Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

The fundamental corneal properties of mechanical rigidity, maintenance of curvature and optical transparency result from the specific organisation of collagen fibrils in the corneal stroma. The exact arrangement of stromal collagen is currently unknown but several structural models have been proposed. The purpose of the present study is to investigate inconsistencies between current x‐ray derived structural models of the cornea and optically derived birefringence data. Firstly, the thesis reviews the current understanding of corneal structure, particularly in relation to corneal birefringence. It also reviews and develops the different analytical approaches used to model optical biaxial behaviour, particularly as applied to predict corneal optical phase retardation. The second part develops a novel technique of elliptic polarization biomicroscopy (EPB), enabling study of corneal birefringence in vivo. Using EPB, the pattern of corneal retardation is recorded for a range of human subjects. This dataset is then used to investigate both central and peripheral corneal birefringence as well as the corneal microstructure. A key finding is that the central parts of the cornea exhibit a retardation pattern compatible with a negative biaxial crystal, whereas the peripheral corneal regions do not. Furthermore, within the central regions of the cornea, orthogonal confocal conic fibrillar structures are identified which resemble the analytically derived contours of equal refractive index of an ideal negative biaxial crystal. The third part of this work presents a synthesis of previous published experimental, anatomical and theoretical findings and the experimental results presented in this thesis. Based on these findings, a novel corneal structural model is proposed that comprises overlapping spherical elliptic structural units. Finally, ensuing biomechanical and clinical consequences of the spherical elliptic structural model and of the EPB technique are discussed including their potential diagnostic and surgical applications.

Stereophotogrammetric imaging systems produce representations of surfaces (two-dimensional manifolds in three-dimensional space) through triangulations of a large number of estimated surface points. Traditional forms of analysis of these surfaces are based on point locations (manually marked anatomical landmarks) as described in Chapter 1. An advanced application of these types of landmarks will be thoroughly examined in Chapter 2 through the concept of Ghost Imaging. The results of this chapter necessitated a reliability study of stereophotogrammetric imaging systems which is discussed in Chapter 3. Given the results of the reliability study, an investigation info new definitions of landmarks and facial shape description is undertaken in Chapter 4. A much richer representation is expressed by the curves which track the ridges and valleys of the dense surface and by the relatively smooth surface patches which lie between these curves. New automatic methods for identifying anatomical curves and the resulting full surface representation, based on shape index, curvature, smoothing techniques, warping, and bending energy, are described. Chapter 5 discussed new and extended tools of analysis that are necessary for this richer representation of facial shape. These methods will be applied in Chapter 6 to different shape objects, including the human face, mussel shells, and computational imaging comparisons. Issues of sexual dimorphism (differences in shapes between males and females), change in shape with age, as well as pre- and post-facial surgical intervention will be explored. These comparisons will be made using new methodological tools developed specifically for the new curve and surface identification method. In particular, the assessment of facial asymmetry and the questions involved in comparing facial shapes in general, at both the individual and the group level, will also be considered. In Chapter 7, Bayesian methods are explored to determine further ways in which to understand and compare human facial features. In summary, this thesis shows a novel method of curve and full facial mesh identification that is used, successfully, in pilot case studies of multiple types of surfaces. It then shows a novel proof of principle for using Bayesian methods to create a fully automatic process in facial shape characterisation. In order to view this thesis in full, please view in Adobe Reader.

