Low Intensity Pulsed Ultrasound (LIPUS) is used clinically to promote wound healing. In vivo studies show that LIPUS is effective in a wide range of tissue types, and in vitro experiments show that multiple cell types respond to LIPUS stimulation. Despite this, there is no unifying mechanism of how LIPUS stimulation is sensed by cells, and it is unknown what the early signalling events are. The LIPUS signal is a mechanical one; therefore I hypothesised that mechanosensitive organelles, called focal adhesions, would be essential for the induction of cellular signalling events in response to this type of stimulation. Proteins within these structures (such as vinculin and talin) link the actin cytoskeleton to the extracellular matrix via integrins, and are known to be sensitive to mechanical forces, capable of generating intracellular signalling events in response to mechanical stimulation. The purpose of this work was to identify the early signalling events occurring within minutes of LIPUS stimulation; determine the molecular mechanisms behind such events; and to investigate whether such events require integrin-mediated adhesions. In the first part of the work, I established the use of live-cell imaging together with LIPUS stimulation to directly observe the cellular response. I determined rapid reorganizations of the actin cytoskeleton, which led to increased cell velocity. These effects were found to be Rac dependent, and, using FRET-based probes, I measured rapid increases in Rac activity occurring within minutes of LIPUS stimulation. The second part of this work identified an increase in the number of early endosomes in cells stimulated with LIPUS. This phenotype was also Rac dependent, as well as requiring the early endosomal regulator protein Rab5. In this chapter, I observed an increase in the association between Rac and Rab5 in response to LIPUS stimulation, and this contributes to Rac activation. Using substrates to block integrin-mediated adhesion, I determined that cell-matrix adhesions are required for the effects of LIPUS stimulation. Using vinculin-deficient cells, I determined that this mechanosensitive protein is vital for co-ordinating Rac activation in response to LIPUS. In particular, the actin binding tail is needed for mechanosensing of this LIPUS signal. In the final chapter I established the use of photoactivatable (PA) GFP to assess adhesion protein turnover. This technique was used to show that LIPUS stimulation directly affects the turnover of vinculin. Overall, this work shows that the mechanosensitive protein vinculin is crucial for sensing the mechanical stimulation provided by LIPUS, orchestrating downstream Rab5-mediated Rac activation to enhance cell motility.

A large number of bacteria are able to secrete extracellular proteases that play vital roles in nutrient acquisition and biofilm formation but are also important biochemical mediators in bacterial virulence, facilitating host invasion. Microorganisms within chronic wounds have been hypothesised to form biofilms, which result in perpetuated inflammation and delayed wound closure. Whilst it is thought that exaggerated secretion of host-derived proteases within chronic wounds prevents successful wound closure, the contribution of bacterial proteases secreted from planktonic microorganisms and biofilms in chronic wound pathology has yet to be elucidated. It is therefore the primary research aim of this thesis is to assess the proteolytic activity of Pseudomonas aeruginosa and Staphylococcus aureus isolated from equine and human chronic wounds and determine whether there is a difference in activity between planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM). The next aim will be to identify these bacterial-derived proteases using zymography and mass spectrometry, and determine whether these proteases reduce wound closure in in vitro scratch wound models. More specifically, the effect of P. aeruginosa and S. aureus PCM and BCM from equine and human clinical isolates, and also purified proteases of these preparations, on the in vitro wound closure of equine normal fibroblasts (NFs), equine chronic wound granulation tissue fibroblasts (GTFs) and human dermal fibroblasts (HDFs), shall be investigated. In these wound models, zymography will be utilised to determine the release of host-derived matrix metalloproteases (MMPs). In this thesis I have identified, for the first time, high protease activity in equine chronic wound-derived P. aeruginosa PCM and BCM, by which 52kDa and 42kDa proteases were detected. P. aeruginosa PCM and BCM were shown to significantly reduce the wound closure of NFs (P < 0.0001) and GTFs (P < 0.0001). Furthermore, the soluble products of P. aeruginosa biofilms, but not planktonic P. aeruginosa, elicited the release of metalloproteases from equine NFs and GTFs. In human studies, high protease activity specific to P. aeruginosa BCM but not PCM (P < 0.0001) was determined. P. aeruginosa BCM significantly reduced the wound closure of HDFs (P <0.0001) and cell viability (P < 0.0001), and further induced the release of metalloproteases from HDFs. P. aeruginosa in biofilm form secreted 62kDa and 52kDa proteases, however, through the partial purification of these proteases, it was determined that these proteases did not play a role in the reduction of wound closure in HDFs. The P. aeruginosa-derived partially purified proteases did however reduce HDF cell viability (P < 0.05). The results in this thesis support the theory that bacterial-derived proteases may contribute to the emphasised proteolytic environment of human chronic wounds. Furthermore, the presence of bacterial biofilms within chronic wounds may induce the release of host-derived proteases. However the role of these specific P. aeruginosa-derived bacterial proteases in chronic wound pathology remains undetermined.

Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

Background and Aims: Regular physical activities have shown to have various health benefits however there is always an accompanying risk of developing musculoskeletal soft tissue injuries. Indeed, damaged tendons account for 30-50% of sports-related injuries where the lifetime risk of Achilles tendon pathology (ATP) approaches 50% in runners, and that of patellar tendon pathology (PTP) is 21% in football players. The exact aetiology and mechanisms of tendon pathologies are still under investigation, however extrinsic and intrinsic risk factors (of which genetic) have been identified. Recent genetic association studies found that gene variants within the TNC, COL5A1, MMP3, GDF5 and CASP8 were associated with ATP in a Caucasian Australian and South African population. Furthermore, epigenetic mechanisms such as DNA methylation and microRNA (miRNA) activity have been implicated in a range of diseases but were never investigated for their role in human tendinopathy. Based on the aforementioned information, this thesis aimed at investigating novel candidate genes that may be associated with ATP and to replicate previously conducted studies in a newly recruited case-control population. Additionally, this thesis aimed at investigating potential differences in DNA methylation profiles and miRNA expression levels between healthy and damaged Achilles and patellar tendons. One hundred and thirty six UK Caucasian participants with clinically diagnosed ATP and 131 asymptomatic, unrelated, physically active control participants were recruited for this study. Furthermore, the previously recruited 173 clinically diagnosed ATP participants and 238 asymptomatic, unrelated, physically active control participants from Australia and South Africa (AUS+SA) were also included in the studies of this thesis. Participants within the combined AUS+SA were genotyped for the ELN rs2071307, FBN2 rs331079, ADAM12 rs3740199, ADAMTS2 rs1054480, ADAMTS5 rs226794, ADAMTS14 rs4747096, and TIMP2 rs4789932 variants, and the UK participants were genotyped for the COL5A1 rs71746744, FBN2 rs331079, GDF5 rs143833, MMP3 rs679620, TIMP2 rs4789932 variants using fluorescent based TaqMan® technology. Furthermore, the UK cohort was genotyped for the COL5A1 rs12722 variant using polyacrylamide gel electrophoresis. Moreover, 10 healthy and 10 diseased patellar tendon tissue samples in addition to 4 healthy, and 1 diseased Achilles tendon samples were obtained for the epigenetic studies. The DNA methylation profile within the TIMP2 and GDF5 promoter regions were analysed for all samples using pyrosequencing technology. Furthermore, the expression levels of TIMP2, miR-21, miR-155, and miR-191 were determined by RT-PCR using TaqMan technology. Results and Discussion: The genetic association studies conducted showed that the FBN2 rs331079 GG genotype was over-represented among the tendinopathy (TEN) group and that the ELN rs2071307 AA genotype was over-represented in the rupture (RUP) group within the AUS+SA population. Furthermore, the COL5A1 rs12722 and rs71746744 were associated with RUP (TT genotype over-represented in the RUP group, p=0.004; OR=4.2; 95% CI 1.58-11.97) and ATP (DEL allele over-represented in the CON group, p=0.046; OR=1.61; 95% CI 1.01-2.56) respectively in the male UK cohort. The GDF5 rs143833, on the other hand, was not associated with ATP (p=0.538) and showed no sign of gender-specific association (female p=0.737; male p=0.319) in the UK population. Furthermore, the CT genotype for the TIMP2 rs4789932 variant was over-represented (p=0.004; OR=1.77; 95% CI 1.20 - 2.64) in the ATP group of the combined AUS+SA population and the CC genotype was over represented (p=0.016; OR=2.36; 95% CI 1.16 – 5.81) in ATP of the UK male cohort. It was also reported that the MMP3 rs679620 GG genotype was over represented (p=0.027; OR=2.51; 95% CI 1.11 – 5.64) in the UK RUP group. The ADAM12 rs3740199 (p=0.633), ADAMTS2 rs1054480 (p=0.316), ADAMTS5 rs226794 (p=0.342), and ADAMTS14 rs4747096 (p=0.849) gene variants were not associated with ATP in the AUS+SA population. Interestingly, individuals carrying the ADAMTS14 rs4747096 GG genotype within the AUS+SA population and the ELN rs2071307 AA genotype within the AUS population developed their injuries at a significantly (p=0.024; p=0.005, respectively) later stage than other participants. Moreover, UK males diagnosed with tendinosis and carrying the GG genotype at the MMP3 rs679620 locus developed significantly (p=0.003) thicker tendons than other participants with the AA, and AG genotypes. The preliminary epigenetic DNA methylation studies showed no differences in the average methylation profiles of the investigated regions within the TIMP2 (p=0.885) and GDF5 (p=0.333) genes in the patellar tendon samples. Moreover, no DNA methylation differences (p=0.617) were observed in the investigated region of the TIMP2 gene in the Achilles tendon samples. Interestingly, the single ATP sample showed a lower GDF5 average methylation profile than the CON samples. Furthermore, the expression of TIMP2 was up-regulated, and miR-191 was down-regulated in the ATP tissue sample compared to the CON group. The expression levels of miR-21 and miR-151, however, were not different between the two groups. Conclusion: This thesis provides evidence that novel genes coding for structural and ECM regulatory enzymes are associated with ATPs in Caucasians. The findings of this thesis should to be replicated in new and larger cohorts from different ethnic backgrounds before being incorporated into multifactorial risk assessment models aiming at reducing the incidence of human tendinopathy.

