Chemotaxis underlies many physiological processes and is also hijacked by metastatic cancer cells to enter blood or lymphatic vessels enabling travel around the body. Using a novel migration assay we sought to determine the molecular mechanisms by which vesicle trafficking regulates chemotactic invasion. We show that dynamin is necessary for EGF-dependent migration and proceeding RNAi studies found clathrin-mediated endocytosis, but not caveolar endocytosis, to be necessary for migration in MDA-MB-231 cells. Using TIRF microscopy to investigate a role for endocytosis of integrins during chemotactic invasion we found no significant colocalisation of focal adhesion markers with endocytic markers. Inhibiting endocytosis also had no effect on the number or localisation of focal adhesions. Studies investigating EGFR showed that during EGF-directed chemotactic invasion of human breast cancer cells, EGFR endocytosis is polarised to the front of migrating cells and occurs via clathrin-mediated endocytosis. Analysis of exocytic trafficking of EGFR found this to be polarised towards the front of cells during chemotactic invasion. Finally we used FLIM-FRET microscopy to show that cells migrating in an EGF-dependent manner have increased signalling of Scr and Akt Biosensors. These experiments begin to investigate the link between endocytosis and signalling, an area not yet very well studied.

Musculoskeletal disorders are among the most important health problem affecting the quality of life and contributing to a high burden on healthcare systems worldwide. Understanding the molecular mechanisms underlying these disorders is crucial for the development of efficient treatments. In this thesis, musculoskeletal disorders including muscle wasting, bone loss and cartilage deformation have been studied using systems biology approaches. Muscle wasting occurring as a systemic effect in COPD patients has been investigated with an integrative network inference approach. This work has lead to a model describing the relationship between muscle molecular and physiological response to training and systemic inflammatory mediators. This model has shown for the first time that oxygen dependent changes in the expression of epigenetic modifiers and not chronic inflammation may be causally linked to muscle dysfunction. Bone and cartilage deformation observed in ageing, arthritis and multiple myeloma (MM) patients have also been investigated by using a novel modularization approach developed within this thesis. This methodology allows integration of multi-level dataset with large interaction networks. It aims to identify sub-networks with genes differentially expressed between experimental conditions that are co-regulated across samples in different biological systems. This study has identified several potential key players such as Myc, DUSP6 and components of Notch that could enhance osteogenic differentiation in MM patients. In conclusion, this thesis present the effectiveness of systems biology approaches in understanding complex diseases and these approaches could be applied for studying other systems and datasets.

A third of over 65s have at least one fall per year whilst a quarter of over 45s endure foot pain. Footwear is associated with both fall risk and foot pain hence its investigation is of great importance. This thesis explores the potential benefits of minimalist footwear for the older adult population. Chapter 2 ascertained the kinematic and kinetic differences between walking barefoot versus in footwear whilst highlighting the limited research on minimalist footwear, older adults and muscle activity differences. Accordingly, Chapter 3 outlined that minimalist footwear is kinematically more similar to barefoot, irrespective of age, thus offering a viable alternative. Similarly, Chapter 4 showed walking in minimalist footwear and walking unshod exhibit similar lower leg muscle activation patterns whilst differences exist to conventional footwear. Chapter 6 demonstrated how increasing intrinsic foot strength improved functional and static balance whilst Chapter 7 showed promise for minimalist footwear improving foot strength, functional balance, balance confidence as well as reducing foot and joint pain in a sample of older adults. In conclusion, this thesis highlights the need for future work to continue to investigate minimalist footwear in both older adults and other age groups for benefits to stability, foot health and joint pain.

