Atomistic numerical investigation of single-crystal copper nanowire with surface defects

Based on the embedded atom method (EAM), a molecular dynamics (MD) simulation is performed to study the single-crystal copper nanowire with surface defects through tension. The tension simulations for nanowire without defect are first carried out under different temperatures, strain rates and time steps and then surface defect effects for nanowire are investigated. The stress-strain curves obtained by the MD simulations of various strain rates show a rate below 1 x 10(9) s-1 will exert less effect on the yield strength and yield point, and the Young's modulus is independent of strain rate. a time step below 5 fs is recommend for the atomic model during the MD simulation. It is observed that high temperature leads to low Young's modulus, as well as the yield strength. The surface defects on nanowires are systematically studied in considering different defect orientations. It is found that the surface defect serves as a dislocation source, and the yield strength shows 34.20% decresse with 45 degree surface defect. Both yield strength and yield point are significantly influenced by the surface defects, except the Young's modulus.

Free surface flow into a horizontal slot with zero gravity

The two-dimensional free surface flow of a finite-depth fluid into a horizontal slot is considered. For this study, the effects of viscosity and gravity are ignored. A generalised Schwarz-Christoffel mapping is used to formulate the problem in terms of a linear integral equation, which is solved exactly with the use of a Fourier transform. The resulting free surface profile is given explicitly in closed-form.

Asset health prediction using condition indicators and operating environment indicators : a case study

Prognostics and asset life prediction is one of research potentials in engineering asset health management. We previously developed the Explicit Hazard Model (EHM) to effectively and explicitly predict asset life using three types of information: population characteristics; condition indicators; and operating environment indicators. We have formerly studied the application of both the semi-parametric EHM and non-parametric EHM to the survival probability estimation in the reliability field. The survival time in these models is dependent not only upon the age of the asset monitored, but also upon the condition and operating environment information obtained. This paper is a further study of the semi-parametric and non-parametric EHMs to the hazard and residual life prediction of a set of resistance elements. The resistance elements were used as corrosion sensors for measuring the atmospheric corrosion rate in a laboratory experiment. In this paper, the estimated hazard of the resistance element using the semi-parametric EHM and the non-parametric EHM is compared to the traditional Weibull model and the Aalen Linear Regression Model (ALRM), respectively. Due to assuming a Weibull distribution in the baseline hazard of the semi-parametric EHM, the estimated hazard using this model is compared to the traditional Weibull model. The estimated hazard using the non-parametric EHM is compared to ALRM which is a well-known non-parametric covariate-based hazard model. At last, the predicted residual life of the resistance element using both EHMs is compared to the actual life data.

An enriched radial point interpolation method (e-RPIM) for analysis of crack tip fields

In this paper, an enriched radial point interpolation method (e-RPIM) is developed the for the determination of crack tip fields. In e-RPIM, the conventional RBF interpolation is novelly augmented by the suitable trigonometric basis functions to reflect the properties of stresses for the crack tip fields. The performance of the enriched RBF meshfree shape functions is firstly investigated to fit different surfaces. The surface fitting results have proven that, comparing with the conventional RBF shape function, the enriched RBF shape function has: (1) a similar accuracy to fit a polynomial surface; (2) a much better accuracy to fit a trigonometric surface; and (3) a similar interpolation stability without increase of the condition number of the RBF interpolation matrix. Therefore, it has proven that the enriched RBF shape function will not only possess all advantages of the conventional RBF shape function, but also can accurately reflect the properties of stresses for the crack tip fields. The system of equations for the crack analysis is then derived based on the enriched RBF meshfree shape function and the meshfree weak-form. Several problems of linear fracture mechanics are simulated using this newlydeveloped e-RPIM method. It has demonstrated that the present e-RPIM is very accurate and stable, and it has a good potential to develop a practical simulation tool for fracture mechanics problems.

