Muscle sarcopenia and weakness are common health problems accompanying ageing. As the proportion of people older than the age of 60 in our population increases, it becomes important to understand systems implicated in the regulation of muscle strength in ageing. This thesis investigates biochemical changes occurring within skeletal muscles of ageing individuals. A proteomic method of identifying biomarkers was developed and optirnised for discovery and verification of protein biomarkers from small skeletal muscle biopsies from mice. This was an integrated proteomics platform using 2D-DIGE, LC-MS-MS and SELDI-TOF-MS. The method was adapted to find biomarkers from human muscle biopsies. Four human subject groups (10 old females, 5 young females, 4 old males and 7 young males (old: 65-80 years old; young: 20-30 years old» were recruited and completed three-months regime of strength training employing leg press and leg flexion and extension weight training machines. Biopsies from the vastus lateralis (used for isometric strength testing) were taken on day 0 and day 90 of training. An initial screen for differentially expressed proteins between female young/old pre- and post- training was performed using 2D-DIGE (n=3). Muscle ageing in females was associated with significant differential expression of 21 protein biomarkers. 19 of these protein biomarkers were differentially expressed after three months of training were identified, including RKIP/PEBP1. SELDI-TOF was used to study all 52 samples (10 old females, 5 young females, 4 old males and 7 young males pre- and post- trained the three-month strength training). Because of its central role in a number of key intracellular signalling processes including suppressing cancer cell metastasis, RKIP/PEBPI was investigated further. Western blots (n=4) of mouse muscles showed absolute levels of both RKIP/PEBP1 and its phosphorylated isoform (phospho-RKIPfPEBPl) were more highly expressed in slow contracting soleus compared with fast contracting digitorum longus muscles, The relative proportions of phospho-RKIPfPEBPl and total RKIP were the same in both types of muscles suggesting that the balance in the effect of signalling was unchanged. To test that if the increased RKIPfPEBPl in ageing skeletal muscle acts to suppress RAF-l kinase activation of MAPK (ERKl and ERK2) through MAPK/ERK pathway, Western blots assay of ERKl and ERK2 in young and older female's muscle biopsies (n=3) were performed. The results show that they increased proportionately in aging muscles. Thus the increased RKIP/PEBPl in ageing skeletal muscle may not act to suppress RAF-l kinase activation of MAPK (ERKl and ERK2). This implies a different role for RKIPIPEBPl III I skeletal muscle. To test if RKIlPEBPl expression was neurally regulated and if denervation would therefore eradicate RKIP expression, western blot analysis (n=4) was used to assay the levels of total RKIP and phospho-RKIP/PEBPl in soleus muscles of rats following 5 weeks of irreversible muscle denervation. The level of Phospho-RKIP/PEBPl (Ser-153) expression was reduced 2.5-fold but the level of total RKIPIPEBPl was unaffected by irreversible denervation. In summary, it is demonstrated for the first time that both total RKIPIPEBPl and phospho- RKIPIPEBPl (Ser-153) are higher in older muscles than in young muscles pre-training. RKIPIPEBPl may play an important role in modulating skeletal muscle phenotype during ageing.

Marked changes in the plasma activities of the intracellular enzyme creatine kinase (CK) have been observed in broiler chickens subjected to a range of stressful stimuli. Using a novel anion exchange chromatography technique to separate the various CK isoenzymes, it was demonstrated that the skeletal muscle form of CK (MM-CK) was predominant in plasma (96.8%) and that its activity increased 65.4% (p<0.05) in response to an episode of acute heat stress (2 h, 30<SUP>o</SUP>C/80% relative humidity). An investigation of mechanisms that might mediate the release of CK from this tissue was undertaken using a novel validated isolated <I>in vitro</I> chicken skeletal muscle preparation (<I>m. tensor patigialis</I>) and incubation system. CK loss from the muscle preparation under optimised control incubation conditions (150 min at 41.5<SUP>o</SUP>C, pH 74) was negligible, constituting less than 0.003% of total muscle CK content. Incubation at 45<SUP>o</SUP>C had no effect on the rate of CK loss above that observed under control conditions. Incubation under conditions of reduced O<SUB>2</SUB> availability (anoxia) was without effect on CK loss from the preparation. Limiting ATP availability by impairing muscle mitochondrial oxidative metabolism induced a 4-fold increase (p<0.05) in the rate of loss of CK. Promoting external ionic calcium (Ca<SUP>2+</SUP>) entry into the muscle cells using 4 Br-A23187 calcium ionophore (25μM) caused a 60-fold increase (p<0.001) in the release rate of CK. Incubation with the ionophore induced a significant increase in Ca<SUP>2+</SUP> accumulation (79.7%; p<0.05) as measured by the uptake of radio-labelled <SUP>45 </SUP>calcium and was associated with a 8.6 fold greater total loss of CK.

