To investigate the lip reed, artificial mouths have recently been employed to facilitate scientific study of the behaviour of the reed. These devices model the human lips using natural latex rubber tubes filled with water. This study uses one such device to experimentally investigate the relationships between the resonances of the reed and the resulting self-sustained oscillation when driving an acoustic resonator - a trombone in this case. The resonances of the reed are initially investigated by measuring the area of opening between the lips as they are driven by a known oscillating acoustic signal. Additionally, the resonances of the reed are investigated in two dimensions using a laser doppler vibrometer.  Motion of the lips in two dimensions is analysed using a high speed digital video camera. Using these results, computational investigations of lip models provide a great deal of insight into the basic mechanics of the reed. Linear stability analysis shows that a model with at least two degrees of freedom is required to reproduce experimentally observed threshold playing frequencies. Time domain simulations are used to investigate the response of the models, and comparisons are made with the experimentally obtained response data. Strong similarities are found between the area of opening function of a two degree of freedom model and the area function recorded in experimental studies, when the opening area is described by an appropriate non-linear functions of the reed position. These results greatly improve the understanding of the merchants of the lip reed, and pave the way for the development of fully capable physical sound synthesis models.

These observations on the development of the human foot arose from the discovery of a bipartite medial cuneiform bone. The investigation of literature concerning this anomaly led to a survey of certain facts set out below in the ontogeny and phylogeny of the human foot. It seems suitable at the outset, therefore, to state the differences between the human foot and its nearest relative, the anthropoid foot. These are clearly and concisely stated by Professor Wood Jones (1929) as follows: "The outstanding features of the human foot are as follows: (a) The innermost (tibial) digit is the dominant digit: it is directed in line with its fellows, and as a rule its tip is in advance of that of any of the others. (b) Its plantar and dorsal surfaces are directed in the same Y7 as those of other digits. (off It is 'webbed' or rather included in the common integuaentary covering of the foot to approximately', the aid point of its basal phalanx, its metatarsal being wholly incorporated within the tissues of the foot. (d) Its metatarsal is firmly bound to the metatarsals of the remaining digits by the powerful transverse metatarsal ligament. (e) Its basal articulation with the ento-cuneiform is effected by a flattened articular surface.

Cortisol (C) and testosterone (T) are commonly suggested as markers of overreaching and the unexplained underperformance syndrome (UPS) as taken together they highlight the body s state of stress by indicating the body s catabolic/anabolic balance. Research in this area has focused on the resting concentrations of these hormones and provided inconsistent findings with increases, decreases and no changes reported when individuals are compared in an overreached state with a normally trained state. Little attention has been given to the exercise-induced responses of these hormones and whether this could be a reliable marker of overreaching/UPS. Overreaching will only occur with an intensification of training so the aims of the studies in this thesis were to determine the effects of intensified training on the exercise-induced responses of salivary and plasma C and T concentrations. Study 1 (Chapter 4) determined the salivary and plasma C, T and plasma adrenocorticotrophic hormone (ACTH) concentration responses in physically fit, healthy males to a double-bout cycle to fatigue protocol devised by Meeusen et al. (2004). They reported blunted exercise-induced hormonal responses to this protocol when well-trained cyclists were overreached compared with a normally trained state. Study 1 concluded that the exercise-induced responses of the salivary and plasma C and plasma ACTH concentrations were unaffected by a 4-day intensified training period. Blunted exercise-induced salivary and plasma T concentrations were found post-training but were due to blunted resting, basal T concentrations post-training compared with pre-training. The double-bout cycle to fatigue protocol did not elicit large C or T responses and so was not ideally suited to highlight alterations in the exercise-induced hormone responses. A high-intensity, short-duration exercise protocol (called the 55/80 bout) was established in Chapter 5 which induced robust elevations of salivary and plasma C and salivary T concentrations when in a normal trained state. Such a protocol could highlight any adaptations in the exercise-induced responses of C and T concentrations. It was also concluded that salivary and plasma C concentrations positively correlated if the peak post-exercise values were compared but not so with the salivary and plasma T concentrations. Chapter 6 and Chapter 7 concluded that blunted responses of the salivary C (Chapter 6) and T (Chapter 6 and Chapter 7) concentrations to a 55/80 bout occurred after an intensified endurance training period (~10 days). These results indicate that the 55/80 bout could be a useful detection tool of exercise-induced alterations in salivary C and T concentrations caused by an elevation of training loads in both recreationally active and elite athlete populations. The reproducibility of the salivary hormonal responses to the 55/80 bout needed to be established before it could be concluded that this was indeed a useful tool. Chapter 8 concluded that the responses of both salivary C and T concentrations to the 55/80 bout were reasonably reproducible with intra-individual variations of 12% (salivary C) and 7% (salivary T) reported. Chapter 8 also concluded that a familiarisation 55/80 bout was needed to reduce the variation in the responses of both salivary C and T concentrations. The final experimental chapter examined the response of salivary C and T over a competitive season in elite male triathletes and concluded that the 55/80 bout was unable to highlight any adaptations in the salivary C and T exercise-induced responses. This was suggested to be due to the low numbers of participants in this study and the ability of the triathletes to cope well with the elevations in training loads over the season. In conclusion, the studies in this thesis suggest that the exercise-induced responses of salivary C and T do alter due to an intensification of training loads. This alteration presents as a blunting of the exercise-induced responses of these salivary hormones. The 55/80 cycle bout can highlight this blunted response in both recreationally active and elite athlete male populations and therefore may be a useful tool to examine exercise-induced adaptations in salivary C and T concentrations caused by periods of intensified training.

