The skin is the outermost organ vital for human life. Its unique structure creates a formidable barrier, one that must be overcome for the delivery of pharmaceutical and cosmetic actives. Ibuprofen is often used for the short term management of musculoskeletal conditions and is applied topically for local delivery. Retinyl palmitate is a vitamin A derivative commonly included in cosmetics as an anti-ageing ingredient. Both model actives were formulated based on their physiochemical properties and solubility or miscibility with selected excipients. Clinically relevant or finite doses were used throughout to simulate point of use conditions. Ibuprofen was prepared in volatile formulations and after evaporation of alcohol only ibuprofen and the excipient remained on the skin. The in vitro permeation of ibuprofen using Franz diffusion cells was initially studied through silicone membranes and porcine skin. Neither of these model membranes were comparable to human skin. Permeation and mass balance studies using human skin were carried out for ibuprofen and the vehicle. This was to determine the vehicle fate and to establish its role during the permeation process. The use of human skin mounted in Franz diffusion cells was also used to determine the penetration of retinyl palmitate from single and binary vehicle formulations. Mass balance studies were primarily used to determine the penetration of both retinyl palmitate and the vehicle. Analysis was also performed for retinol, a metabolite of retinyl palmitate. Finally an in vivo study using confocal Raman spectroscopy was performed on human subjects. Volatile ibuprofen formulations were applied and the delivery of ibuprofen and vehicle to the stratum corneum was determined. A good relationship between in vitro and in vivo data was observed. Overall the formulation strategy adopted demonstrates that a rational selection of the vehicle can deliver enhanced efficacy of topical formulations.

Here, the effects of macrophage-stimulating protein (MSP) and γ-synuclein on two systems in the developing brainstem involved in controlling movement have been studied: a) the cranial motoneurons and b) the dopaminergic neurons of the substantia nigra and the ventral tegmental area. MSP exerts a variety of biological actions on many cell types, but has no known functions in the brain. To investigate whether MSP is also capable of acting as a neurotrophic factor, hypoglossal motoneurons were purified from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. The study shows that MSP promotes the in vitro survival of these neurons during the period of naturally occurring neuronal cell death and enhances the growth of neurites from these neurons. Furthermore, MSP mRNA was detected in the developing tongue which is the target tissue for hypoglossal neurons. These studies demonstrate that MSP is a neurotrophic factor for a distinct population of developing motoneurons. γ-synuclein is a recently discovered member of the synuclein family. Another member of this family, α-synuclein has been implicated in the pathogenesis of Parkinson’s disease. However, little is known about the function of γ-synuclein and it has not yet been directly implicated in the genesis of neurodegenerative conditions. Here, brainstems of transgenic mice lacking γ-synuclein have been analysed by means of immunohistochemical and histological techniques. The data obtained shows that γ-synuclein is expressed in the murine substantia nigra and in most cranial motor nuclei and that the localization of the protein undergoes a shift during development from a cytosomal to an axonal and synaptic localization. Mice lacking γ-synuclein have a deficit of neurons in these structures. In the context of recent studies which have revealed in vivo and in vitro interactions between the synucleins, this data suggests that a fine balance between α- and γ-synuclein seems critical to prevent the demise of certain neurons during the period of naturally occurring cell death. It also indicates that γ-synuclein may play a role in the pathogenesis of Parkinson’s disease.

Since the finding that NO released from smooth muscle vascular endothelium has vasodilatory properties, there has been a surge in research designed to further elucidate the biological roles of this free radical. Enzyme-dependent and enzyme-independent mechanisms of NO generation have been observed in human skin. NO generation by both mechanisms increases following ultraviolet radiation (UVR). <i>In vivo</i> murine data suggests that NO has an anti-apoptotic role in human skin following UVR. In the work presented in this thesis I have shown that zeolite NO (Ze-NO) is an inert topical NO donor which releases physiologically relevant concentrations of NO. The NO released by Ze-NO induces minimal cutaneous inflammation, in contrast to earlier NO donors investigated. I provide evidence to show that NO is stored in the form of nitrosospecies in human skin and sweat on the skin surface. NO release from nitrosospecies stores occurs within 30 minutes of exposure to UVR. I provide preliminary <i>in vivo</i> human data which suggests that NO is acting anti-apoptotically in human skin following UVR. NO had no significant effect on DNA damage or repair following UVR. Finally, I provide evidence that a control mechanism for the upregulation of iNOS exists in human skin in the form of arginase and the urea cycle. In order to develop our understanding of the role of NO in human skin, all the work presented in this thesis has been carried out <i>in vivo</i> in human subjects. NO has complex pleiotropic actions which depend upon the concentration of NO, the target cell and the microenvironment. <i>In vivo </i>clinical research, using physiologically relevant parameters, is therefore essential if we are to further our knowledge of this free radical.