Several studies using a sociological approach identified the existence of a culture of risk in sport. These works studied professional, amateur, male and female sport figurations and concluded that this culture of risk enmeshes athletes to practice and compete while in pain and when injured. Particularly, studies about gender acknowledge the existence of similar male and female experiences of pain and injury. However, these studies separately studied male and female sports. No work to date has studied within the same research design athletes and coaches perceptions about pain and injury experiences and how they are negotiated and socialized. Thus, this study sought to explore whether male and female experiences of pain and injury really are similar, or whether differences would become evident through a study which involved a more direct comparison. The research design of this study was informed by figurational sociology. Data for this work were firstly gathered during 9 months of overt-observation. By including observational notes from the interactions between 11 male athletes, 13 female athletes, 3 coaches of the male team, 3 coaches of the female team and 2 physiotherapists, this research provides a more adequate understanding of the gymnastics figuration, its interdependences and power fluxes. Additionally, 9 male athletes, 8 female athletes, 3 male team coaches and 2 female team coaches were interviewed. Data collected in this research is in accordance with sociological literature about pain and injury in sport. All athletes revealed a willingness to continue training and competing even when injured and in pain. However, data also revealed gendered differences about how male and female athletes are enmeshed in the culture of risk. Particularly, gendered differences were found in the training environment, coach-athlete power differences, body control, socialization processes and in male and female athletes expressions of pain and injury. Thus, this research raises several questions about the value of sociological studies of gender in sport that approach male and female experiences separately, as gender is sociologically created through male and female interdependence.