Symptoms of gastro-oesophageal reflux disease are equally common in both sexes and at all ages. However, complications of gastro-oesophageal reflux disease such as reflux oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma, although more common in men, increase sharply in older women, suggestive of a protective effect of female sex hormones in menstruating women. Oestrogen has anti-inflammatory properties, improves healing in oral and skin wounds and may therefore reduce the severity of reflux-induced oesophageal mucosal injury, consequently protecting women from developing severe reflux oesophagitis. Long-term oestrogen treatment with hormone replacement therapy seems to be additionally associated with a reduction in the risk of oesophageal cancer. Moreover, there are gender-specific genotypic differences in the response of oesophageal mucosa to chronic acid reflux suggestive of multiple factors that may play a role in explaining the male predominance of oesophageal adenocarcinoma. Finally, oestrogen has no association with the severity of acid reflux once adjustment is made for the influence of increasing body mass index in women undergoing oesophageal pH monitoring. The gender difference in the prevalence of gastro-oesophageal reflux disease and its complications may thus be related to the effect of female sex hormones, particularly oestrogen and its 'protective' effect in pre-menopausal women.

The vestibular system provides vital information about head position and motion; which is used for the control of balance through vestibulospinal reflexes. Chapter 2 explores the process of transforming head position to body coordinates, with and without vision. The results show when vision is available, the evoked response is less precise. Chapter 3 explores the transformation process before and after 60 days of bedrest. After this period of inactivity, participants swayed more, and their EVS-evoked sway response was less precise. This decrement in precision appears to begin recovery 6 days postbedrest. Chapter 4 focuses on vestibulo-ocular reflexes rather than postural reflexes. Electrical vestibular stimulation is used to evoke measurable torsional eye-movements. The magnitude of the response is modulated by stimulus frequency. Results suggest that CNS interprets electrical vestibular stimulation as a velocity signal rather than a position or acceleration signal. This technique is an ideal measure of pure vestibular function, Chapter 5 utilised the technique in a clinical environment. Vestibular schwannoma patients, with known unilateral vestibular deficit, were tested to identify if the proposed technique can detect this deficit. Results showed that asymmetries could be detected, and, the test may be more sensitive than previously used measures of vestibular asymmetries.

Homocysteine is an amino acid formed by the metabolism of methionine. Increased plasma homocysteine concentrations are associated with cardiovascular disease, and it has been suggested that homocysteine lowering therapy may reduce cardiovascular risk. Plasma homocysteine measurements are frequently requested by clinicians investigating patients with vascular disease. A mechanism for homocysteine causing vascular disease has not yet been proven, but one possibility is that an elevated plasma homocysteine concentration may lead to the accumulation of asymmetric dimethylarginine (ADMA), a naturally occurring amino acid that inhibits nitric oxide synthase, resulting in impaired nitric oxide production, and therefore vascular dysfuntion. The aim of this project was to develop analytical methods suitable for the measurement of homocysteine and related metabolites in a routine clinical laboratory, and two methods have been established; i) for homocysteine, cysteine and methionine and ii) for asymmetric dimethylarginine, symmetric dimethylarginine (SDMA, a sterioisomer of ADMA), monomethylarginine (MMA) and arginine. A novel feature of the method for ADMA is that the use of unique daughter ions allows the determination of both ADMA and SDMA without the need to separate the isomers chromatographically. In addition, the synthesis and application of isotopically labelled SDMA, for use as an internal standard, is described for the first time. When the methods were applied to the analysis of routine clinical samples no association was detected between plasma total homocysteine and plasma ADMA concentrations. Measurements were also performed on samples from patients enrolled in a clinical trial investigating the progression of vascular dysfuntion, as measured by carotid-femoral pulse wave velocity (CF-PWV), in chronic kidney disease. Again no association could be found between plasma total homocysteine and plasma ADMA concentrations. In addition plasma total homocysteine was not a determinant of CF-PWV. These findings do not support the hypothesis that hyperhomocysteinaemia causes vascular disease by increasing ADMA concentrations.