Introduction: Quantitative data of spinal range of motion in vivo is essential to improve clinicians’ understanding of spinal pathologies, procedure of assessment and treatment. Accurate knowledge of physiological movement of lumbar spine regions, hip and the behaviour of each regional movement is important. Spine and hip motion play an essential role in daily functional activities, such as self-caring or performing occupational duties. Measuring the regional breakdown of spinal motion in three planes and describing the relative motion of different regions of the thoracolumbar (TL) spine can provide useful clinical information, which can be used in clinical procedure for spinal assessment. The relationship between the forward flexion (i.e. cardinal motion) and more functional tasks, such as lifting, stand-to-sit and sit-to-stand, as well as dividing the lumbar spine into more than one region, relative to the hip during these tasks, have not yet been established. Measuring the regional breakdown of spinal motions in three planes, as well as the relationship between lumbar spine and hip in sagittal plane, requires a multi-regional analysis system. Aims and objectives: The fundamental aim was to explore range of motion and velocity magnitudes in flexion, extension lifting, stand-to-sit and sit-to-stand tasks, using three lumbar regions relative to the hip, and to determine correlations and differences between flexion and other dominant functional tasks. An objective was to obtain an appropriate measurement system that is capable of measuring dynamic movement in ‘real time’ and examine its validity against a “gold standard” system and its reliability, by measuring the range of motion of multi-spinal regions. Also, to demonstrate the relative contribution of five regions from within the thoracolumbar and head-cervical regions in 3D. Methods: The selected system (tri-axial accelerometer sensors-(3A sensors)) was validated against a “gold standard” system (roll table (RT)) to demonstrate a correlation and root mean square errors (RMSEs) between the two devices. Reliability of the 3A sensors and the contribution of multi-spinal regions was assessed on 18 healthy participants. Two protocols were applied: in protocol one, two sensors were placed on the forehead and T1, to measure cervical ROM; in protocol two, six sensors were placed on the spinous processes of T1, T4, T8, T12, L3 and S1 to measure thoraco-lumbar regional range of motion. It also divided the lumbar spine as one single joint (S1 to T12) and as two regions (the upper (T12-L3) and lower (L3-S1)) and hip region. Data was gathered from 53 participants using four sensors attached to the skin over the S1, L3, T12 and lateral thigh. Two different statistical analyses were applied: one for analysing each particular region’s contribution, relative to the hip; and another to analyse the correlation between the kinematic profiles of flexion and three sagittally dominant functional tasks (lifting, stand-to-sit and sit-to-stand). Results: Validation of 3A accelerometer sensors system against the roll table revealed a strong correlation between the two systems average (ICC=.998 (95% CI=.993-.999)) and an acceptable rate of errors ranged from (2.54º (0.70%) to 5.01º (1.39%)). It also demonstrated the reliability of this system, when the ICC values for all regions were high with relatively small errors associated with a novel multi-regional clinical spinal motion system. The ICC values for all regions were found to be high, ranging from .88 and .99 with 95% CI ranged from .62 to .99 while errors values ranged from 0.4 to 5.2°. The additional movement information, gathered from a multi-regional breakdown, adds insight into the relative contributions to spinal movement. Significant differences existed between ROM of LLS and ULS across all movements (p < 0.05). A significant difference also existed between ULS-hip and LLS-hip ratio for the majority of tasks (p < 0.05), and between ULS and LLS velocity for the majority of tasks (p < 0.05). The findings from the lumbar spine as one region, underestimates the contribution of the lower lumbar and overestimates the contribution of the upper lumbar spine. Strong correlations for ROM are reported between forward flexion tasks and lifting for the LL spine (r = 0.83) and all regions during stand-to-sit and sit-to-stand (r = 0.70-0.73). No tasks were strongly correlated for velocity (r = 0.03-0.55). Conclusion: The validity and reliability of the accelerometer sensors system is evidence of its ability to measure spinal movement. Since it is inexpensive, small, portable and relatively easy to use, it could be a preferable system for clinical application. The data, from multi-spinal regions, provides a novel method for practitioners to focus on a greater number of regions, rather than measuring only the three main areas of the spine (cervical, thoracic and lumbar). Investigating the lumbar spine as only one region risks missing out important kinematic detail. Further, the methodology provides the potential to measure functionally unique kinematics from more complex functional tasks, rather than generalised findings from clinical assessments of simple flexion.

