This thesis has investigated the mechanisms by which re-epithelialisation occurs with regard to TG2. In the first stage of wound healing, an influx of growth factors and metalloproteinases occurs, that through the initial interaction of TG2 are able to stabilise the matrix and stimulate keratinocyte cells to migrate and proliferate to close the wound. Experiments have indicated that TG2 is able to stimulate proliferation and migration of keratinocyte cells both directly and indirectly by modulating metalloproteinase signalling, leading to the activation of the EGFR by EGF ligands liberated from the ECM. Furthermore, through the course of altering TG2's conformation and activity experiments have determined that TG2 must be in an open and active conformation in order for it to affect keratinocyte signalling leading to proliferation and migration. Finally, a G protein coupled receptor has been investigated as to whether it may be involved in TG2 driven proliferation and migration in keratinocytes. Previous work by Xu et al., 2006 had shown TG2 binds to GPR56 and this GPCR has been shown to be involved in proliferation and migration of cells located in the brain and heart. Although investigations of GPR56 are at this time inconclusive regarding keratinocytes proliferation and migration this thesis confirms the presence of GPR56 in keratinocytes. Therefore GPR56 may be involved in keratinocyte migration and proliferation.

Type X collagen is a member of the family of network-forming collagens, it contains a triple helical domain flanked by two non-collagenous (NC) domains, NC2 at the N-terminal and NCI at the C-terminal. It is expressed and synthesised by hypertrophic chondrocytes of the epiphyseal growth plates during the process of endochondral ossification (EO). This process involves replacement of a cartilaginous anlagen by bone, the coordination of chondrocyte proliferation, maturation and hypertrophy are followed by calcification of hypertrophic cartilage, vascular invasion and deposition of a bone matrix. A precise functional role for type X collagen has not been defined, although its temporal and spatial expression has linked it to endochondral ossification. A family of Small Leucine Rich Proteoglycans (SLRPs) have been demonstrated to be important for collagen biology. In particular, the well characterised decorin and biglycan, interact with numerous collagen types, non-collagenous proteins and growth factors. Decorin and biglycan have also been linked to the mineralisation process. This led to the hypothesis that type X collagen interacts with decorin and biglycan in the hypertrophic cartilage extracellular matrix. Interactions of type X collagen with decorin and biglycan were investigated using a solid phase assay and surface plasmon resonance (SPR). The interaction of type X collagen with decorin and biglycan was found to be of high affinity, with dissociation constants in the nanomolar range. Through using different domains of the type X collagen molecule the NCI domain, the triple helical region or whole type X collagen, it was demonstrated that the interactions are likely to be mediated by the NCI domain. The interaction of type X collagen with decorin and biglycan was found to be independent of the presence of the glycosaminoglycan chain(s) on decorin and biglycan, indicating the protein cores of decorin and biglycan are involved. Negative staining and transmission electron microscopy were used to visualise the interactions of type X collagen with decorin and biglycan labelled gold particles. The localisation of gold particles to the ends of type X collagen molecules supports the finding that the interactions are mediated by NCI. Recombinant fragments of the NCI domain were synthesised in an attempt to further determine regions of NC 1 important for the interactions with decorin and biglycan, these fragments have to date provided no additional information. Using RT-PCR decorin was demonstrated to be co-expressed with type X collagen by hypertrophic chondrocytes. Immunohistochemistry was utilised to study the localisation of type X collagen, decorin and biglycan in the hypertrophic cartilage extracellular matrix. All of the interacting components were shown to co-localise. The co-expression and co-localisation studies indicate that these interactions could occur in vivo. The characterised interactions of type X collagen with decorin and biglycan are likely to have functional roles in EO. Potentially they may be involved in regulation and assembly of a type X collagen pericellular matrix, they may adopt a structural role providing mechanical stability to an extracellular matrix undergoing dynamic remodelling, they could be involved in sequestering growth factors in the hypertrophic cartilage and hence be involved in modulating interactions of growth factors with their signalling receptors. These postulated functions are all possible in the normal development and growth of a long bone. Type X collagen, decorin and biglycan have all been implicated in pathology the interactions could alternatively be involved in pathological calcification. Molecular interactions that contribute to the molecular assembly of the growth plate are fundamental to its functions. Characterisation of such interactions will aid in defining precise roles for molecules such as type X collagen, decorin and biglycan during the process of EO.

