Mutations in LMNA gene have been linked to a number of age-related tissue-specific diseases and premature ageing syndromes termed laminopathies. The finding that A- type lamins affect longevity and maintenance of a number of somatic tissues makes them potential candidates in ageing of normal tissues. At the cellular level, ageing is manifested by an accumulation of irreversibly arrested senescent cells. Premature senescence is also hallmark of premature ageing syndromes. A-type lamins and lamina- associated polypeptide 2 α (LAP2a) bind to and tether retinoblastoma protein (Rb) in the nucleus, a main regulator of senescence pathway. In order to explore the role of A- type lamins and LAP2a in normal ageing and disease, I studied these proteins during ageing of wild type human fibroblasts in vitro. My results show that protein expression level of lamins and LAP2s does not change during ageing of wild-type fibroblasts in vitro. Nonetheless, fibroblasts aged in vitro acquire a range of nuclear morphological changes characteristic of laimnopathy fibroblasts. These nuclear 'laminopathy' phenotypes are accompanied by changes in peripheral heterochromatin, accumulation of lamins А/С in the nucleoplasm, aggregation of LAP2a and decreased Rb phosphorylation. Interestingly, acquisition of such abnormal nuclear changes correlates with premature senescent arrest in five laminopathy cell lines studied. In contrast, although a down-regulation of LAP2a expression and Rb dephosphorylation accompany a reversible quiescent arrest, quiescent fibroblasts do not acquire alterations in nuclear morphology and instead show increased association of lamins at the NE. A-type and B- type lamms become more insoluble in quiescent cells, which may be a cause of a loss of nuclear anchorage of LAP2a and Rb as well as an increased solubility of LAP2ß. On the contrary, during biochemical fractionation of aged fibroblasts, lamin A, but not lamin B2， becomes altered in a manner which makes it more unstable (i.e. prone to degradation) which leads to accumulation of a soluble lamin A proteolytic fragment whilst lamin с and LAP2a become lost from detergent/salt-resistant nucleoskdeton. Protein sequence analysis revealed that both lamm A and LAP2a contain number of cysteine residues in their C-terminal specific tails. Glutathione blot assay and immunoprecipitation under non-reducing conditions showed that A-type lamins and LAP2a become s-glutathiolated in senescent cells, which may cause their decreased binding seen on immunoprecipitation. Both lamin A and LAP2a show an age-dependent accumulation of monomeric protein under non-reducing conditions, which տ early passage cells leads to formation of non-native disulphide cross-links in these proteins. Interestingly, neither of the four laminopathy cell lines studied show an increased accumulation of monomeric lamin A as compared to their age-matched controls. Aged and lamin A/C-deficient laminopathy fibroblasts display a targeted loss of nucleoplasmic but not speckle-associated forms of Rb. These Rb speckles associate with sites of DNA damage in both senescent and laminopathy cells and may be involved in DNA damage signalling or post-transcriptional regulation of gene expression. sRNAi knockdown of LAP2a also leads to decreased phosphorylation and a loss of nucleoplasmic forms of Rb, which correlates with cell cycle arrest. I propose a model for ageing of human fibroblasts in vitro whereby oxidative modification of A-type lamin filaments induces changes in their structural conformation leading to destabilisation and a less efficient assembly at the nuclear envelope. In addition, aberrant telomere targeting of oxidatively modified LAP2a in senescent cells or aggregated LAP2a m laminopathy cells would lead to destabilised telomere structures, telomere uncapping and/or chromosome fusions which would trigger Rb-mediated DNA damage-induced senescent arrest.