Rupture of the medial head of the gastrocnemius muscle (GM) is a common injury of the calf muscles. Magnetic resonance imaging (MRI) and ultrasound (US) are the medical imaging modalities that are usually used to assess such injuries. Texture analysis is a digital image processing technique that quantifies the relationship between pixel intensities (grey levels) and pixel positions. Texture can reveal valuable information that cannot be perceived by the naked eye. Dedicated image processing software is required to extract texture parameters. Texture analysis has been implemented for medical imaging modalities such as MRI, US and computed tomography (CT) for the evaluation sports muscle injury. Peripheral quantitative tomography (pQCT) is an adaptation of conventional CT. In this project, texture analysis was implemented on MRI and pQCT images of the gastrocnemius muscle (GM). MRI is an expensive technique that requires specialised facilities. Conversely, pQCT utilises a small-bore, low-dose X-ray scanner, which is portable and less costly than MRI. It has traditionally been used mainly for bone analysis. The aim of this study was to assess the suitability of pQCT for GM tissue characterisation using texture analysis compared with MRI. The study is novel in that it is the first to apply texture analysis to GM images using pQCT Texture analysis was done on image data acquired from MRI (GE, 1.5T) and pQCT (Stratec XCT 2000) in a group of healthy human subjects and an injured subject. A water phantom was also scanned with pQCT. An existing standard imaging protocol was observed for MRI acquisition, while pQCT image acquisition parameters were explored and optimised to yield a standard protocol. The pQCT scanner was shown to be capable of acquiring calf muscle images and distinguishing calf muscle boundaries. Texture parameters (grey level, variance, skewness, kurtosis, co-occurrence matrix, run length matrix, gradient, autoregressive (AR) model and wavelet transform) were extracted from the acquired images. The repeatability of these quantities for pQCT in a healthy human subject and a water phantom was assessed by calculating the coefficient of variation (%CV). The effect of pQCT parameters (scan speed and pixel size) was tested using multiple variate II analysis of variance (MANOVA). The effect of region of interest (ROI) area and anatomical position were evaluated using simple linear regression. The t-test was used to compare the mean values of the texture features in the right and left leg for both MRI and pQCT in a group of healthy human subjects. Neither MRI nor pQCT showed significant differences between the two legs for any of the texture features. In addition, there was no significant difference between the two modalities for the AR model and wavelet transform texture parameters. Reference ranges for the medial head of the GM were defined for both modalities. A study of a single injured subject revealed that the values of the AR model texture parameter fell outside the reference ranges for both MRI and pQCT, and so the AR model was identified as the most sensitive texture parameter for distinguishing injured from uninjured GM. The principal conclusion from this work is that pQCT has the potential to be used for imaging the gastrocnemius muscle and that GM images from both MRI and pQCT scanners can be objectively characterised by texture analysis. In addition, the autoregressive model texture parameter may be the most appropriate for muscle characterisation.

No description available.

The major aim of this project was to develop a biologically active bone scaffold that could induce vascularization in critical-size defects (CSD) and hence bone formation. In this study, functionalization of three-dimensional (3D) printed biphasic calcium phosphate (BCP) scaffolds was investigated. The first functionalization approach involved printing scaffolds with two different pore geometries and sizes; square (400μ) and round (800μ). The second was by coating scaffolds with DAR16-II; a self-assembly peptide that forms a hydrogel nanostructure mimicking extracellular matrix (ECM). A rabbit model was used to study these functionalization methods; square and round pore scaffolds with and without DAR16-II coating were implanted into experimental rabbit calvaria bone CSD defects. After 8 weeks, animals were killed and tissue was processed for histomorphometric analysis. Histological evaluation showed that bone formation was pore size and geometry independent while DAR16-II was successful in inducing bone formation compared to non-coated scaffolds. The following in vitro studies aimed towards understanding the basic cell response that enhanced bone formation in vivo. Human mesenchymal stem cells (MSCs) were used to identify the osteogenic potential of DAR16-II. Molecular analysis and mineralization staining showed that DAR16-II lacks osteoinductive properties. However, DAR16-II preserved cell viability when used as a BCP coating in vitro. In addition, DAR16-II exhibited angiogenic potential upon culturing with human umbilical vein endothelial cells (HUVECs) in vitro. DAR16-II induced the spreading of endothelial cells, activation and tubular-structure formation. Angiogenesis Real time-2 (RT2) polymerase chain reaction (PCR) array was used for gene expression analysis and showed that DAR16-II angiogenic effect was regulated by overexpression of endoglin (ENG or CD105), a clade E member of the serine protease inhibitor-1 (SERPIN-1) and β-Actin (ACTB) and down-regulation of VEGF receptor I (Flt1) and VEGF receptor II (KDR) Flt1. Furthermore, DAR16-II enhanced attachment of monocyte THP-1 cells. Results have demonstrated that DAR16-II add a proactive factor to BCP scaffolds. The proposed functionalization methodology increases the potential of enhancing vascularization and bone formation within ceramic scaffolds.