When desirable retardation of skin penetration of toxic compounds such as pesticides and chemical warfare agents was evaluated using model permeants, retardation was observed under certain conditions, but was not predictable. It was concluded that generic penetration modulation was not a realistic goal. Investigations into skin surface sampling techniques highlighted the difficulties of minimising variability, and comparison of in vitro tape stripping with published in vivo data for a clobetasol propionate cream demonstrated the significance of formulation inhomogeneity. Determination of orally administered doxycycline in the stratum corneum using in vivo skin surface biopsies suggested that it was unlikely that this technique could be of value for most orally administered drugs. Evaporative loss of volatile permeants, such as fragrances, during skin penetration studies makes it impossible to achieve full mass balance. Direct capture of evaporating material was occlusive and significantly affected the amount that permeated through the skin membranes. A simple novel method was therefore developed to allow estimation of evaporative loss under the experimental conditions. Pre-study skin water permeability coefficient (Kp) is frequently used as a membrane integrity check and it is generally assumed that skin of higher water permeability will be of higher permeability for subsequently applied compounds, regardless of differing physiochemical properties. However, it was shown that Kp did not correlate with subsequent test compound permeation for twelve compounds of moderate to high lipophilicity.

Collectively, the data demonstrated that HA can serve as a signal integrator by facilitating TGF-beta11-mediated CD44-EGF-R-ERK interactions and ultimately regulate fibroblast phenotype. I propose a model to explain this novel mechanism and the functional consequence of age-dependent dysregulation. This mechanism may have direct implications for modifying the wound healing response, particularly for developing therapeutic strategies to improve healing in the elderly.

No description available.

Increased levels of blood glucose are associated with the vascular complications of diabetes. Microvascular complications lead to delayed wound healing in patients suffering from diabetes. Hypoxia and hyperglycaemia characterise a wound environment of a person with diabetes. Angiogenesis is central to restore the supply of oxygen and nutrients to the wounded tissue. Endothelial cell migration is central to angiogenesis which is aided by hypoxia and attenuated by hyperglycaemia. However, the molecular mechanisms underlying the disruption to angiogenesis of diabetic wounds are not completely understood. The effect of hypoxia and/or high glucose concentration on the endothelial cell migration in vitro was studied and an anti-oxidant, silymarin formulated as freeze dried wafer discs was tested for its beneficial effect. A radial migration and a wound healing assay were developed, validated and used to assess the effect of hypoxia and/or high glucose concentration on the migration of human endothelial cells of dermal origin. Circular and semi-circular monolayers of endothelial cells were used for the measurement of the migration by radial migration and wound healing assay respectively. Net migration was calculated by subtracting the radii at a specified time point from that measured at time zero. The migration was studied under normal (20%) or below (5%) normal oxygen tension in combination with normal (5mM) or high (20mM) glucose concentration. Endothelial cells were treated with an anti-proliferative agent, intracellular signal inhibitors and silymarin. Results demonstrated that hypoxia and high glucose concentration have opposing effects of increase (p<0.001) and decrease (p<0.001) respectively on the migration of endothelial cells. The results of the wound healing assay revealed that re-endothelialisation occurs faster (p<0.001) than endothelialisation. The effects of hypoxia and high glucose concentration appeared to be mediated via PI3K-Akt and PKCβII pathways respectively. Further investigations revealed the possibility of HIF-1α being involved in both the pathways. High glucose concentration-induced decrease in cell migration was successfully restored (p<0.001) by the use of an anti-oxidant silymarin. This could be due to anti-oxidant activity of silymarin on glucose-induced overproduction of reactive oxygen species. Silymarin formulated as freeze dried wafer discs, sterilised by gamma irradiation was successful in retaining its effect (p<0.001) against the high glucose impaired cell migration compared to control wafers. In conclusion, delayed wound healing due to disrupted endothelial cell migration was reaffirmed to be due to elevated glucose concentration. Silymarin was successful in restoring glucose-induced attenuation of cell migration. Freeze dried wafers show promising potential as a topical application for the treatment of chronic wounds for people with diabetes.