The human fetal adrenal cortex is highly active, producing large amounts of Δ5 androgens. Together with the placenta, the fetal adrenal cortex regulates circulating progesterone, estrogen and corticosteroids in the fetus and has a major effect on maternal steroid levels. At birth, production of adequate aldosterone and cortisol are essential for survival, and shortly after birth in the human the cortex undergoes rapid remodelling. Catecholamines, produced by the adrenal medulla are also vital to the body’s ability to respond to stress, and regulate metabolic processes and blood pressure. Normal development of both the cortex and medulla of the human fetal adrenal are therefore critical for post-natal human endocrinology and health. Despite this, adrenal development and function during fetal life is poorly understood due both to the inherent difficulties in obtaining and studying human samples and to the unique nature of human adrenal/endocrine developmental which means animal models are of limited value. While poorly understood, the processes involved in fetal development are highly regulated although they can be disrupted by exposure to environmental chemicals or endocrine disruptors. Exposure to maternal smoking, for example, can disrupt normal fetal programming and has been linked with an altered postnatal stress response in the offspring, as well as a higher risk of developing cardiovascular disease, diabetes, and metabolic syndrome in adult life. These programming effects point to the possible involvement of the human fetal adrenal gland in the post-natal pathologies associated with maternal smoking. The aims of the studies described here were to develop a method to measure 26 steroids by liquid chromatography mass spectrometry (LC/MS) from samples, from which RNA and protein were also extracted, in order to make optimal use of small amounts of human tissue and use this method, and others, to examine the development and steroidogenic capacity of the human fetal adrenal cortex as well as the migration and development of the pheochromoblast cells which will go on to form the adrenal medulla. The influence of maternal smoke-exposure was also examined in these tissues. Additional aims of a set of in vitro studies were to examine the effects of cigarette smoke extract (CSE) and its components on the steroidogenic capacity of a human pluripotent adrenocortical cell line, as well as the influence of placental steroids and their withdrawal. 109 human fetal adrenals were obtained from elective terminations (REC 04/S0802/21) of second trimester fetuses between 11-21 weeks of gestation. Fetuses were grouped according to sex, gestational age and maternal smoking. Cortical steroids extracted from these adrenals were quantified by LC/MS. Cortical and medulla development was examined by measurement of key elements of the steroidogenic, catecholaminergic, and angiogenic pathways using real-time PCR (RT-qPCR), western blot and immunohistochemistry. Statistical analysis of the data was carried out using generalised linear models with age, sex and maternal smoking status as covariates. For in vitro smoke-exposure experiments, H295R cells were cultured for 3 full days in the presence of cotinine, nicotine, or CSE, and stimulated with forskolin. For examinations of the effects of placental steroids, cells were cultured in the presence of estrogen and/or progesterone for 4 days followed by an additional 4 days of culture without added steroids. Steroids and mRNA transcript levels from cells were measured by ELISA, LC/MS and RT-qPCR. Data was analysed using mixed-effects general linear models. The most abundant steroid (ng/mg of tissue) in the human fetal adrenal was pregnenolone, followed by dehydroepiandrosterone-sulphate and 17-hydroxyprogesterone and levels of these steroids were similar between male and female fetuses. Cortisol was present in all adrenals examined although aldosterone was undetected. This data suggests adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the 2nd trimester while lack of aldosterone probably explains salt-wasting disorders frequently seen in extreme preterm neonates. Fetal plasma levels of ACTH, intra-adrenal levels of progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone and adrenal transcript levels of the transcription factors GATA-6 and NR5A1 were increased by maternal smoking. However, plasma and intra-adrenal cortisol, and intra-adrenal DHEAS were unaffected. The enzymes involved in catecholamine biosynthesis were all highly expressed in the developing medulla. Migrating prechromoblast cells were clearly visible in the adrenal fetal zone through H&E staining and could be categorised into noradrenaline- and adrenaline-producing cells by immunohistochemistry for tyrosine hydroxylase and phenylethanolamine-N-methyl transferase. Maternal smoking was also associated with increased levels of PHOX2B transcript, a transcription factor involved in the maturation of chromaffin cells. In cell culture experiments, levels of CYP11A1, CYP17A1, CYP21A2, HSD3B, PGR and ESR2 transcripts were all significantly reduced in cells exposed to CSE. The effects of CSE-exposure on steroid production was variable but pregnenolone and progesterone production was highly stimulated under basal conditions, indicating that CSE has both cell-stimulatory and cell-inhibitory effects. Estrogen and progesterone exposure also had variable effects on steroid production by the cells, depending on other stimulatory factors, and results indicate that other placental factors, in addition to placental steroids, are likely to play a role in cortical remodelling after birth. In conclusion, the studies described here have comprehensively examined the steroidogenic capacity of the human fetal adrenal cortex during the second trimester and have begun to characterise the development and migration of the pheochromoblasts. The results described here provide an understanding of normal development of the steroidogenic capacity of the human fetal adrenal during the second trimester, as well as some of the pathologies associated with abnormal adrenal development. The effects of maternal smoking on the processes examined were not marked, however the disruption of steroid production or dysregulation of transcription factors may lead to changes in adrenal function in postnatal life.