Although the mechanical properties of human bone and many of the tissue characteristics upon which they depend have been investigated in detail, the relationship of ageing to these variables is not clear. In this study, an ageing population of human femoral tissue was examined <i>in vitro</i> in order to characterize the mechanical changes and determine the relative significance of the many tissue variables. Cortical and cancellous bone specimens were machined from 47 human femora ranging in age from 20 to 102 years. The cortical specimens underwent uni-axial tensile testing to failure after which the porosity (volume fraction), mineralization and microstructure were determined. Linear regression analysis demonstrated a decrease in ultimate stress (strength), ultimate strain and fracture energy absorption by respectively 5, 9 and 12% per decade while elastic modulus was unaffected. Bone porosity and microstructure changed significantly with age while mineralization was not affected. Both bivariate and multivariate analyses demonstrated the significance of age-related changes in porosity (volume fraction) to the decline in mechanical competence with porosity accounting for 76% of the reduction in tensile strength. Microstructural changes were highly correlated to porosity and had little independent effect. The cancellous specimens were tested in compression to failure in the weight-bearing axis. After determination of apparent density, standard histomorphometric techniques were used to characterize the trabecular architecture. Compressive strength decreased by 8.5% per decade. Apparent density (similarly volume fraction) accounted for 92% of the variance in strength seen with ageing. Architectural changes were highly correlated to apparent density and provided little additional explanatory power to the models. In conclusion, in cortical and cancellous bone, the quantitative changes occurring in ageing bone tissue, rather than the qualitative changes, have the greatest influence on the mechanical competence of bone. Other tissue variables not examined, perhaps collagen structure, are necessary to explain the remaining changes in cortical bone strength. The results indicate that the mechanical properties of cortical and cancellous bone can be estimated <i>in vivo</i> by non-invasive bone density determinations, thereby allowing the prediction of fracture risks and a further understanding of fracture mechanics in the elderly. The data also provide information relevant to the design of fracture and endoprosthetic implants whose clinical success is dependent on the implant-bone interface.

The work reported in this thesis was carried out to investigate the mechanisms of ligamentous action in the human foot during the functions of standing and walking. A comprehensive literature review of the relevant subject areas was presented and a need for further work demonstrated. Ligament strain patterns in cadaveric feet were measured for a number of foot positions and static and dynamic loading patterns, including simulations of standing and gait. Levels of functional strain have been established for each ligament and it has been shown that the plantar ligaments of the tarsus are especially sensitive to the motions of inversion and eversion. Maximum strain levels were witnessed during the toe-off phase of the gait cycle where values of approximately 4 times those seen during standing were found. The action of the extrinsic musculature was able to supplement passive support mechanisms by reducing strain in the subject ligaments. Uniaxial tension testing of isolated preparations of the subject ligaments was then carried out utilising measurements of local strain. In addition to providing a record of the load-deformation responses of the tissues tested, these experiments allowed derivation of a range of functional force estimates for intact normal feet. Stress-strain relationships were also derived and these were compared to contemporary models of ligament mechanics. Functional values of stress were also reported. A mathematical model representing the foot during stance was developed. Forcedeformation data and measured anatomical data provided the necessary information to solve the model for the stance condition. The effect of simulated ligament rupture through injury and the effects of surgical ligament release on the strain and force patterns of the unaffected ligament structures were identified. The results of this investigation served as a record of the quantitative biomechanics of the ligaments in the human foot.

This study was concerned with the measurement and modelling of postural work load and recovery. Five stationary stooped postures were examined and the data derived therefrom were used in the development of some predictive mathematical models of postural work recovery. The models were found to fit the data very well and, when tested in two different situations, were found to be about 70% reliable, at the worst. The models tend to refute the view that a knowledge of the forces being exerted by the various muscles is necessary before postural work recovery can be predicted. The models showed, at least for the five postures studied, the duration of work was the primary factor influencing recovery, rather than rest. Rest pauses of about 1200% of work were used for some of the subjects, despite which full and complete recovery was not achieved. The models lend support, albeit obliquely, to some studies at ~he micro-level involving muscle-biopsies, performed to elucidate the possible mechanisms which underlie the fatigue and recovery processes. In keeping with the multi-disciplinary nature of ergonomics, this study has taken an eclectic course, drawing upon the theories and methods of such areas as statistics and communication engineering to produce two new techniques for the analysis of subjective assessment data. These-two techniques make subjective assessment data more amenable to quantitative analysis, thereby affording some precision in the handling of data which are susceptible to personalistic or idiosyncratic influences. Bayes Theorem and Shannon's Information Theory have been the basis of these two techniques.