Exercise-induced muscle damage (EIMD) is a commonly experienced phenomenon, yet its effect on the human response to dynamic exercise is poorly understood. Therefore the intention of this thesis was to provide empirical evidence to advance the scientific knowledge and understanding of the phenomenon of EIMD; principally by investigating the physiological, perceived exertion and metabolic responses to the performance of dynamic exercise with EIMD. The eccentric, muscle-damaging exercise protocol employed for all four studies involved participants completing 100 squats performed as 10 sets of 10 repetitions with the load on the bar corresponding to 70% of the individual’s body mass. Measures of markers of muscle damage were taken before and after the eccentric exercise protocol in each of the four studies. The markers used were plasma creatine kinase activity, isokinetic peak torque and perceived muscle soreness. Cycling rather than running was used as the dynamic exercise mode in studies 1, 2 and 4 in order to avoid the confounding influence of alterations in gait subsequent to EIMD. The dynamic exercise in study 3 was performed inside a whole body scanner and was therefore limited to knee extension and flexion. These four studies have provided novel insights into the influence of eccentric, muscle-damaging exercise on the human response to the performance of dynamic exercise. We have demonstrated for the first time that following EIMD, the enhanced ventilatory response to dynamic exercise is provoked by stimuli unrelated to the blood lactate response, and that this enhanced ventilation may provide an important cue to inform the perception of effort. Furthermore, we have shown that the reduced time to exhaustion observed following EIMD is associated with an elevated perception of exertion and increases in [Pi] during dynamic exercise. Finally, we have demonstrated that the kinetic response is unaltered during the transition to high intensity dynamic exercise. Changes in [HHb] kinetics indicate that compensatory mechanisms act to preserve blood-myocyte O2 flux in the face of microvascular dysfunction, resulting in the unaltered observed across the rest-to-exercise transition.

Following exercise, a rapid return to optimal performance is desirable for athletes. Some types of physical activity can result in exercise-induced muscle damage (EIMD), an occurrence which is well documented in adult populations. Symptoms of EIMD include increases in perceived muscle soreness, decrements in muscle strength and power, alterations in limb proprioception, increased myoprotein release, increased perceived exertion during exercise, and reduced endurance performance. Many strategies are currently employed in attempts to manage the symptoms of EIMD, though the majority are based on anecdotal or equivocal evidence. The aim of this thesis was to investigate a range of contemporary recovery strategies to determine their effectiveness and appropriateness for use with athletic populations. This thesis comprises eight chapters: • Chapter 1: Introduction • Chapter 2: Review of Literature • Chapter 3: Study 1 – Cold water immersion therapy as a recovery strategy following exercise-induced muscle damage • Chapter 4: Study 2 – Manual massage as a recovery strategy following exercise-induced muscle damage • Chapter 5: Study 3 – The use of compressive clothing as a recovery strategy following exercise-induced muscle damage • Chapter 6: Study 4 – Does the use of manual massage therapy have an added beneficial effect on symptoms of exercise-induced muscle damage when used in conjunction with compressive clothing? • Chapter 7: Study 5 – Muscle strength loss characteristics at optimal and short muscle lengths following exercise-induced muscle damage • Chapter 8: General discussion • Chapter 9: Conclusion and recommendation for further study Chapter 3 considers the efficacy of a single bout of cold water immersion therapy following strenuous exercise as a recovery strategy. The results indicate that this treatment protocol has no significant effect on moderating increases in soreness or creatine kinase activity, or reducing decrements in muscle function (p > 0.05). Although this type of recovery strategy is popular in elite sporting fields, this study is consistent with a number of others, indicating that a single bout of cold water immersion therapy is an ineffective recovery strategy following EIMD. Chapter 4 focuses on manual massage a post-exercise recovery strategy. Similar to cold water immersion therapies, the use of massage as a treatment is widespread within the athletic field, though few studies have considered its efficacy following EIMD. This study indicates that although no effect of massage was apparent on perceived soreness or creatine kinase activity, individuals receiving a thirty minute treatment immediately following damaging exercise demonstrated reduced decrements in muscle strength as assessed by isokinetic dynamometry (p < 0.05). This chapter concludes that immediate manual massage may be an effective treatment strategy to manage some of the symptoms of EIMD. Chapter 5 examines the use of clothing with specific compressive qualities as an increasingly popular method of managing symptoms of muscle damage, which is largely unsupported by scientific evidence. Following damaging exercise, the use of lower limb compression reduced decrements in isokinetic muscle function and jump performance, while moderating increases in perceived soreness. This chapter concludes that the use of lower limb compression clothing is an effective, non-invasive method of managing symptoms of EIMD. Data collected from chapters 4 and 5 indicated that manual massage and lower limb compression may manage some of the symptoms of EIMD. Chapter 6 investigates whether these two recovery strategies used in combination have an additive beneficial effect on symptoms of muscle damage. Data from study 3 (chapter 5) were used to compare the combined treatment with the compressive treatment as an independent recovery modality. A significant beneficial effect was observed on perceived soreness as a result of combining the massage and compression treatments, in comparison with compression alone. No consistent benefits on muscle function, above those offered by compression independently were derived as a result of the combined treatment. This chapter concludes that although some benefits are afforded by the addition of massage to the compressive treatment with regard to perceived soreness, logistically, this combination of treatments is unlikely to provide a substantial enough benefit to warrant use with groups of athletes. Exercise-induced muscle damage leads to losses of strength and alterations in the muscle length-tension relationship. Data collated from chapters 5 and 6 were used to investigate muscle strength loss characteristics following damaging exercise. Consistent with previous research, a disproportionate loss of muscle strength was observed at shorter muscle lengths, attributable to mechanical changes within the affected muscle. The use of isokinetic dynamometry was able to detect a shift in length-tension relationship in favour of a longer optimal muscle length for tension generation. The shift in length-tension relationship has been observed in previous studies, although no study has observed this shift in the knee extensor muscle group, and those considering the knee extensor group have inferred the shift as a result of the disproportionate nature of muscle strength loss. The final chapter of this thesis uses data collected from chapters 3-6 to determine the variations in response to different types of eccentrically biased exercise. Typically, plyometric drop jumps elicited greater decrements in isokinetic muscle function and higher perceptions of soreness following exercise in comparison with either downhill running or vertical plyometric jumps. Downhill running resulted in greater cellular permeability as indicated by creatine kinase activity.