Individuals working in physically demanding occupations can experience high levels of heat strain due to the physical demands of the job, harsh environmental conditions and/or the wearing of personnel protective equipment (PPE). The ability to monitor body core temperature (Tc) could reduce the risk of heat illness. The main purpose of this thesis was to examine non-invasive methods of Tc measurement. Studies 1, 2 and 3 investigated the validity of insulated skin temperature (Tis) as a non-invasive measure of Tc, in emergency service (ES) and military personnel. In Study 1, a model including Tis and micro-climate temperature (Tmc) was developed to predict rectal temperature (Tre) for ES personnel wearing PPE. The resulting standard error of the estimate (SEE=0.20 °C) was within the a-priori pre-defined SEE limit (0.20 °C), providing encouragement for the further investigation of Tis as a surrogate measure of Tc. Studies 2 and 3 sought to determine the validity of Tis in predicting Tre in military personnel, wearing PPE in temperate conditions (Study 2) and in a desert environment (Study 3). Although the SEE was outside the acceptable SEE in Study 2 (0.22 °C), the sensitivity (97 %) for predicting Tre values over 38.5 °C provided scope for a military application for Tis to predict Tc. In Study 3, Tis could not predict Tre in a desert environment, with simulated solar radiation directly affecting Tis and invalidating the prediction (SEE = 0.29 °C). Study 4 involved a series of experiments performed under six conditions in a thermal chamber with two clothing types, to determine whether the addition of other physiological and/or environmental factors might improve the prediction of Tre. A model including Tis and Tmc resulted in an SEE of 0.26 °C; with heart rate (HR) and work significantly reducing the SEE (0.23 °C) (p<0.05). Although the SEE achieved in the validation (0.27 °C) was larger than in Studies 1 and 2, these results provide novel information regarding the measures that explain the variance when predicting Tre in a wide range of heat stress conditions. To our knowledge this is the most detailed analysis of Tc prediction based on non-invasive sensors, with the inclusion of all the parameters that are likely to be relevant. The main conclusion from the work thus far was that it is unlikely a reliable prediction of Tc can be achieved, using Tis, for validity under different types of heat stress conditions. However, predictions of Tre for more specific conditions using Tis are achievable. Having collected a vast amount of data on participants demonstrating high levels of heat strain, it was considered valuable to analyse the drop-outs in more detail. More specifically the goal of Study 5 was to see whether measurements of individual heat strain (Tc, HR) and the combination of these in the Physiological Strain Index (PSI) had predictive power for individual drop-out. There were no differences in PSI between individuals who stopped from heat exhaustion (HE) (7.9 ± 0.8) and those who completed the trial (C) (8.3 ± 0.9). The only differences between these two groups were rate of rise of Tre, (C 0.03 ± 0.01 °C min-1 and; HE 0.04 ± 0.01 °C min-1), chest temperature (Tchest) (C 38.1 ± 1.0 °C and; HE 39.0 ± 0.6 °C) and the temperature gradient between Tchest and Tre (C 1.04 ± 1.07 °C, and; HE -0.05 ± 0.59 °C) (p<0.05); It was therefore concluded that PSI did not provide a good personal heat strain measure that would predict tolerance of the individual. In conclusion, Tis (with Tmc) is promising as a non-invasive measure of Tre, in ES and military personnel wearing fully-encapsulated PPE, based on the resulting SEE and sensitivity and specificity. With the current methodology, it is not valid in conditions with a solar load. The addition of HR and work together improve the prediction of Tre. The PSI does not enable identification of individuals who are approaching heat exhaustion, requiring the inclusion of other physiological responses which determine tolerance.