It is well accepted that inter-individual variability exists in muscle strength (more specifically maximal voluntary contraction torque), and many of its determinants. The extent of this inter-individual variability, however, has yet to be quantified despite many researchers suggesting genetic factors contribute. The aims of the present thesis were to first quantify the inter-individual variability within skeletal muscle phenotypes in a homogenous population, and secondly to investigate the contribution of multiple genetic polymorphisms to the inter-individual variability within these phenotypes. Genotype and phenotype data was collected from 120 untrained Caucasian males (aged 18-39 yr). Considerable inter-individual variability in muscle strength phenotypes and many of its determinants was observed. Subsequently, polymorphisms in the CNTF, TTN, PTK2, TRHR and ACTN3 genes demonstrated significant associations with one or more of the skeletal muscle phenotypes, but neither ACE nor COL5A1 were found to associate with any of the measured phenotypes. Adopting a polygenic approach that incorporated all of these genetic polymorphisms did not account for the inter-individual variability observed within VL muscle size (inter-individual variability = 13-20%; P ≥ 0.166) or strength (14-19%; P ≥ 0.220). The results identified novel genetic associations between TTN, CNTF and skeletal muscle architecture, in addition to providing the first independent replications of associations between PTK2 and specific force, and TRHR and lean mass. In conclusion, there appears to be a genetic influence on skeletal muscle phenotypes, however, further research is necessary to replicate the associations observed within the current thesis in comparable and different populations. Nonetheless, the work presented here has applications for improving physical performance, in addition to enhancing our understanding of skeletal muscle disorders, which may have implications for how individuals exercise and how skeletal muscle disorders are treated and/or prevented in future.

The introduction to this thesis comprises two parts. A comprehensive review of the mechanisms and control of human skeletal muscle metabolism with emphasis on anaerobic metabolism and the effects of training, and a review of atherosclerosis and peripheral vascular disease with special mention of skeletal muscle metabolism in vascular disease together with an account of areas in which drugs may help in its treatment. There follows a detailed account of the technique for biopsying human skeletal muscle together with the analytical methods for the fluorimetric assay of high energy phosphates, glycogen, glyclolytic intermediates and succinic dehydrogenase activity. Three experimental chapters are then presented. The first describes the Anaerobic Work Test (AWT) and the metabolic changes that occurred in the quadriceps muscle of man after 6 and 30 s of supra-maximal exercise. This showed that within 6 s of exercise of this magnitude more than 50% of ATP was already being supplied by glycolysis. Over a 30 s sprint of the same intensity almost 70% of the energy was supplied by glycolysis and marked reduction in ATP occurred in the presence of significant amounts of PCr. The second experimental chapter examined the influence of high intensity training on the 30 s AWT and a trend for the resulting improvement in performance to be associated with an increased glycolytic capacity was revealed. The final experimental chapter combined a study of metabolic changes in the gastrocnemius muscle of patients with ischaemic rest pain together with a trial of intravenous naftidrofuryl in its treatment. Changes in muscle metabolism observed in patients with rest pain (low glycogen, ATP, PCr and SDH levels) were felt to represent disuse rather than a direct effect of muscle ischaemia and the drug naftidrofuryl conferred no obvious metabolic benefit although it did appear to possess analgesic properties. In the final discussion an hypothesis on fuel supply as a mechanism for fatigue is presented in the light of the metabolic changes reported.

No description available.