Articular cartilage (AC) is a soft connective tissue that lines the ends of synovial bones in joints. It is responsible for absorbing impact loads and resisting shear associated with joint articulation. Pathologies such as osteoarthritis have symptoms including degradation and sometimes complete loss of the articular cartilage, which can lead to poor load support and considerable pain. There has been considerable work in the field of cartilage mechanics. The transient deformation behaviour of loaded cartilage has been examined using several different experimental approaches, and complex models have been developed to accurately describe this behaviour. The microstructural response of AC to load is less well understood, however. The aim of this work was to investigate the anisotropy and heterogeneity of cartilage and its effect on the load bearing characteristics. Firstly, existing experimental data were used to create a layered finite element model of cartilage under load. Using this model, three sets of material parameters were evaluated for their suitability in reproducing experimentally observed strains, as well as minimising peak stresses. It was found that only by including the heterogeneity associated with collagen fibre orientation could the layer boundary deformations be exactly modelled, whilst preventing potentially damaging interfacial shear. Tensile testing of cartilage from the equine metacarpophalangeal joint, using samples obtained from each individual layer, was performed to validate findings from the model as well as to determine the variation in mechanical properties in regions of different weight bearing characteristics. It was found that the tensile stiffnesses varied with depth as predicted by the model, demonstrating an average value of 31.3 MPa at the surface and 9.4 MPa in the radial zone, although there was considerable variation. Polarised light microscopy was used to determine the preferential collagen orientation, as well as qualitatively assess the angular spread and other patterns in collagen organisation. It was found that the appearance of the collagen network varied both with depth and location on the joint, with high weight bearing regions showing more isotropic fibre distributions below the surface than low weight bearing regions. To directly probe the microstructural response of AC to load, the tensile loading rig was modified to allow simultaneous imaging of the sample using two photon fluorescence microscopy. This allowed the relative displacement of cells and elastin fibres, which are intrinsically fluorescent, to be observed at increasing levels of strain. From locations and orientations of these features, the strain field could be calculated at two length scales: in the vicinity of specific elastin fibres (microns) and intercellular strains averaged over whole stacks (hundreds of microns). The strains at the two different scales did not correlate, suggesting that the microscopic strain environment varies considerably. The elastin fibre network was also investigated, and it was found that fibres appear to interconnect both at pericellular matrices, as well as at 'nodes' in the extracellular matrix.

No description available.