No description available.

The aims of this thesis were to fully characterise the surface properties of titanium alloy treated with a novel process of electrochemical etching and to assess the effect of these surfaces on osseointegration and the behaviour of osteoblasts. In vitro investigation was performed using rat osteoblasts and human mesenchymal cells. Significant differences were observed in cell polarity and cell area between cells cultured on 3V and 5V and those treated at 9V. Significantly greater mean focal adhesion area and mean number of focal adhesions per unit cell area were also observed between the 3V and 5V treated surfaces compared to the 9V treated surfaces. Longer term experiments revealed no significant changes in the levels of alkaline phosphatase activity between cells cultured on the different surfaces. After 28 days in culture, cells were stained for mineralised deposits and no significant differences were seen in the total area of bone nodules found on the experimental surfaces. To assess the influence of the treated surfaces on bone cell biology, immunofluorescence was used to localise cadherin-11 and activity and inhibition assays were performed for small GTPases of the Rho family. On the 9V treated surfaces, significantly increased staining for cadherin-11 was seen, as well as significantly greater RhoA activity. In vivo experiments were performed with electrochemically treated titanium implants in a rat tibia model. Analysis of micro-CT images of retrieved tibias revealed a significantly greater area of mineralised bone matrix in contact with the implant surface for the 9V treated surfaces and the same trend was observed on histomorphometric analysis. High variability in the mechanical testing data meant that no significant differences were seen, but a trend for greater load to failure of the bone-implant interface was observed for the 3V treated surfaces.

Touch perception is important in most living organisms and extremely sensitive detection systems have evolved to meet this need. Pacinian corpuscles (PCs) are primary mechanoreceptors. In the human, they are found in the skin (where they act as touch receptors), in the joints, in muscles and in many organs (where they act as motion sensors). The purpose of the work described in this thesis is to investigate how the performance of the PC is achieved, with reference to structure, mechanical properties and possible transduction mechanisms. PCs were obtained from the equine hoof and their distribution and clustering were investigated. Corpuscles were located in the frog area of the hoof (the digital cushion); they were found to be surrounded by adipose tissue and often closely associated with blood vessels. The physiological implications of these observations are discussed. The structure and composition of corpuscles was investigated using confocal microscopy with histological stains for collagen, proteoglycans and lipids. Nonlinear microscopy was also used to investigate the distribution of collagen (by second-harmonic generation), elastin (by intrinsic two-photon fluorescence) and membrane 4 lipids (by coherent Raman imaging). These techniques provided novel insights into the three-dimensional structure of the intact corpuscle, demonstrating: (i) three clearly distinguishable zones – the outer zone, the inner zone, and the core; (ii) blood vessels running through the outer lamellae and the core; (iii) the presence of proteoglycans – less in the outer zone than in the inner zone; (iv) two types of collagen fibres (one type associated with the lamellae and the other forming a complex fibre network through the inter-lamellar spaces); (v) occasional elastic fibres; (vi) a sheath of adipose tissue closely associated with the corpuscle’s outer surface. Mechanical testing by micro indentation, micropipette aspiration and osmotic challenge showed that the outer zone was stiff and able to quickly restore its original shape after distortion. Dynamic mechanical properties were investigated over a range 50 to 400 Hz. Observations of lamellar displacement (amplitude and phase) were consistent with the predictions of the Loewenstein-Skalak model. This model includes 30 lamellae; however, the same overall frequency response could be replicated in a single-lamella model with suitably chosen parameters. The benefits of a lamellar structure for transduction of mechanical signals therefore remain unresolved. 5 The permeability of the corpuscle to water and solutes was investigated using osmotic swelling and fluorescence tracer techniques. Both revealed unexpected complexity in the pathways of uptake to the inner core and demonstrated the presence of an impermeable boundary between the inner and outer zones, whose implications for mechanotransduction and nutrition in the corpuscle remain to be determined.