More than 90% of BRAF mutations in human cancer are represented by a valine to glutamic acid mutation at residue 600 known as the V600EBRAF mutation. Our laboratory has generated mouse models expressing a conditional knock-in allele of BrafV600E and in previous work we have shown that expression of the oncogene in a range of tissues including embryonic fibroblasts (MEFs), lung tissue, melanocytes and gastrointestinal crypts induces cell proliferation followed by senescence. Previous work from our laboratory showed that in the transition from proliferation to senescence, a decrease in Braf protein levels occurred in lung adenomas from BrafV600E mice. I have further investigated the mechanisms underpinning this behavior in two different cellular systems: primary mice embryonic fibroblasts conditionally expressing endogenous V600EBraf and HEK293T cells transiently expressing V600EBRAF. My findings show that in a similar way to BrafV600E lung adenomas, a reduction in Braf expression is observed in both cellular systems in addition to a decrease in Craf protein levels. I found through qRTPCR experiments that Braf and Craf mRNA levels are not downregulated in BrafV600E MEFs. However, a reduction in the stability of BRAF and CRAF proteins in the soluble fraction and a simultaneous accumulation of these proteins in the insoluble fractions was observed in both cellular systems. Inhibition of the Erk pathway at the level of Braf and Mek rescued Braf and Craf expression in BrafV600E MEFs indicating that the expression of these proteins is dependent on Braf and Mek kinase activities. LC-MS/MS analysis of ectopic WTCRAF when co-expressed with ectopic V600EBRAF showed that the accumulation of ectopic WTCRAF in the insoluble fraction was associated with the phosphorylation of six novel serine residues in CRAF. The mutation of these phospho-residues to non-phosphorylatable alanines rescued ectopic WTCRAF expression in the soluble fraction and prevented the accumulation of this protein in the insoluble fraction. Although phosphorylation of Ser675 in ectopic V600EBRAF was also specifically detected in the insoluble fraction, mutation of this site did not alter the distribution of this protein. Thus, I propose that the post-translational regulation of BRAF and CRAF expression is an alternative mechanism for the regulation of the MAPK pathway output in cells expressing the V600EBRAF oncogene.

Initial work carried out in this study, set out to develop a suitable cellular model for Bcl-2 over-expression that could be employed in later studies to characterise the interaction between Bcl-2 and calcium signaling. An established anti-apoptotic effect of Bcl-2, is reduction of calcium release from the ER. As a consequence of this reduction, mitochondrial calcium uptake and apoptosis is suppressed. These effects of Bcl-2 were observed in my experimental model of stable over-expression. Examination of the mechanism responsible, for this reduction in calcium release, revealed that it was not due to a decrease in ER store loading, but due to a decrease in sensitivity of the calcium release process. In agreement with previous studies which demonstrate that calcium release from the ER is inhibited as a result of a functional interaction between Bcl-2 and the inositol 1,4,5-trisphosphate (InsP₃) receptor, Bcl-2 was shown to interact with the InsP₃R in HEK-293 cells. Since lumenal calcium concentration was shown to be unaffected by Bcl-2, there is a high likelihood that this interaction was contributing to the inhibition of ER calcium release observed in this study. In addition to regulating ER calcium signalling, Bcl-2 modulates mitochondrial calcium homeostasis. Bcl-2 has been shown to increase the calcium buffering and storage capacity of the mitochondria, allowing higher calcium concentrations to be reached before mitochondrial calcium overload occurs. Results presented in this thesis, showed that Bcl-2 also functions to reduce the sensitivity of mitochondrial calcium uptake. Over-expressed Bcl-2 increased the cytosolic calcium concentration required to trigger mitochondrial calcium uptake, in response to ER calcium release and store operated calcium entry.

No description available.

The dietary lipid Conjugated Linoleic Acid (CLA) has been the subject of a large body of research in relation to its anti-inflammatory and immunoregulatory role in many cell types and animal models. This study aimed to investigate the effect of a mixture of the 9:11 and 10:12 isomers of CLA as well as the effect of the 9:11 isomer alone on the human monocytic cell-line THP-1 cells and human endothelial cells (HUVEC). The effect of supplementation with 2g/day CLA (a 50:50 mixture of 9:11 and 10:12 CLA) and 9:11 CLA in a cross-over trial in subjects with metabolic syndrome was also investigated. Neither CLA nor 9:11 CLA demonstrated any significant endoplasmic reticulum stress or induced apoptosis when cells were treated with the lipids over a concentration range of 20-200μM. Both lipids were able to upregulate gene expression of the nuclear transcription factor PPARγ in both cell types, with CLA having the most marked effect. A significant increase in the PPARγ dependant gene CD36 was also observed and a significant increase in PPARα mRNA was seen with CLA. However, no significant increase in the PPARα dependant CXCL2 gene was observed. PPAR activity in these cells did not appear to be mediated by the induction of endogenous ligands by Cycloxygenase-2. Both lipids were capable of upregulating another transcription factor LXRα, suggesting an important role for both lipids in cholesterol homeostasis. Both CLA and 9:11 CLA increased mRNA expression for the receptor for advanced glycated end-products (RAGE) and induced solubilisation of the receptor that was not mediated by increasing activation of the metalloproteinase enzyme ADAM10. There was significant induction of RAGE splice variants secretion in particular esRAGE after CLA supplementation. The clinical trial demonstrated that dietary supplementation with both isomers induced increased sRAGE secretion, and improved markers of endothelial function and diastolic blood pressure. Improved platelet function was also observed and a non-significant but beneficial trend was observed for HDL-cholesterol and LDL-cholesterol. Multiregression analysis would suggest that the most significant predictor of the reduction in DBP was the increase in sRAGE secretion induced by both CLA and 9:11 CLA. In conclusion, this study would support a beneficial role for CLA and its 9:11 isomer in mediating the pro-atherogenic effects observed in inflammatory disorders such as type 2 Diabetes Mellitus.