The natural history of scar maturation in humans has been described by Bond et al. (2008b) in a male European Continental Ancestry Group (ECAG). It is important that the natural history of scar maturation in humans is established for all skin types. This study therefore aims to describe clinically and histologically the maturation of scars in male volunteers from the African Continental Ancestry Group (ACAG).This study was performed as a single centre, methodology trial. Three incisions and a punch biopsy were carried out on each arm. Monthly assessments of the resultant scars included: investigator scar assessments; scar photography; VAS scoring by an Independent External Scar Assessment Panel; and objective measures of colour and scar mechanics. At various time points scars were excised for histology. Sixty male subjects of African Continental Ancestry between the ages of 18-56 years were recruited to take part in the study. The clinical appearance of a scar in the ACAG improves with time. Scar colour mismatch decreases and the mechanical properties of scars improve with time. Scar width increased over the 12 months. With the exception of scar contour and scar redness, a steady state was not achieved. Volunteer skin type was shown to influence the resulting scar appearance and not age. The histology of scar maturation in the ACAG over 12 months was described and scars classified into three groups each displaying a different rate of longitudinal progression of scar maturation. The process of collagen maturation is still ongoing at month 12; many scars demonstrated a prolonged high turnover state of collagen synthesis and degradation, rete ridge restoration and angiogenesis were still ongoing with persistent inflammation identified in scars up to Month 12. There is a strong correlation shown between the Clinical VAS scores and the Histology VAS scores for the papillary dermis which is of better quality than the reticular dermis. There is some evidence that young people (ACAG) and volunteers with darker skin have poorer scar histology. The spectrophotometry data indicated that the Fitzpatrick Skin Type Classification is a useful method of classifying the varying skin colours of this group of volunteers. In conclusion, scar maturation in the ACAG occurs as a series of defined macroscopic and microscopic stages over the course of 1 year. The process of scar maturation is not complete at 12 months. All scars showed evidence of improvement over the course of the study influenced in part by volunteer skin type and age. Results suggest that scar maturation in this study group occurs at a different rate and is of a different quality, compared to current knowledge of scar maturation in the ECAG.

For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.

No description available.

Infection after trauma complicates the patients clinical course. Infection leads to longer critical care and hospital stays, has been associated with increased mortality rates and places considerable cost pressures on health economies. The predictors of infection after severe injury are not known, and the effects on outcomes other than mortality are under-reported. The overall objective of this research was to characterise factors predictive of infection in severely injured patients admitted to critical care. A prospective cohort study of 271 patients investigated admission factors predictive of the development of infection. A second study of 280 patients evaluated post-injury immune cell changes and the association with infection. Thirdly the relationship between early coagulopathy and infections was investigated in 158 patients. Finally a study of 385 patients examined the use of Tranexamic Acid (TXA) and its association with infection and other outcomes. Infection was a significant burden for severely injured patients. Admission hypoperfusion was the only early characteristic associated with the development of infection, and a dose dependent relationship was observed between severity of shock and increased percentage of infection (p<0.01). Lymphopenia prolonged to day four post injury was strongly predictive infection (OR 0.10, CI 0.02-0.48, p<0.01). At 24 hours, the anticoagulant Protein C was lower in those with infection (Infection: 70.2 iu/dL vs. No infection: 83.3 iu/dL p=0.02), and increased fibrinolysis was also associated with infectious complications (Infection: 6156 μg/L vs. No infection: 3324 μg/L p=0.03). There was a trend to a beneficial relationship between TXA and infection, and it was independently associated with reduced organ failure (OR 0.27, CI: 0.10 – 0.73, p=0.01) and mortality (OR 0.16 CI 0.03 - 0.86, p=0.03). In severely injured patients, admission shock, prolonged lymphopenia and early coagulation dysfunction post severe injury were independent predictors of infection. Timely modulation of these responses after trauma may help to reduce the burden of infection.