Adhesion and dispersion of salmeterol xinofoate from lactose interactive mixtures for inhalation - can surface roughness of carriers determine performance

The objective was to understand the influence of the surface roughness of lactose carriers on the adhesion and dispersion of salmeterol xinafoate (SX) from interactive mixtures. The surface roughness of lactose carriers was determined by confocal microscopy. Particle images and adhesion forces between SX and lactose particles were determined by Atomic Force Microscopy. The dispersion of SX (2.5%) from interactive mixtures with lactose was determined using a twin-stage impinger (TSI) with a Rotahaler® at an airflow rate of 60L/min. SX was analysed using a validated HPLC assay. The RMS Rq of lactose carriers ranged from 0.93-2.84μm, the Fine Particle Fraction (FPF) of SX ranged between 4 and 24 percent and average adhesion force between a SX and lactose particles ranged between 49 and 134 nN. No direct correlation was observed between the RMS Rq of lactose carriers and either the FPF of SX for the interactive mixtures or the adhesion force of a SX on the lactose particles; however, the presence of fine lactose associated with the carrier surface increased the FPF of SX. Dispersion through direct SX detachment from the carrier surface was not consistent with the poor correlations described and was more likely to occur through complex particulate interactions involving fine lactose.

Dryness : the aesthetic condition of landscape design in Australia

Despite having a band of greenness around the edge, Australia is fundamentally a dry country. Australian vegetation has developed a high range of mechanisms to cope with the dryness, but after 200 years of white settlement, Australians still have not really come to terms with the real dryness of their country, and still exploit European paradigms that attempted to transplant European aesthetic conditions, greenness, to the brown land of Australia. Australia is going through serious water shortages that are still and will continue with the Greenhouse effect, to become a major factor in the location and extent of urbanisation, and also Australia's carrying capacity. While such aesthetic concerns might seem ornamental, until the population changes its attitude to the real condition of the country, it will keep using water and operating unsustainably. The design of the public landscape, however, offers the opportunity to contribute to changing people's aesthetic perception of the country, which might in turn help to redirect their water use practices. This essay develops a language for discussion dryness based around the experiences of water. After having developed this sensibility it then discusses a range of different approaches that landscape design in Australia has used to try to develop geographically appropriate design languages, including the Bush Garden and the Mediterranean Garden. It then discusses four design projects, one from the 1970's, the other three from the last five years that demonstrate what such an aesthetic might look like.

Development of an online condition monitoring system for slow speed machinery

One of the main challenges of slow speed machinery condition monitoring is that the energy generated from an incipient defect is too weak to be detected by traditional vibration measurements due to its low impact energy. Acoustic emission (AE) measurement is an alternative for this as it has the ability to detect crack initiations or rubbing between moving surfaces. However, AE measurement requires high sampling frequency and consequently huge amount of data are obtained to be processed. It also requires expensive hardware to capture those data, storage and involves signal processing techniques to retrieve valuable information on the state of the machine. AE signal has been utilised for early detection of defects in bearings and gears. This paper presents an online condition monitoring (CM) system for slow speed machinery, which attempts to overcome those challenges. The system incorporates relevant signal processing techniques for slow speed CM which include noise removal techniques to enhance the signal-to-noise and peak-holding down sampling to reduce the burden of massive data handling. The analysis software works under Labview environment, which enables online remote control of data acquisition, real-time analysis, offline analysis and diagnostic trending. The system has been fully implemented on a site machine and contributing significantly to improve the maintenance efficiency and provide a safer and reliable operation.

A probability-based approach for assessing the relationship between crash accidents and pavement properties

Skid resistance is a condition parameter characterising the contribution that a road makes to the friction between a road surface and a vehicle tyre. Studies of traffic crash histories around the world have consistently found that a disproportionate number of crashes occur where the road surface has a low level of surface friction and/or surface texture, particularly when the road surface is wet. Various research results have been published over many years and have tried to quantify the influence of skid resistance on accident occurrence and to characterise a correlation between skid resistance and accident frequency. Most of the research studies used simple statistical correlation methods in analysing skid resistance and crash data.----- ------ Preliminary findings of a systematic and extensive literature search conclude that there is rarely a single causation factor in a crash. Findings from research projects do affirm various levels of correlation between skid resistance and accident occurrence. Studies indicate that the level of skid resistance at critical places such as intersections, curves, roundabouts, ramps and approaches to pedestrian crossings needs to be well maintained.----- ----- Management of risk is an integral aspect of the Queensland Department of Main Roads (QDMR) strategy for managing its infrastructure assets. The risk-based approach has been used in many areas of infrastructure engineering. However, very limited information is reported on using risk-based approach to mitigate crash rates related to road surface. Low skid resistance and surface texture may increase the risk of traffic crashes.----- ----- The objectives of this paper are to explore current issues of skid resistance in relation to crashes, to provide a framework of probability-based approach to be adopted by QDMR in assessing the relationship between crash accidents and pavement properties, and to explain why the probability-based approach is a suitable tool for QDMR in order to reduce accident rates due to skid resistance.