How do we know where objects are in the environment and how do we use this information to guide our actions? Recovering the three-dimensional (3D) structure of our surroundings from the two-dimensional retinal input received from the eyes is a computationally challenging task and depends on the brain processing and combining ambiguous sources of sensory information (cues) to depth. This thesis combines psychophysical and computational techniques to gain further insight into (i) which cues the brain uses for perceptual judgments of depth and motion-in-depth; and (ii) the processes underlying the combination of the information from these cues into a single percept of depth. The first chapter deals with the question which sources of information the visual system uses to estimate the time remaining until an approaching object will hit us; a problem that is complicated by the fact that the variable of interest (time) is highly correlated to other perceptual variables that may be used (e.g. distance). Despite these high correlations we show that the visual system recovers a temporal estimate, rather than using one or more of its covariates. In the second chapter I ask how extra-retinal signals (changes in the convergence angles of the eyes) contribute to estimates of 3D speed. Traditionally, extra-retinal signals are reputed to be a poor indicator of 3D motion. Using techniques to isolate extra-retinal signals to changes in vergence, we show that judgments of 3D speed are best explained on the basis that the visual system computes a weighted average of retinal and extra-retinal signals. The third and fourth chapters investigate how the visual system combines binocular and monocular cues to depth in judgments of relative depth and the speed of 3D motion. In chapter three I show that differences in retinal size systematically affect the perceived disparityde defined depth between two unfamiliar targets, so that a target with a larger retinal size is seen as closer than a target with a smaller retinal size at the same disparity-defined distance. This perceptual bias increases as the retinal size ratio between the targets is increased but remains constant as the absolute sizes of the targets change concurrently while keeping the retinal size ratio constant. In addition, bias increases as the absolute distance to both targets increases. I propose that these findings can be explained on the basis that the visual system attempts to optimally combine disparity with retinal size cues (or in the case of 3D motion: changing disparity information with looming cues), but assumes that both objects are of equal size while they are not. In chapter 4 these findings are extended to 3D motion: physically larger unfamiliar targets are reported to approach faster than a smaller target moving at the same speed at the same distance. These findings cannot be explained on the basis of observers' use of a biased perceived distance, caused by differences in the retinal size (as found in chapter 3). I conclude that, in line with contemporary theories of visual perception, the brain solves the puzzle of 3D perception by combining all available sources of visual information in an optimal manner, even though this may lead to inaccuracies in the final estimate of depth.

The work described in this thesis concerns the synthesis, DNA binding and cytotoxicity studies of new Rh(III) supramolecular complexes. Chapter 1 reviews DNA molecular recognition by synthetic agents; exploring the different DNA binding modes and their importance in the anticancer properties of several metallodrugs. Special attention is given to the exciting cylinder agents, which underpin the work in this thesis and to the work with rhodium complexes and their studies with DNA and as anticancer drugs. Chapter 2 describes the synthesis, purification and characterization of Rh(III) mononuclear, dinuclear single, double and triple stranded complexes. NMR, MS, UV‐Vis, elemental analyses and in some cases X‐ray crystallography are discussed in detail. In Chapter 3, DNA binding properties of the Rh(III) complexes are explored by CD and LD spectroscopy. Gel Electrophoresis experiments are also carried out using plasmid DNA (pBR322). The dinuclear complexes are found to bind to ct‐DNA and to have more dramatic effects than the mononuclear analogues. In addition they were found to cleave plasmid DNA. Chapter 4 presents cytotoxicity studies for some of the complexes synthesized against breast and ovarian cancer cell lines. A PCR study with the Rh(III) double stranded isomers is also carried out demonstrating that these complexes are able to inhibit and block DNA transactions as represented by PCR DNA replication.

In this thesis, the design, fabrication and characterisation of a bio-inspired microelectromechanical systems (MEMS) based tactile sensor array is presented. A vast amount of research has been carried out in the area of tactile sensing and various transduction methods have been explored. However, currently no device exists with a performance comparable to that of the biological tactile sensors of the human fingertip in terms of robustness, sensitivity, spatial resolution and dynamic performance. The sensors developed in this work employ the principles of electrical capacitance and are fabricated from commercially available siliconon- oxide wafers using simple process steps. Each sensor is formed from two plates of highly conductive silicon separated by an air-gap formed from sacrificial etching of the oxide layer. Deflection of the 2 \(\mu\)m thick upper plate of the sensor as a result of applied mechanical stimulus, causes a change in capacitance which is the output of the sensor. Within the array, the individual sensors are spaced 150 \(\mu\)m apart (centre-centre pitch of 570 \(\mu\)m) and therefore offer the potential for high spatial resolution. To protect the sensor array from mechanical shock and provide skin like compliance, the use of suitable packaging materials was explored. The use of poly dimethyl siloxane (PDMS) as a suitable skin-like material was demonstrated. Modification of the surface topography of the packaging layer to include ’fingerprint’ like features was explored and its benefits highlighted. Sensor characterisation experiments revealed that the sensing device was sufficiently sensitive to allow the discrimination of different textures (with feature spacing down to 0.2 mm) through tests conducted using gratings varying in spatial periodicity and fabrics. Based on the results, the sensors can be used as an analogue of the slowly adapting tactile receptors (Merkel disks) for robotic finger applications.