Resurfacing arthroplasty is gaining popularity as an alternative method of hip joint reconstruction for younger individuals. Hip resurfacing has several perceived benefits over the conventional total hip arthroplasty (THA), including a greater range of motion offered by the larger diameter bearings and improved abductor muscle function due to femoral neck preservation. Literature reports that hip resurfacing often provides a 'natural' feel and allows greater functional return, which may be related to improved proprioception. However, existing studies have failed to fully substantiate the functional merits of resurfacing arthroplasty over those provided by the traditional stemmed THA. Outcomes were evaluated for 31 patients with primary unilateral resurfacing or standard stemmed THA at 3 and 12 months following surgery. Kinematic and kinetic data were collected while subjects completed level walking. stair ascent and stair descent activities. 3-dimensional hip moments and angles were compared between the arthroplasty groups. Threshold motion detection sense of the hip was tested to quantify hip joint proprioception, by administering passive abduction and flexion motion stimuli using a validated test rig and comparing threshold detection angles. Hip moments showed no statistically significant difference due to arthroplasty type. Slightly greater peak hip angles were achieved by those with standard THA. The resurfacing group showed greater walking velocities. Threshold detection angles were statistically similar. Resurfacing and standard THA demonstrated equivalent proprioceptive and functional outcomes. Motion detection sense did not differ due to arthroplasty type. Preserving the femoral offset did not appear to benefit abductor function and the greater diameter resurfacing bearings did not result in greater functional range of motion. The increased offset and greater head-neck ratio provided by the prosthetic neck may benefit abductor function and increase motion to an extent which meets the benefits of hip resurfacing. Given that there are greater risks and difficulties associated with hip resurfacing, standard THA may be viewed as the more desirable alternative for young active patients.

Paracoccioides brasiliensis (Pb) is the causative agent of the disease Paracoccioidomycosis (PCM), which is one of the most prevalent systemic mycoses in Latin Amercia (BorgesWalmsley et al., 2002). P. brasiliensis is a thermally dimorphic fungus which undergoes morphological changes from a mycelial form at 26°C (environment) to a pathogenic yeast form at 37°C (human body) after inhalation of spores/conidia into the lungs of a human host (Nemecek et al., 2006). The cAMP pathway controls this morphological transformation in several fungi (Borges-Walmsley and Walmsley, 2000; Kronstad et al., 1998). G proteins are guanine-nucleotide (GDP or GTP) binding proteins that are generally associated with the cytoplasmic si~e of the plasma membrane. They receive signals from G protein-coupled receptors (GPCR). Adenylyl cyclase acts downstream of these G proteins. Ga subunits are required to regulate the activity of adenylyl cyclase (AC), which controls the level of cellular cAMP (Ivey and Hoffman, 2005). Protein Kinase A (PKA), which is activated by cAMP, is required for morphogenesis and virulence (Durrenberger et al., 1998; Sonneborn et al., 2000). The cAMP pathway in P. brasiliensis is poorly understood. However, recently the genes encoding a number of the components of the cAMP pathway have been cloned in our lab: these include the genes encoding three Ga proteins, Gpa1-3, a Gp protein, Gpb1; a Gy protein, Gpg1; Ras; adenylyl cyclase, Cyrl; and the catalytic subunit of PKA, Tpk2. Twohybrid analyses confirmed that Gpa1 and Gpg1 interact with Gpbl. These data indicate the formation of a Gapy trimer complex. A GST pull-down assay confirnled that Gpa1 and Gpb1 interacted with the N-telminus of adenylyl cyclase. Our hypothesis is that Gpal and Gpb1 modulate the activity of the AC/Tpk2 signalling pathway. Consistent with this hypothesis, we I found changes in intracellular cAMP levels during the mycelium to yeast transformation that correlated with changing transcript levels of the signalling genes (Chen et al., 2007). We have established that Tpk2 interacts with the N-terrninus of adenylyl cyclase, the G protein Psubunit Gpb1 and with the co-repressor Tup1 by both two-hybrid and GST pulldown analyses. This suggests that Tpk2 activity is required for feedback regulation of adenylyl cyclase to reduce cAMP levels. P. brasiliensis Tpk2-C-terrninal 226-583-GFP and Tpk2 full length (FL) complemented the growth defect of a S. cerevisiae tpk2 temperature sensitive mutant strain SGY446 and induced the formation of pseudohyphae in the S. cerevisiae tpk2 mutant diploid strain XPY5a/a. Tpk2 C-terrninus has been over expressed in E. coli and in vitro PKA activity was measured. On the other hand we have also analysed the second catalytic subunit Tpkl, which failed to induce pseudohyphae in S. cerevisiae tpk2 mutant strain and is localised to the cytoplasm. Interestingly, the Pb Gp subunit Gpbl inhibited the development of pseudohyphae in TPK2 FL transformed yeast cells. Tpk2 C-temlinus and Tpk2 FL co-transformed with GpbGFP were localized in the nucleus. Our hypothesis is that Gpb1 down regulates the activity of Tpk2, because Gpbl binds to the catalytic C-terrninal domain of Tpk2.

