflection system with an LED light source and is suitable for the analysis of the birefringent cornea structures in vivo. A further instrument based upon a synthetic aperture approach has been created with the purpose of measuring the three dimensional birefringence structure of the cornea. The concept of the instrument is a combination of the parallax between individual lenses and the numerically generated planes of focus to visualise the phase structure.

Tendon injuries have, for many years, frustrated clinicians and patients alike due to their longevity and resistance to therapy. In recent years there has been good response in the extensor tendons of the lower limb to an intense painful eccentric exercise protocol. As yet there is no established reason known why a tendon should develop degeneration within its structure or why it should respond to the eccentric exercises. We do however know that, like bone, tendons are biologically active and rapidly adapt to the mechanical environment to which they are exposed. Recent investigations have revealed that within a tendon such as the Achilles or the patellar tendon there may be regions that experience different strains to the rest of the tendon. Much of this work has been in vitro and an ultimate goal would be the development of a non-invasive method by which intra-tendinous strain might be measured. The basis of this thesis is the validation of an existing grey-scale speckle pattern matching software programme developed for tracking motion through serial ultrasound images. Through in vitro and in vivo work we have developed its use for tracking the unique type of speckle found in tendons. By verifying, in vitro, that the displacements tracked in phantoms and tendons alike are representative of reality we provide confidence in the use of an exciting tool for measuring tendon motion in vivo. Furthermore, we have established the method by which the tracking can be adapted to accurately represent tendon strain in vitro which again provides assurance for its reliability when applied to examine tendon strain in vivo. The methods of data collection and analysis developed in this study provide the foundations for an exciting avenue of research into tendon biomechanics.

Articular cartilage can be damaged directly through injury or osteoarthritis (OA). This tissue is very poor at regenerating itself due to its avascular nature and the immobility of chondrocytes within the tissue. There are a range of surgical techniques to repair cartilage lesions. Cellular therapies such Autologous Chondrocyte Implantation (ACI) and later modifications have been used to repair cartilage lesions for the past two decades. However, there is currently no completely successful treatment of cartilage lesions, with the newly generated cartilage often possessing very poor mechanical properties. Also, cell-based therapies require large numbers of chondrocytes which have to be expanded in monolayer. A consequence of this expansion is a loss of the chondrocyte phenotype (dedifferentiation). The overall aim of this thesis was to develop a greater understanding of chondrocyte dedifferentiation and redifferentiation in vitro using canine chondrocytes. Dogs can also suffer with OA and have been used extensively as a model for OA. Firstly, canine chondrocytes were expanded in monolayer up to P5 to confirm dedifferentiation. These cells were shown to have lost their typical chondrocytic phenotype through decreased expression of collagen type II and increased expression of collagen type I and CD44. A considerable part of this element of the thesis also involved identifying antibodies that would cross-react with the target canine antigens. The next aim of the thesis was to redifferentiate dedifferentiated chondrocytes through three-dimensional (3D) culture. Initial problems with high density pellet culture led to the selection of a supporting material. Alginate was chosen as it is a naturally occurring polymer which has previously been used to culture chondrocytes in 3D. After making several adjustments to the set-up and downstream analysis of the beads, chondrocytes from different passages were seeded into them. Alginate beads seeded with P2 chondrocytes appeared to contain cells with a more chondrocyte-like phenotype compared to P3- and P4-seeded beads. However, expression of collagen type I was still relatively high in P2-seeded beads, indicating 3D culture alone is not enough to induce complete redifferentiation. Therefore, the final aim of this thesis was to enhance the redifferentiation of dedifferentiated canine chondrocytes. Two initial conditions were selected; addition of 25μg/ml ascorbate to the culture medium and incubating the beads under reduced oxygen conditions (2.4%). Culturing the beads under reduced oxygen conditions (2.4%) appeared to enhance redifferentiation. However addition of ascorbate to the culture medium had mixed results. This culture system can now be further adapted and modified to better enhance chondrocyte redifferentiation. This work could include combining the two conditions already tested as well as adding growth factors to the culture medium. More successful maintenance of the chondrocyte phenotype in vitro, could potentially lead to better articular cartilage regeneration both in vitro and in vivo.

The thymus is organised into distinct microenvironments, and trafficking through these regions enables thymocytes to receive essential signals for the generation of a diverse and self-tolerant T-cell repertoire. Thymic epithelial cells (TEC) represent a key stromal cell type during defined stages in T cell development, yet the mechanisms regulating their development are only partly understood. An ontogenetic approach was employed to study stages of cortical thymic epithelial cell (cTEC) development. This study identifies a previously unreported population of cTEC progenitors expressing CD205 and 5T and has defined distinct checkpoints in the development of the cTEC lineage. Furthermore, the importance of thymic crosstalk during specific stages of cTEC development and also the requirement for RANK-RANKL signalling for the development of various medullary thymic epithelial cell (mTEC) subsets has also been defined. Additionally, the importance of chemokine-mediated signalling for the establishment and compartmentalisation of the thymus has been highlighted by employing laser capture microdissection and studying thymus microenvironments in mice deficient for particular chemokine related signalling pathways. Overall this study has provided novel insight into the development of the thymic cortex and will help to understand how these cells become specialised in their ability to support positive selection of developing T cells.