It is very rare, even in degraded listening environments, that we might confuse speech with a dog bark or vice versa, despite the fact that both are complex acoustic signals. Despite the solid assumption of left lateralisation in speech processing from clinical and anatomical observations, the results from brain imaging studies have been inconsistent. One possible cause for this controversy may come from the use of different imaging system. Using inadequate baselines, however, may bring more critical problem. In brain imaging studies, especially when cognitive subtraction is used, images of cognitive processes are generally derived by subtracting a control stimulus/task from an experimental counterpart. The two stimuli/tasks to be compared are expected to differ only in one factor/process and the difference in brain activations is thus considered to come from the particular difference between the two. This thus makes it difficult to find baseline stimuli/tasks that activate all but the process of interest. By far, spectrally rotated speech stands as a most satisfying control against intelligible speech as it is equally complex as speech but totally unintelligible. However, the spectral rotation so far has been a total rotation regardless of the source and the filter of sound, which are independent and heterogeneous by nature. A series of behavioural studies performed in this thesis showed that the source rotation did not significantly affect speech intelligibility whilst filter drastically decreased the intelligibility. Another possibility can be different brain imaging paradigms used. With carefully designed parametres using functional magnetic resonance imaging (fMRI), we confirmed that intelligible speech recruited predominantly the left superior temporal area, replicating the results from previous positron emission tomography (PET) and fMRI studies. Since the intervention of scanner noise has been an issue in auditory research using an MRI system, four imaging paradigms were compared and it is concluded that a sparse sampling with 8 seconds of repetition time had a clear advantage over longer repetition time with 16 seconds and a continuous sampling. This paradigm was used in the study investigating the effects of channel number and presence/absence of tonal variation on speech intelligibility. Intelligibility increased together with increasing number of band channels and showed drastic improvement especially in the range of 2 – 6 numbers of frequency channel bands. A brain imaging study followed with mixed subtraction and parametric designs and revealed that the right superior temporal gyrus responded most when pitch variation was provided in the speech, regardless of intelligibility, unlike the pitch variation in non-speech (spectrally rotated speech here). Increasing intelligibility with increased spectral detail showed linear increase in percent signal change in hemodynamic response in the left superior temporal gyrus. The current result supports a streamed hierarchical model, in which speech comprehension occurs predominantly in the left hemisphere.

This thesis describes ultrastructural observations on normal dendritic cells (melanocytes and Langerhans cells) of human epidermis, and changes in their fine structure after various forms of physical stress and malignant transformation. The historical background and previous research on the structure and function of the melanocyte and Langerhans cell are reviewed. During a holiday in Madeira the grave of Paul Langerhans was found neglected and overgrown, but arrangements made with a chance acquaintance should ensure that it is looked after in perpetuo. The unique fragility of melanocyte mitochondria was the only new finding noted in studies of normal skin. Ultrastructural dopa and tyrosine studies were made on skin exposed to ultraviolet radiation. They highlight the central role of the Golgi apparatus and surrounding system of smooth endoplasmic reticulum (GERL) in the transport of tyrosinase from ribosome to melanosome, and indicate that Stage I melanosomas arise from all parts of the Golgi apparatus as well as from GERL. The concept that the fine structure with periodicity in early melanosomes is the supporting structure of tyrosines was not confirmed. After a single exposure to ultraviolet radiation (6 X minimal erythome done), reaction product was not noted in melanocytes until twenty four hours after irradiation. It was then seen within the Golgi apparatus and neighbouring GERL; seventy two hours after irradiation it was seen mainly in peripheral cytoplasmic vesicles. Langerhans and mast cells were consistently dopa negative. Suction and friction stress to the epidermis have a profound effect on keratinocytes but cause minimal damage to dendritic cells, probably due to their lack of attachment with surrounding cells. Suction experiments indicate that, in contrast to keratinocytes, there is no communication between the extracellular space and system of smooth endoplasmic reticulum in dendritic cells. Subcellular observations on a single case of histiocytosis X were similar to those of others, and support the view that this condition is due to reactive proliferation of the epidermal Langerhans cell. Ultrastructural studies were carried out on forty specimens of invasive malignant melanoma. The findings support the concept that the fine structure of lentigo maligna melanoma is usually characteristic, and differs from that of superficial spreading and nodular malignant melanoma. The melanosomes in lentigo maligns melanoma are usually ellipsoidal and resemble those of normal melanocytes, whereas the melanocomes in superficial spreading and nodular melanoma are most often spheroidal and abnormal in appearance. Superficial spreading and nodular melanomas cannot be reliably distinguished by their ultrastructure, though vascular melanosomes are more common in superficial spreading melanoma. There was no support for the hypothesis that superficial spreading melanomas and nodular melanomas develop from pre-existing naevi. Ultrastructural dope reactions carried out on ten malignant melanoma specimens indicated that malignant transformation effects the structural protein of the EB/U melanosome rather than the distribution and passage of tyrosinase. Vacuolar melanosomes seem to represent an arrest in the development of Stage I melanosomes. A single case of a breast carcinoma invading the overlying epidermis and causing gross proliferation and activity of the melanocytes is described. Hyperactive melanocytes, which had migrated from the epidermis, were seen surrounding dermal tumour cell, but little evidence of phagocytosis, either by the melanocytes or tumour cells, was noted. This curiosity illustrates the close relationship which exists between melanocytes and cells of epithelial origin.