The natural angiogenic response of the vasculature to cardiovascular disease has been shown, at least in part, to involve circulating endothelial progenitor cells (EPCs). However, the native response is often insufficient to restore vascularity without additional intervention. In this study the angiogenic activity of EPCs, demonstrated by in vitro tubule formation, confirmed the suggested potential of EPCs to be used therapeutically. However, as EPCs are found in limited circulating numbers, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were also investigated as sources of donor EPCs for transplantation. Here ESCs, but not iPSCs, were shown to generate cells with a genetic and proteomic profile, as well as an angiogenic potential, identical to natural EPCs. Using an in vivo mouse model of hindlimb ischemia, this investigation illustrated the preferential binding of transplanted EPCs at sites of angiogenic stimulation, and revealed the importance of platelets in the recruitment of circulating EPCs. In particular, using in vitro aggregation and flow-based adhesion assays, the adhesion molecule P-selectin was shown to play a significant role in this recruitment mechanism. In conclusion, this study has demonstrated that EPC transplantation has abundant potential for development into a viable and efficiacious therapeutic angiogenic treatment.

Tendon and ligament damage causes extreme pain and decreased joint functionality. Current repair methods cannot restore original joint biomechanics nor promote regeneration of native tissue. Recent advances in tendon and ligament repair have involved engineering tissue using cell-seeded scaffolds. Self-aligned cellular structures, similar to those in ligaments and tendons, have been successfully formed, albeit with weak attachment between construct and bone. Calcium phosphates form an intimate bond with both soft and hard tissues and have successfully been used in tissue engineering bone, whilst hydrogels have often been used as cellular scaffolds. This thesis explores agarose, gelatin, carrageenan and fibrin hydrogels as potential soft tissue scaffolds. Fibrin gel exhibited high cellular compatibility with highest metabolic activity on day 14. Although the cellular gel contracted significantly, it was found that the dry weight remained stable in both the acellular and cellular forms. 3D powder printed calcium phosphate scaffolds remained structurally stable after immersion in cell culture media with immersion in protein-rich sera promoting tenocyte attachment. Bracket designs were developed to enhance grip of the cell-seeded fibrin. Ligament constructs were selfsupporting and exhibited structural characteristics similar to native connective tissue. Tenocyte density peaked on day 14, with added L-proline and ascorbic acid inducing a constant level of glycosaminoglycans and 7.4 ± 1.5 % w/w collagen. This research may significantly enhance the clinical application of tissue engineered ligaments and tendons.

Factors contributing towards falls in older age during overground walking have been widely studied. Stepping behaviour, balance and head posture control during stair negotiation in young adults (YA) and older adults with either lower (LROA) or higher (HROA) risk of falling during midstair negotiation have not been investigated. The aims of the thesis were threefold. Firstly, age-related changes in gaze behaviour were investigated. The main finding was that older adults fixate stair edges for longer than YA. Secondly, the effect of manipulating visual information on stepping parameters and balance control was compared between YA, LROA and HROA. For stair ascent, stepping and balance control was preserved in LROA and HROA and highlighted stair edges led to increased foot clearance in all groups. For stair descent, HROA demonstrated smaller foot clearance than LROA and highlighted stair edges improved balance in LROA and HROA. Thirdly, head posture was studied in YA, LROA and HROA. Compared to walking, LROA and HROA demonstrated more variable head posture than YA. Overall the findings suggest that adults use visual and probably proprioceptive information about stair edge locations to negotiate stairs and HROA benefited from highlighted stair edges. HROA should be included in future stair negotiation studies.

Perhexiline is an anti-anginal drug that is thought to shift myocardial metabolism from \(\beta\)-oxidation of fatty acids to glucose utilisation. An associated improvement in energy efficiency may be beneficial in ischaemia-reperfusion as an adjunct to established techniques for myocardial protection during cardiac surgery. In this thesis, I conduct a prospective double-blind randomised placebo-controlled trial of oral perhexiline in patients undergoing coronary artery surgery, obtaining samples of serum, right atrium and left ventricle. I measure the concentration of perhexiline using high performance liquid chromatography and find that although highly concentrated in the heart, it may not have reached steady-state in the ventricular myocardium. I perform enzymatic colourimetry and ultra-high resolution mass spectrometry to detect changes in carbohydrate and lipid metabolism; however, the myocardial metabolic profiles of patients on perhexiline are indistinguishable from controls. On analysing the results of the clinical trial, I find no improvement in the primary endpoint, the incidence of a low cardiac output episode, or any secondary outcomes. I conclude that preoperative oral perhexiline does not improve clinical markers of myocardial protection and despite significant accumulation in the myocardium, it has no significant effect on the measurable metabolic profile of the heart at the time of surgery.