The epidermal growth factor receptor (EGFR) and its family of ligands may be associated with the progression of atherosclerosis by mediating the proliferation and migration of endothelial cells and vascular smooth muscle cells. However there is some evidence that EGFR is expressed by cells ofthe monocytic lineage. This study was undertaken to investigate the expression of the EGFR on monocytes and macrophages and explore its potential function. The expression of the EGFR was studied using the monoclonal antibody ICR62.Flow cytometry revealed that the monocytic THP-l cell line expressed EGFR on its surface and specific EGFR reactivity was also observed by western blotting analysis. Whilst PMA differentiated THP-l cells failed to show cell surface expression of the receptor,immunocytochemical analysis revealed the receptor was localized to the nucleus. Human peripheral blood:-derived monocytes and monocyte-derived macrophages showed minimal cell surface expression of EGFR.

This thesis presents the development of a measurement system, based on Opto- Thermal Transient Emission Radiometry (OTTER), for studying the static and dynamic properties of human axillar skin in-vivo in this project. OTTER has been well known as a non-invasive hydration sensitive measurement technique, however, due to the restriction of infra-red laser and measurement optics, the bench-top apparatus restricts the skin measurements only on volar forearms and hands. Also, the data processing programs are getting obsolete and isolated from the measurement system, which cause most of the inconvenience of the post processing to reveal skin properties from OTTER measurements. Therefore, the main role of this project is to develop OTTER into an automatic and complete measurements system, including the portable probe, data acquisition and real-time analysis, To achieve the aim of this project, the new opto-thermal measurement system needed to be developed and optimized as follows: 1. Development of a mobile measurement head suitable for any skin site all over the human body, using fibres to deliver laser pulses. 2. Mathematical models development to improve data analysis speed and accuracy by optimizing and implementing more algorithms for the purpose of real-time analysis.

No description available.

The lasting integrity of the bond between cement and bone defines the long-term stability of cemented acetabular replacements. So far experimental evidences seem to suggest that debonding of the bone-cement interface is responsible for the fixation failure in the cemented acetabular replacements. Both mechanical and biological effects may jointly contribute to implant loosening (Huiskes 1993, Sundfeldt et aI., 2006). However, mechanical analysis of pelvic bone with cemented acetabular replacement and quantitative assessment of fatigue behaviour under physiological condition on acetabular replacement have been difficult, due to the complexity of the pelvic bone geometry and the associated loading conditions. In this work, the mechanical analysis of the pelvic bone with cemented acetabular replacements was carried out using the finite element method, and the fatigue behaviour of the cement fixation in acetabular replacements under physiological condition was studied on a specially designed hip simulator. A series of finite element models of pelvic bone with cemented acetabular replacements were developed and validated, including a 3D simplified model, 3D half pelvic bone model and 3D whole pelvic bone model. The basic mechanical characteristics of the pelvic bone and pelvic bone with cemented acetabular replacement were analysed to examine the influence of cemented acetabular reconstruction. Stress and strain distributions in the cement mantle, especially at the bone-cement interface, were obtained and analysed. Parametric studies were performed, including parameters such as cup size, cement thickness, cup orientations, and the influences of load variations and insertion of muscle forces on the cement mantle. Numerical results seem to suggest that cement thickness and cup orientation have a significant effect on the stress distributions in the cement mantle and at the bone-cement interface. The stress distribution in the cement mantle seems to be most favourable in the standard cup position, as opposed to open and retroverted cup positions. The hip simulator for fixation endurance testing was utilized to study the long-term mechanical response of cemented acetabular reconstructs, i.e. damage initiation and development, under a combined loading blocks representative of patient typical routine activities (Bergmann et aI., 2001), as well as stair climbing and normal walking loading conditions. A micro-CT scanner was used to detect and monitor damage development at regular intervals of the experiments. Microscopic studies post testing were carried out to verify the damage detected by the CT scanning. The results show that debonding at the bone-cement interface defined the failure of the cement fixation in all cases, and debondings initiated near the dome of the acetabulum in the superior-anterior quadrant, consistent with the high stress regions identified from the finite element analysis of implanted acetabular models. An environmental chamber that mimics in vivo condition was also developed. Preliminary results show that bone-cement interfacial debonding was identified in all Delee zones in implanted acetabula tested with an environmental chamber. The introduction of simulated body fluid into the hip simulator seems to be significant in that a much reduced survival life of the bone-cement interface was reported, due to a combination of mechani~al loading and environmental effects. This combination might also be responsible for early bone-cement interfacial debonding, more consistent with the clinical observations. Results from the present study may be useful for orthopaedic surgeons towards improving surgical procedures to achieve long-term stability of acetabular cement fixation.