Biodynamic models have been developed to predict the dynamic spinal forces induced by whole-body vibration but the effects of sitting posture and backrest conditions on these forces are unclear. The main objectives of the research reported in thesis were to advance understanding of: (i) how sitting posture and contact with a backrest affect the biodynamic responses of the human body, and (ii) the effects of sitting posture and backrests on spinal forces during exposure to vertical whole-body vibration. Experimental measurements found that the apparent mass of the body and transmissibilities to the spine (to the pelvis, L5, L3, and T5) are affected by the presence of a vertical backrest or an inclined backrest (inclined by 10º, 20º, and 30º). An inclined backrest induced a broad peak, or even two peaks, around 4 to 8 Hz in the vertical apparent mass at the seat pan, probably because the backrest separated the body modes contributing to the principal resonance around 5 Hz evident when sitting with no backrest. Sitting with either vertical or inclined backrests increased vertical motions of the pelvis and the spine. Leaning forward in an ‘anterior leaning’ sitting posture increased the frequency of the principal resonance in the vertical apparent mass at the seat, possibly due to increased tension in back muscles. Leaning forward in ‘anterior leaning’ or ‘kyphotic leaning’ postures induced a resonance around 2.5 Hz in the vertical apparent mass at the seat pan, due to excitation of body modes at frequencies less than 5 Hz associated with fore-and-aft motions of the pelvis and the spine. Changing sitting posture changes muscle activity. Tensing muscles in the lower body (including the lower lumbar spine, pelvis, and thighs), or tensing muscles in the whole body (the lower and upper torso), produces similar increases in the frequency of the principal resonance in the vertical apparent mass at the seat around 5 Hz. This suggests tensing muscles in the lower body causes a greater increase in the frequency of the principal resonance than tensing muscles in upper body. Biodynamic models of the seated human body that included forces from muscles were developed to fit the measured responses of the body (apparent mass and transmissibilities) in the various sitting conditions (normal and leaning forward, vertical and inclined backrests). The spinal forces in the vertical and fore-and-aft directions at the L5/S1 intervertebral disc were estimated from the sum of the predicted static and dynamic forces in both directions. In each sitting condition, a linear model was used to predict the frequency-dependent transfer function between the vertical seat acceleration and the dynamic forces in the spine. For the sitting conditions studied in this research, the contributions from the muscles to static spinal forces were comparable to the forces from gravity of the body mass supported on the intervertebral disc. Dynamic muscle forces were predicted to contribute significant dynamic spinal forces in the vertical and fore-and-aft directions during vertical whole-body vibration. Varying the sitting conditions varied the spinal forces predicted by the models, both with and without exposure to vibration. Transfer functions between vertical seat acceleration and dynamic spinal forces showed one or two resonances around 4 to 8 Hz. The resonance frequency in the transfer function between vertical seat acceleration and dynamic vertical spinal force increased with increasing inclination of a backrest, similar to the effect of backrest inclination on the vertical apparent mass at the seat pan. Compared to a normal sitting posture, sitting with 20º-inclined backrest increased the predicted static and dynamic spinal forces in the spine in the vertical and fore-and-aft directions, due to increased forces at the backrest and increased motion of the spine in both directions. Forward leaning sitting postures increased the fore-and-aft motions of the spine and increased the fore-and-aft dynamic spinal forces predicted by the model. It is concluded that sitting posture and contact with vertical or inclined backrests alter the biodynamic responses of the seated human body. The changes arise from several mechanisms including the backrest supporting some of the body mass, changes in static muscle activity, changes in dynamic muscle activity, and changes in the modes of vibration in the body. These mechanisms are also responsible for predicted changes in the forces in the spine during vertical whole-body vibration.

The biomechanical responses (i.e. apparent mass and transmissibility) to whole-body vibration have been found to be nonlinear - resonance frequency decreases with increasing vibration magnitude. The main mechanism causing the nonlinearity has been recently found to be caused by the soft tissue at the excitation-subject interface (i.e. buttock). Despite the experimental evidence of the cause, mathematical formulation of the dynamic forces at the interface and the prediction of the body movement at different magnitudes of excitation remain absent. The principal objective of the research reported in this thesis was to examine the dynamic behaviour of excitation-subject interface of the whole-body vibration using a scaled rigid mass-soft tissue system. The research was also designed to investigate the single degree of freedom (SDOF) linear and nonlinear models in time domain to predict the responses of the whole- body vibration at different magnitudes. The preliminary study looked into the time domain modelling of whole-body vibration (WBV) responses using four different SDOF linear and nonlinear viscoelastic models found that all four models failed to predict the responses at different magnitudes. In the first experiment, the dynamic characterisation of the silicone rubber was accomplished by means of uni-axial cyclic compression test. The experiment was conducted using different excitation frequencies (i.e. 2, 5, 10, 15, 20 and 40 Hz) and magnitudes (i.e. 25, 50, 75 and 100 N). The effect of the excitation frequency and the magnitude on the mechanical properties was found to be significant. The stiffness and elastic modulus was observed to be increased with increasing excitation frequency and the magnitude. The thixotropic or memory dependent behaviour observed in WBV is missing in this experiment with silicone rubber. In the second experiment, scaled rigid mass-silicone rubber system was studied using base excitation with broadband (2 to 80 Hz) random vibration at four different magnitudes (i.e. 0.5, 1.0, 1.5 and 2.0 ms-2 r.m.s.). The silicone rubber specimens with three different thickness (i.e. 10, 15 and 20 mm) and three different diameters (i.e. 50, 75 and 100 mm) were tested with three different sprung masses (i.e. 1.5 kg, 2.5 kg and 5.0 kg) and two different sprung mass contact contour (i.e. flat and hip-borne). A dominant single resonance was observed for the rigid mass-silicone rubber system in the present study and also in most whole-body vibration studies with vertical vibration. Although the frequency range at which the resonance occurred were different: 20 to 33 Hz for current study and below 10 Hz for WBV studies, the mode of sprung mass might be similar in both scenarios. The effect of thickness, diameter, sprung mass and sprung mass contact contour on resonance frequency and stiffness was found to be significant. However the magnitude dependency was observed to be absent in the present study. In the third experiment, Impulse responses of the scaled rigid mass-soft tissue system were studied for the first time using silicone rubber and the porcine muscle. A linear SDOF viscoelastic model was utilised to extract stiffness and damping from the measured accelerance and receptance frequency response functions. With a porcine muscle exposed to vertical impact, a repeatable bimodal resonance was observed at 25 Hz and 40 Hz and the similar size silicone rubber specimen showed a single resonance at 20 Hz for 20 mm, 22 Hz for 15 mm and 26Hz for 20 mm thick specimens - a higher frequency range than those observed in vertical WBV. A repeatable principal resonance is observed for the horizontal impact tests at around 3 Hz in both silicone rubber and porcine muscle specimens – similar to that observed in horizontal WBV. The effect of thickness, diameter, sprung mass and sprung mass contact contour on resonance frequency and stiffness of the silicon specimens was found to be significant in both vertical and horizontal impacts. However the porcine specimen thickness has no clear effect on the parameters extracted. It is concluded that the thixotropic or memory effect of the human body is missing in the SDOF rigid mass-soft tissue system studied in this thesis.