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic effector cells. Cryptococcus can survive and proliferate within macrophages, and is also capable of escaping into the intracellular environment via a non-lytic mechanism (‘expulsion’) and can be transferred directly from one cell to another (lateral transfer). In the first part of this thesis, I demonstrate that enhanced Th2, but not Th1, cytokine levels lead to increased intracellular cryptococcal proliferation but lower levels of cryptococcal expulsion. In the second part, I describe the generation and characterisation of GFP-expressing derivates of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Furthermore, I have developed a method to effectively and rapidly investigate macrophage parasitism by flow cytometry that preserves the accuracy of current approaches but offers a four-fold improvement in speed. The final part dissects the regulation and induction of mitochondrial tubularisation in hypervirulent C. gattii strains and describes the first steps towards a comparative mitochondrial genome sequencing approach to identify the underlying molecular mechanisms.

Arboreal travel for large apes is energetically demanding and risky due to the complexity of the forest canopy. Careful selection of supports is therefore essential for safe and efficient locomotion. This thesis investigates the factors involved in route and support selection in bonobos (Pan paniscus) and in modern human (Homo sapiens) tree climbers. Naturalistically housed bonobos were given a choice of two ropes, one that provided easy access and another that required more demanding postures, with which to access a hard-to-reach food goal. The bonobos selected a rope based on its distance from the goal and its flexibility. Decision making in human tree climbers was investigated using a novel combination of qualitative (participant interviews) and quantitative (observations of behaviour) data. Participants were asked to collect goals from within a tree crown three times each. Interviews revealed that participants either considered risk avoidance or ease/efficiency as the main factor influencing their decisions whilst climbing. Those considering risk took longer to complete each climb, but became quicker after their first climb. These studies demonstrate that the demands of the arboreal environment require knowledge of the functional properties of supports and that memory of specific routes may increase the efficiency of arboreal locomotion.

Hepatocytes can capture dead cells. This phenomenon is called efferocytosis. Furthermore, our lab previously observed live CD4+ T cells captured by hepatocytes. This was reminiscent of entosis. This project aimed to further the understanding of the mechanisms and consequences of these processes. In vitro experimentation showed that efferocytosis could be modulated through cytokine treatment and using macropinocytosis inhibitors. Captured cells were also shown to associate with the uncharacterised receptor, SCARF2. Furthermore, efferocytosis was shown to cause multinucleation in hepatocytes. This was demonstrated in vitro, in vivo using mouse models of acute injury, and ex vivo with cauterised donor human tissue. Increased multinucleation was also associated with hepatocellular carcinoma and vascular invasion. Live CD4+ T cell capture occurred at a lower frequency than efferocytosis and required alternative membrane rearrangements. This process also did not share defining characteristics of entosis, such as E-cadherin association or susceptibility to Rho-kinase inhibitors. Furthermore, anti-inflammatory T-regulatory cells were more likely to enter acidic compartments within their captors in vitro. This project has unearthed novel aspects regarding the regulation and molecular processes of hepatocyte cell-in-cell structure formation. Further understanding into the mechanisms of these processes may provide future targets for therapeutic intervention of inflammatory disease and cancer.

How a stimulus, such as an odour, is represented in the brain is one of the main questions in neuroscience. It is becoming clearer that information is encoded by a population of neurons, but, how the spiking activity of a population of neurons conveys this information is unknown. Several population coding hypotheses have formulated over the years, and therefore, to obtain a more definitive answer as to how a population of neurons represents stimulus information we need to test, i.e. support or falsify, each of the hypotheses. One way of addressing these hypotheses is to record and analyse the activity of multiple individual neurons from the brain of a test subject when a stimulus is, and is not, presented. With the advent of multi electrode arrays (MEA) we can now record such activity. However, before we can investigate/test the population coding hypotheses using such recordings, we need to determine the number of neurons recorded by the MEA and their spiking activity, after spike detection, using an automatic spike sorting algorithm (we refer to the spiking activity of the neurons extracted from the MEA recordings as MEA sorted data). While there are many automatic spike sorting methods available, they have limitations. In addition, we are lacking methods to test/investigate the population coding hypotheses in detail using the MEA sorted data. That is, methods that show whether neurons respond in a hypothesised way and, if they do, shows how the stimulus is represented within the recorded area. Thus, in this thesis, we were motivated to, firstly, develop a new automatic spike sorting method, which avoids the limitations of other methods. We validated our method using simulated and biological data. In addition, we found our method can perform better than other standard methods. We next focused on the population rate coding hypothesis (i.e. the hypothesis that information is conveyed in the number of spikes fired by a pop- ulation of neurons within a relevant time period). More specifically, we developed a method for testing/investigating the population rate coding hypothesis using the MEA sorted data. That is, a method that uses the multi variate analysis of variance (MANOVA) test, where we modified its output, to show the most responsive subar- eas within the recorded area. We validated this using simulated and biological data. Finally, we investigated whether noise correlation between neurons (i.e. correlations in the trial to trial variability of the response of neurons to the same stimulus) in a rat's olfactory bulb can affect the amount of information a population rate code conveys about a set of stimuli. We found that noise correlation between neurons was predominately positive, which, ultimately, reduced the amount of information a population containing >45 neurons could convey about the stimuli by ~30%.