Proximal forearm fractures account for over 10% of all upper limb fractures. There is limited epidemiological data available and much of the literature focuses on the more complex fracture patterns, with the role of non-operative management for the isolated proximal forearm fracture still to be defined. Prospective short and long-term patient reported outcome data for simple isolated fractures of the radial head and olecranon would help define the indications for the non-operative management of these injuries. This thesis aims to test the hypothesis that non-operative management provides a comparable outcome to operative intervention for defined fractures of the proximal forearm. A large prospective database of 6872 fractures collected over a one-year period was used to define the epidemiology of proximal forearm fractures. A separate large prospective study carried out over an eighteen-month period using a pre-defined management protocol for all isolated radial head and neck fractures was analysed to determine the short and long-term outcome. Additional retrospective databases were collected and analysed to determine the short and long-term outcome for the non-operative and operative management of olecranon fractures, as well as the operative management of complex radial head fractures. Finally, two prospective randomised controlled trials (PRCTs) of isolated displaced fractures of the olecranon were carried out to compare 1) tension band wire (TBW) versus plate fixation in younger patients (< 75 years) and 2) operative versus non-operative management in elderly patients (≥75 years). The primary outcome measure for these studies was the upper limb specific patient reported Disabilities of the Arm, Shoulder and Hand (DASH) score. Secondary outcome measures included surgeon reported outcome scores, complication rates and cost. The incidence of proximal forearm fractures was 68 per 100,000. Radial head fractures fit a type D distribution curve (unimodal young man, bimodal woman) and radial neck type A (unimodal young man, unimodal older woman). Proximal ulna and olecranon fractures were both a type F (unimodal older man, unimodal older woman), with an increasing incidence after the 6th decade. Over 90% of proximal radial fractures were isolated stable fractures. Prospective analysis of 201 isolated proximal radius fractures found that the patient and surgeon reported outcome following primary non-operative management for Mason type 1 and type 2 (n=185) fractures was excellent in the short and long-term, with < 2% of patients undergoing secondary surgical intervention. At a mean of 10 years post injury (n=100), the mean DASH score was 5.8 and 92% of patients were satisfied. Factors associated with a poorer short and long-term patient reported outcome included increasing fracture displacement (≥5mm) and socio-economic deprivation. Retrospective analysis of 105 acute unstable complex radial head fractures found that the mean short-term functional outcome was good (mean Broberg and Morrey Score 80) following radial head replacement. In the long-term (mean 7 years), 28% of patients required removal or revision of the prosthesis, with younger patients and silastic implants independent risk factors (both p < 0.05). Retrospective analysis of 36 operatively managed isolated displaced olecranon fractures found satisfactory short and long-term outcomes, with the symptomatic metalwork removal rate 47% and the mean DASH 2.5 at a mean of seven years post injury. In the PRCT of plate (n=34) versus TBW (n=33) fixation, comparable functional and patient reported outcomes (DASH 8.5 vs 13.5; p=0.252) were found at one year following injury. Complication rates were significantly higher in the TBW group (63.3% vs 37.5%; p=0.042), predominantly due to a significantly higher rate of symptomatic metalwork removal (50.0% vs 21.9%; p=0.021), resulting in equivocal costs for both techniques (p=0.131). In older lower-demand patients, short and long-term retrospective analysis found very satisfactory outcomes following non-operative management of isolated displaced fractures of the olecranon, with patient satisfaction 91% and no patients requiring surgery for a symptomatic non-union. The preliminary results of the PRCT of non-operative (n=8) versus operative (n=11) management demonstrated comparable functional and patient reported outcomes at all points over the one-year following injury (all p≥0.05), with a higher rate of complications (81.8% vs 14.3%; p=0.013) and cost (p=0.01) following surgical intervention. The association found between fragility and the epidemiology of proximal forearm fractures highlighted the importance of considering non-operative management for these injuries. These findings support non-operative management for isolated stable radial head and neck fractures. For more complex injuries when radial head replacement is indicated, there is a high rate of removal or revision, with younger patients most at risk. In younger active patients with an isolated displaced fracture of the olecranon, TBW and plate fixation provide comparable short-term results, with TBW fixation as cost effective despite an increased rate of metalwork removal. In older lower demand patients, this data provides strong evidence for the non-operative management of isolated displaced olecranon fractures.