Skeletal muscle displays a remarkable plasticity to loading and unloading. Such load-dependence is illustrated by the remodelling of metabolic and myofibrillar protein composition. To date the earliest documented molecular adjustments in unloaded human skeletal muscle consists of pre-translational alteration in the expression of factors regulating protein degradation and synthesis and mitochondrial metabolism. In project one I set out to investigate the changes in protein expression in the soleus and vastus lateralis muscle, muscles involved in posture and movement, after three days of unilateral limb suspension. My findings suggest that early unloading alters FAK-pY397 and metavinculin content, and deregulate the expression of factors that set the slow-oxidative phenotype. The underlying mechanisms governing disuse atrophy have not been characterized. Therefore, in the second project I examined muscle fibre morphology and potential factors associated with anabolic signalling as well as markers representative of the catabolic pathways in response to a chronic unloading period of 21 days in humans. Our findings support the proposition based on animal studies that mechanisms responsible for muscle atrophy involve a decrease in protein synthesis accompanied by an increase in protein degradation. It is well known that adjustments in muscle size are driven by changes in the content of myofibrils. However, how such myofibrillar alterations are integrated at the molecular and the architectural level in muscle fibres that undergo adaptive changes has not been resolved. Thus in project 3 I have examined changes in the content of costamere components with increased and reduced loading of human antigravity muscle in relation to changes in muscle size and molecular parameters of muscle size regulation. The findings suggest that load-dependent plasticity of muscle size is integrated through costamere remodelling via a load-regulated process that involves level alterations of FAK and KAK-pY397 concentration.

Peripheral motor nerve injury frequently results in significant morbidity even after repair employing micro-surgical techniques. Following such an injury the trophic effect of the motor nerve is lost, leading to degeneration of the neuromuscular junction, muscle atrophy and eventually irreversible muscle fibrosis.;Several neurotrophic growth factors (including insulin-like growth factor-I and fibroblast growth factor-I) have been identified. These have been shown to facilitate regeneration of peripheral nerve in-vivo, however less is known regarding any trophic action at the neuromuscular junction.;In this series of experiments the ability of insulin-like growth factor-I and fibroblast growth factor-I to facilitate neuromuscular junction regeneration in atrophic muscle was studied. The growth factors were delivered to the nerve/muscle interface of a nerve implant either as a bolus dose using a fibrin glue carrier or by continuous infusion with a mini-osmotic pump. The animal model employed was the rat lower limb model. Functional testing of the regenerated neuromuscular junctions was performed by measuring muscle twitch force following nerve implantation.;Both growth factors were shown to facilitate neuromuscular junction regeneration in atrophic muscle equally effectively. Despite histological evidence of neuromuscular junction regeneration, functional testing did not demonstrate any significant difference between growth factor treatment groups and controls.

Subject-specific forward-dynamics simulation models of human movement incorporating torque generators have typically represented the torque at a joint based on the kinematics of only that joint. Representing the net torque exerted at a joint by considering the kinematics of two joints ought to better represent the torques exerted by the two-joint biarticular muscles. In this study two-joint torque generator representations including monoarticular and biarticular torque components and single-joint torque generator representation of the lower limb were determined for a single male participant using strength measurements collected on an isovelocity dynamometer. For plantar flexion, knee flexion, knee extension torques, a two-joint representation was found to offer better agreement with torques measured on an isovelocity dynamometer than a single joint representation when the joint angle of a secondary joint was changed by 37˚ or more. In addition a two-joint representation should account for the biarticular knee flexor and extensor contributions to hip joint torques. Two subject-specific simulation models of squat jumping, one with single-joint torque generators and another with two-joint torque generators were created to establish the necessity for two-joint torque representation in whole-body simulation models of human movement. Including the two-joint torque generators was shown to provide better agreement with a participant performance than a single-joint torque-driven simulation model. The simulation of a maximal effort human movement should include two-joint torque representations when both of the following characteristics are present in the task to be simulated: a) the task does not have a large initial whole-body momentum; b) the task involves multiple joint kinematics with a large biarticular muscle contribution. A single-joint torque representation was effective when the kinematics of the secondary joint were similar to those which were present during torque measurements. Therefore if the simulated task involves similar kinematics at both joints then a single-joint representation may be sufficient.