The transition from fins to limbs during the colonisation of land was a key innovation in vertebrate evolution. Changes in the limb and shoulder girdle during this event have been investigated extensively, but little attention has been given to the acquisition of the sternum, a feature considered characteristic of virtually all terrestrial vertebrates, and which is mandatory for tetrapod locomotion. The sternum is a thin flat bone lying at the ventral midline of the thorax that provides a crucial attachment site for the pectoral muscles, allowing the forelimbs to raise the body up from the ground. I demonstrate that a sternum completely fails to form in conditional Tbx5 mutant mouse embryos. Consistent with this, sternum defects are a characteristic feature of Holt-Oram syndrome, which is caused by mutations in TBX5. While the role of Tbx5 in the development of the heart and forelimbs has been studied extensively, Tbx5 function in sternum formation is not understood. Using chick and mouse models systems, I set out to investigate the developmental origin of the sternum, and why it fails to form in the absence of Tbx5. Since the function of the sternum is to facilitate forelimb movement, I explored the correlation between forelimb use and sternum morphology by comparing sternum size across different avian species. I then investigated the genetic adaptations that could explain sternum and forelimb reduction in flightless birds, using the emu as a model. I suggest that Tbx5 represents a common node in the molecular pathways regulating forelimb and sternum development.

The primary gene defects of hereditary neuromuscular disorders have not, as yet, been determined. Research has contributed much to our understanding of many aspects of these diseases, but since this has been directed towards the secondary phenomena, the exact role of the nucleus in the pathogenesis of these genetic disorders remains unclear. It was felt that a study of the motor neurone and muscle nucleus in development and disease might reveal the extent of its participation in some of these disease processes. 1. A quantitative histochemical and histometric study of the developing anterior horn cell nucleus during fetal life was undertaken. The result suggested that the 12th to 14th week period is critical for the differentiation of the spinal motor neurone. 2. In contrast to this, a similar study of the deve¬ loping muscle cell nuclei revealed no major changes in muscle nuclear size or composition. During normal post-natal muscle growth an increase in the number of nuclei per muscle fibre seems to be responsible for the maintenance of a constant nucleo-cytoplasmic ratio. 3. The nuclear size of multinucleated myoblasts arising from both normal and dystrophic muscle in tissue culture was measured. Myoblasts from dystrophic muscle exhibited larger nuclei than those from normal muscle. 4. An increase in muscle nuclear size was also detected in a number of muscle samples from male fetuses at risk for Duchenne muscular dystrophy. Both of these findings suggest possible changes in the nucleo-cytoplasmic relationship. 5. In an attempt to elucidate the extent of the parti¬ cipation of the muscle nucleus in the pathogenic process of a number of characteristic neuromuscular disorders, the nuclear size and/or the number of nuclei per cross-sectioned muscle fibre, were estimated. An increased nuclear size was observed in Duchenne muscular dystrophy and an increase in the number of nuclei was detected in chronic neurogenic atrophies, in diabetic neuropathy and in myotonic dystrophy. Such a finding would seem to indicate a fundamental difference in the response of the muscle fibre nucleus to the neurogenic and myopathic processes. 6. In addition, the growth of the human cervical vertebral canal and spinal cord during normal fetal development was studied. The results suggested that these two parts of the developing body exhibit the same developmental pattern. The rate of this parallel growth is lower than that of the body as a whole, but similar to that reported for the brain. In conclusion, although the techniques used did not reveal a direct relationship in the developmental pattern of anterior horn cell nuclei and myonuclei, they did provide normal values for comparison with data obtained from diseased neural and muscle tissue. The finding that in Duchenne muscular dystrophy (from tissue culture, fetuses at risk and juvenile biopsies) the muscle nuclei are enlarged, points to an underlying difference from various neuropathies in which the nuclei are increased in number.