The aim of this project was to develop injectable materials to repair damaged bone and, to simultaneously release antibacterial drugs and genes in a controllable manner. Fluid poly (propylene glycol -co- lactide) dimethacrylate (PGLA-DMA) was first synthesised and then filled with varying levels of β- tricalcium phosphate (β-TCP) and monocalcium phosphate monohydrate (MCPM) to fabricate composite materials. For all formulations (including polymer and composites), full methacrylate conversion was found to occur within 200 s of exposure to blue light. The initial dry polymer modulus was enhanced three-fold by increasing total filler content to 70%. After composite immersion in water, β-TCP and MCPM was found to react and re-precipitate within the set materials as dicalcium phosphate (DCP, i.e. brushite and monetite). At higher MCPM levels there was an increase in DCP formation, composite degradation rate, release of both calcium and phosphate ions and buffering of acidic polymer degradation products. Additionally, bone-like MG-63 cells were found to attach, spread and proliferate on both the polymer and the composite surfaces and, composites implanted into chick embryo femurs demonstrated close apposition to the host tissue. To examine the potential value for drug delivery, both the polymer and the composites were prepared containing 10% of the antibacterial chlorhexidine (CHX). The drug was found to be released from material via diffusion, which increased along with antibacterial activity when the filler content was raised. PGLA-DMA polymer was additionally prepared containing complexes of the commercial cationic lipid MetafecteneTM Pro and green fluorescent protein plasmid DNA. Initial studies demonstrated that the components released from the materials were capable of gene transfection into human bone-forming mesenchymal stem cells in vitro. These studies thus demonstrate that the injectable, rapidly settable PGLADMA materials produced here might have clinical potential as both bone adhesives and drug delivery devices.

Osseointegration is the process of de novo bone regeneration on the surface of an endosseous titanium (Ti) implant in vivo. This neo formation of bone underlies the physical integration of a Ti implant in bone at a level sufficient to restore loss of function; for example, of mastication due to absent teeth. The outer atoms of a bulk of Ti metal form a stable and passive surface oxide layer that serves as a substrate for the amalgamation of tissue reparative components, which entail the formation of an osseous bond between tissue and fixture. This interaction was empirically demonstrated to be highly affected by the characteristics of the surface an implant in experimental studies querying the varied effects of additive or subtractive physical modifications, as well as altered chemical compositions, of Ti implant surfaces on osseointegration. Subsequent clinical and experimental practices have demonstrated that rough surfaced implants perform comparatively ‘better’ than their smooth surfaced counterparts by promoting bone growth on the fixture. Moreover, a particular surface modification that yields high surface energy combined with a widely tested micron scaled topographical roughness (modSLA) has been shown to further promote the timely enhancement of osseointegration compared to its hydrophobic rough counterpart (SLA). The biological mechanisms underlying this apparent enhancement of osseointegration by modified Ti implant surfaces are still subject to intense study due to the materials’ implications in bone related tissue engineering applications. Amongst the several views being opined is a proposition mainly arising from in vitro experimentation, which suggests modified Ti implant surfaces possess an ‘intrinsic’ osteoinductive potential that affects uncommitted reparative cells by inducing a temporal and magnitudinal enhancement in cellular differentiation and function; in turn, implying the early formation of functional osteoblasts and bone tissue matrix in an in vivo scenario. The observations of differential cellular behavior include the apparent modulation by the modified surfaces, of a cell surface receptor tyrosine kinase Axl in human osteoblasts. The proposed role of the receptor in negatively regulating osteogenic mineralisation in uncommitted pericytic cells suggests an association with the altered response of cells to these substrates. The aim of this project was to examine and test the hypothesised differential modulation of Axl in the responses of human marrow derived mesenchymal stromal cells to modified Ti implant substrates.