No description available.

The aim of this programme of research was to add to the existing body of knowledge on the measurement of certain hormones in relation to exercise training, the response to high intensity training, and their potential influence on short-term performance. The initial studies demonstrated agreement between venous and capillary concentrations of an array of hormones, and agreement between venous and saliva concentrations of testosterone and cortisol following scaling of saliva concentrations, suggesting suitability of use in an applied exercise setting. In addition, to ensure accurate measurement of steroid hormone concentrations in saliva, it was shown that samples should be refrigerated immediately, transferred to a freezer within 24 h of collection, and analysed within 28 days. Assessment of the response to two exercise bouts of a different type within the same day indicated that it could be beneficial to perform resistance training in the afternoon preceded by interval exercise in the morning in order to stimulate a hormonal milieu that may be more conducive to stimulating muscle protein turnover. The robust increases seen in testosterone and cortisol following interval exercise performed in the morning in that study were also observed in the same cycling sprint interval protocol performed in females. In this study, the magnitude of change in DHT concentration was related to sprint cadence. In investigating the potential acute effects of hormones on performance, the penultimate study demonstrated a positive association between affect as an indicator mood and percentage testosterone concentration during high intensity cycling. Conversely, in the final study, no postactivation potentiation effect was observed to different exercise stimuli, thus no association was observed between hormone concentrations and strength and power performance. These data may suggest that the acute short-term effects of hormone concentrations on performance may be more related to mood and behaviour in the context of this research.

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases. The cause of AD is multifactoral and it is affected by both genetic and environmental factors. Of all the causes of potential barrier defects, the lowered amino-acid derived natural moisturizing factor (NMF) in the stratum corneum (SC), especially associated with a known filaggrin mutation, shows the strongest link to AD. As a result, quantification of NMF in the SC in both healthy and compromised SC is the principal aim of this thesis. Because tape stripping is a key technique used to harvest the SC, a novel imaging method to measure the amount of SC per tape strip was validated. This method offers rapid, simple and reproducible SC quantification. It shows good correlation with existing gravimetric and infrared absorption methods and may provide a better standard method in the future. The tape-stripping extraction of NMF showed an abundant SC ‘reservoir’ of the constituents in healthy skin. Iontophoretic extraction of NMF was highly dependant upon molecular properties, particularly charge and concentration. In general, charged NMF constituents were easily extracted by reverse iontophoresis, whereas iontophoresis only offered modest enhancement of zwitterionic species. Quantification of NMF at different body sites, specifically forehead and forearm, showed similar profiles. However, forehead SC was thinner, and in general contained a lower total amount of NMF and less-ordered lipids. Forehead SC may therefore be considered a less competent barrier. A 3-week application of 0.1% w/v sodium lauryl sulphate (SLS) to healthy volunteers was used to model damaged skin similar to that in AD and chronic irritant contact dermatitis. The SC barrier post-treatment showed significantly reduced NMF, substantial lipid disordering, and the presence of immature corneocytes. The methods employed were sufficiently sensitive to detect these changes. In particular, the NMF components present at high levels in the SC may be useful, potential markers for skin ‘health’ and for its resistance to irritant chemicals.