In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Fast hole surface conduction observed for indoline sensitizer dyes immobilized at fluorine-doped tin oxide - TiO2 surfaces

The indoline dyes D102, D131, D149, and D205 have been characterized when adsorved on fluorine-doped tin oxide (FTO) and TiO2 electrode surfaces. Adsorption from 50:50 acetonitrile - tert-butanol onto flourine-doped tin oxide (FTO) allows approximate Langmuirian binding constants of 6.5 x 10(4), 2.01 x 10(3), 2.0 x 10(4), and 1.5 x 10(4) mol-1 dm3, respectively, to be determined. Voltammetric data obtained in acetonitrile/0.1 M NBu4PF6 indicate reversible on-electron oxidation at Emid = 0.94, 0.91, 0.88, and 0.88 V vs Ag/AgCI(3 M KCI), respectively, with dye aggregation (at high coverage) causing additional peak features at more positive potentials. Slow chemical degradation processes and electron transfer catalysis for iodine oxidation were observed for all four oxidezed indolinium cations. When adsorbed onto TiO2 nanoparticle films (ca. 9nm particle diameter and ca.3/um thickness of FTO0, reversible voltammetric responses with Emid = 1.08, 1.156, 0.92 and 0.95 V vs Ag/AgCI(3 M KCI), respectively, suggest exceptionally fast hole hopping diffusion (with Dapp > 5 x 10(-9) m2 s-1) for adsorbed layers of four indoline dyes, presumably due to pie-pie stacking in surface aggregates. Slow dye degradation is shown to affect charge transport via electron hopping. Spectrelectrochemical data for the adsorbed indoline dyes on FTO-TiO2 revealed a red-shift of absorption peaks after oxidation and the presence of a strong charge transfer band in the near-IR region. The implications of the indoline dye reactivity and fast hole mobility for solar cell devices are discussed.

The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of ureaplasma parvum

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple banded antigen (MBA) and mba gene. We injected high- or low dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005),and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

Identifying relationship between skid resistance, road characteristics and crashes using probability-based risk approach

Road accidents are of great concerns for road and transport departments around world, which cause tremendous loss and dangers for public. Reducing accident rates and crash severity are imperative goals that governments, road and transport authorities, and researchers are aimed to achieve. In Australia, road crash trauma costs the nation A$ 15 billion annually. Five people are killed, and 550 are injured every day. Each fatality costs the taxpayer A$1.7 million. Serious injury cases can cost the taxpayer many times the cost of a fatality. Crashes are in general uncontrolled events and are dependent on a number of interrelated factors such as driver behaviour, traffic conditions, travel speed, road geometry and condition, and vehicle characteristics (e.g. tyre type pressure and condition, and suspension type and condition). Skid resistance is considered one of the most important surface characteristics as it has a direct impact on traffic safety. Attempts have been made worldwide to study the relationship between skid resistance and road crashes. Most of these studies used the statistical regression and correlation methods in analysing the relationships between skid resistance and road crashes. The outcomes from these studies provided mix results and not conclusive. The objective of this paper is to present a probability-based method of an ongoing study in identifying the relationship between skid resistance and road crashes. Historical skid resistance and crash data of a road network located in the tropical east coast of Queensland were analysed using the probability-based method. Analysis methodology and results of the relationships between skid resistance, road characteristics and crashes are presented.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Non-invasive assessment of tear film surface quality