Skin constitutes a barrier between our body and outside environment providing the first line defence against microbial infection. Epithelial repair and skin wound healing starts with inflammation to clear up invading pathogens and debris followed by cell proliferation and tissue remodelling. The immune response is vital for protecting the body from infection and diseases, however, it remains controversial whether the immune cells contribute to wound closure and tissue repair, or cause scarring and pathology. In this thesis, I investigate the role of Notch signalling in epithelial tissue repair. I demonstrate Notch1 signalling activation in epidermal keratinocytes following acute skin injury recruits innate lymphoid cells (i.e. ILC3s) to the site of injury in a TNF-α/CCL20-dependent mechanism and controls macrophage/monocyte recruitment via ILC3-dependent CCL3. Notch1 also induces epidermal production of IL23 which facilitates ILC3s to produce IL22 for re-epithelialization and skin repair.

The aim of this work was to develop a methodology whereby inertial measurement units (IMUs) could be used to obtain accurate and objective gait parameters within typical developed adults (TDA) and Parkinson’s disease (PD). The thesis comprised four studies, the first establishing the validity of the IMU method when measuring the vertical centre of mass (CoM) acceleration, velocity and position versus an optical motion capture system (OMCS) in TDA. The second study addressed the validity of the IMU and inverted pendulum model measurements within PD and also explored the inter-rater reliability of the measurement. In the third study the optimisation of the inverted pendulum model driven by IMU data was explored when comparing to standardised clinical tests within TDA and PD, and the fourth explored a novel phase plot analysis applied to CoM movement to explore gait in more detail. The validity study showed no significant difference for vertical acceleration and position between IMU and OMCS measurements within TDA. Vertical velocity however did show a significant difference, but the error was still less than 2.5%. ICCs for all three parameters ranged from 0.782 to 0.952, indicating an adequate test-retest reliability. Within PD there was no significant difference found for vertical CoM acceleration, velocity and position. ICCs for all three parameters ranged from 0.77 to 0.982. In addition, the reliability calculations found no difference for step time, stride length and walking speed for people with PD. Inter-rater reliability was found not to be different for the same parameters. The optimisation of the correction factor when using the inverted pendulum model showed no significant difference between TDA and PD. Furthermore the correction factor was found not to be related to walking speed. The fourth and final study found that phase plot analysis of variability could be performed on CoM vertical excursion. TDA and PD were shown to have, on average, different characteristics. This thesis demonstrated that CoM motion can be objectively measured within a clinical setting in people with PD by utilizing IMUs. Furthermore, in depth gait variability analysis can be performed by utilizing a phase plot method.

Bone is a complex heterogeneous composite material with organic and inorganic components. The inorganic component; bone mineral, is a poorly crystalline, non-stoichiometric form of calcium hydroxylapatite. A model for the general structure and composition of bone mineral has been established within the literature. However, the nature and extent of variation in bone mineral composition and structure has, to date, been poorly understood. This situation also applies to the general response of bone mineral to heat treatment and variation in this response. This thesis presents the results of an investigation of inter-species variation in bone mineral characteristics of unheated bone and bone heated to temperatures of 600 °C and 1400 °C. Twelve different animal species were investigated, including human bone. X-ray diffraction analysis was the primary analytical technique employed. The Rietveld method of full profile fitting of diffraction data was used to quantitatively investigate characteristics of unheated and heated bone such as the weight percentages of the thermal decomposition products of bone mineral. Infrared spectroscopy, inductively coupled plasma – atomic emission spectrometry, pyrohydrolysis – ion chromatography and combustion – gas chromatography were also employed to obtain further data regarding the chemical composition of bone. Biological ii control of bone mineral composition and structure and the chemical basis for the variation observed within the results were explored. Significant inter-species variation in bone mineral composition and structure and also, the response of bone to heat treatment have been demonstrated by the results of this thesis. In particular, human bone is significantly different from bone of all other species investigated.