This thesis presents four empirical chapters that challenge current sport concussion research and practice. Chapter 2 measured sport concussion knowledge in the UK general public using an online survey. It showed high sport concussion awareness, but limited and erroneous understanding. Chapter 3 examined the effect of terminology (i.e., concussion; mild traumatic brain injury, mTBI; minor head injury, mHI) on familiarity, injury outcome expectations and symptom self-report in athletes using a questionnaire. The mTBI terminology was the least familiar, reliably more negative conceptualised, but knowledge was more accurate than the other two. Symptom self-report did not vary with terminology or injury history. Chapter 4 compared the late neuropsychological functioning in self-reported sport-concussed to non-concussed athletes using a comprehensive test battery. Injury self-report was associated with worse memory recall and executive function shifting. Chapter 5 piloted a computerised neuropsychological test battery in athletes using a longitudinal control group design. A single case study showed transient deficits in memory recall and executive function at one to six weeks post-concussion. The overall data suggest that (i) education is needed; (ii) the interchangeable terminology use is inappropriate; (iii) sport concussion assessment should be complemented by memory recall and executive function tests; (iv) case studies might be more appropriate than group comparisons.

Minimally invasive surgery (MIS) is fast becoming a preferred choice for patients and surgeons, due to its biological, aesthetic and commercial benefits. The dynamic hip screw (DHS) is the standard implant for the treatment of fractures of the proximal femur, which is considered to be the most frequent injury in the elderly. The aim of this research was to develop MIS for the treatment of these fractures utilising the principle and surgical technique of the DHS implant. During the research, a thorough medical device design process was conducted to develop three new medical devices 13 a new angle guide, a new ergonomic T-handle and a new implant. The design process for each of the new medical devices conformed to requirements of the relevant standards. The designs of the new medical devices were verified using methods such as risk analysis, finite element analysis and mechanical testing of manufactured prototype. Finally, an operative technique applying a minimally invasive approach with the new medical devices was developed to treat the fractures of the proximal femur.

The aim of this project was the study of neutrophil recruitment and reverse transmigration using murine and human in vitro models of inflammation. Murine in vitro models of inflammation were developed using an immortalised microvascular cell line (MCEC-1) and primary murine vascular endothelial cells (mEC) isolated from heart and lung. We found that MCEC-1 could recruit murine neutrophils without the requirement of cytokine stimulation, although efficient transmigration did require such a stimulus. Primary cells required cytokine stimulation to recruit mEC. Interestingly, and in contrast to human EC, mEC were relatively insensitive to TNF-α stimulation, although IL-1β was a good stimulus for adhesion and migration. Using the IL-1β driven system we generated reverse migrated murine neutrophils and their phenotype and prolonged survival were assessed. The effect of shear stress and nitric oxide on the regulation of the process of reverse migration was examined. Using adoptive transfer strategies we investigated the fate of mRPMNs in vivo. A significant part of this work involved the study of human reverse migrated neutrophils at a proteomic level using two-Dimensional Fluorescence Gel Electrophoresis methodology to identify changes in neutrophils associated with reverse migration process. We found that murine reverse migrated neutrophils had a very similar surface phenotype to human reverse migrated cells. They also showed prolonged survival. However, our preliminary data on trafficking in vivo did not give a clear indication about their fate upon adoptive transfer into recipient mice. In vitro studies showed that flow generated shear stress and nitric oxide delayed, but did not inhibit, the process of reverse migration. Finally, the proteomics study revealed a number of metabolic, cytoskeletal and regulatory proteins that were differentially expressed in human reverse migrated neutrophils although the functional significance of these changes is yet to be explored.