The tear film plays an important role preserving the health of the ocular surface and maintaining the optimal refractive power of the cornea. Moreover dry eye syndrome is one of the most commonly reported eye health problems. This syndrome is caused by abnormalities in the properties of the tear film. Current clinical tools to assess the tear film properties have shown certain limitations. The traditional invasive methods for the assessment of tear film quality, which are used by most clinicians, have been criticized for the lack of reliability and/or repeatability. A range of non-invasive methods of tear assessment have been investigated, but also present limitations. Hence no “gold standard” test is currently available to assess the tear film integrity. Therefore, improving techniques for the assessment of the tear film quality is of clinical significance and the main motivation for the work described in this thesis. In this study the tear film surface quality (TFSQ) changes were investigated by means of high-speed videokeratoscopy (HSV). In this technique, a set of concentric rings formed in an illuminated cone or a bowl is projected on the anterior cornea and their reflection from the ocular surface imaged on a charge-coupled device (CCD). The reflection of the light is produced in the outer most layer of the cornea, the tear film. Hence, when the tear film is smooth the reflected image presents a well structure pattern. In contrast, when the tear film surface presents irregularities, the pattern also becomes irregular due to the light scatter and deviation of the reflected light. The videokeratoscope provides an estimate of the corneal topography associated with each Placido disk image. Topographical estimates, which have been used in the past to quantify tear film changes, may not always be suitable for the evaluation of all the dynamic phases of the tear film. However the Placido disk image itself, which contains the reflected pattern, may be more appropriate to assess the tear film dynamics. A set of novel routines have been purposely developed to quantify the changes of the reflected pattern and to extract a time series estimate of the TFSQ from the video recording. The routine extracts from each frame of the video recording a maximized area of analysis. In this area a metric of the TFSQ is calculated. Initially two metrics based on the Gabor filter and Gaussian gradient-based techniques, were used to quantify the consistency of the pattern’s local orientation as a metric of TFSQ. These metrics have helped to demonstrate the applicability of HSV to assess the tear film, and the influence of contact lens wear on TFSQ. The results suggest that the dynamic-area analysis method of HSV was able to distinguish and quantify the subtle, but systematic degradation of tear film surface quality in the inter-blink interval in contact lens wear. It was also able to clearly show a difference between bare eye and contact lens wearing conditions. Thus, the HSV method appears to be a useful technique for quantitatively investigating the effects of contact lens wear on the TFSQ. Subsequently a larger clinical study was conducted to perform a comparison between HSV and two other non-invasive techniques, lateral shearing interferometry (LSI) and dynamic wavefront sensing (DWS). Of these non-invasive techniques, the HSV appeared to be the most precise method for measuring TFSQ, by virtue of its lower coefficient of variation. While the LSI appears to be the most sensitive method for analyzing the tear build-up time (TBUT). The capability of each of the non-invasive methods to discriminate dry eye from normal subjects was also investigated. The receiver operating characteristic (ROC) curves were calculated to assess the ability of each method to predict dry eye syndrome. The LSI technique gave the best results under both natural blinking conditions and in suppressed blinking conditions, which was closely followed by HSV. The DWS did not perform as well as LSI or HSV. The main limitation of the HSV technique, which was identified during the former clinical study, was the lack of the sensitivity to quantify the build-up/formation phase of the tear film cycle. For that reason an extra metric based on image transformation and block processing was proposed. In this metric, the area of analysis was transformed from Cartesian to Polar coordinates, converting the concentric circles pattern into a quasi-straight lines image in which a block statistics value was extracted. This metric has shown better sensitivity under low pattern disturbance as well as has improved the performance of the ROC curves. Additionally a theoretical study, based on ray-tracing techniques and topographical models of the tear film, was proposed to fully comprehend the HSV measurement and the instrument’s potential limitations. Of special interested was the assessment of the instrument’s sensitivity under subtle topographic changes. The theoretical simulations have helped to provide some understanding on the tear film dynamics, for instance the model extracted for the build-up phase has helped to provide some insight into the dynamics during this initial phase. Finally some aspects of the mathematical modeling of TFSQ time series have been reported in this thesis. Over the years, different functions have been used to model the time series as well as to extract the key clinical parameters (i.e., timing). Unfortunately those techniques to model the tear film time series do not simultaneously consider the underlying physiological mechanism and the parameter extraction methods. A set of guidelines are proposed to meet both criteria. Special attention was given to a commonly used fit, the polynomial function, and considerations to select the appropriate model order to ensure the true derivative of the signal is accurately represented. The work described in this thesis has shown the potential of using high-speed videokeratoscopy to assess tear film surface quality. A set of novel image and signal processing techniques have been proposed to quantify different aspects of the tear film assessment, analysis and modeling. The dynamic-area HSV has shown good performance in a broad range of conditions (i.e., contact lens, normal and dry eye subjects). As a result, this technique could be a useful clinical tool to assess tear film surface quality in the future.