Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Is the unigram relevance model term independent? Classifying term dependencies in query expansion

This paper develops a framework for classifying term dependencies in query expansion with respect to the role terms play in structural linguistic associations. The framework is used to classify and compare the query expansion terms produced by the unigram and positional relevance models. As the unigram relevance model does not explicitly model term dependencies in its estimation process it is often thought to ignore dependencies that exist between words in natural language. The framework presented in this paper is underpinned by two types of linguistic association, namely syntagmatic and paradigmatic associations. It was found that syntagmatic associations were a more prevalent form of linguistic association used in query expansion. Paradoxically, it was the unigram model that exhibited this association more than the positional relevance model. This surprising finding has two potential implications for information retrieval models: (1) if linguistic associations underpin query expansion, then a probabilistic term dependence assumption based on position is inadequate for capturing them; (2) the unigram relevance model captures more term dependency information than its underlying theoretical model suggests, so its normative position as a baseline that ignores term dependencies should perhaps be reviewed.

The "free-factor" impact on value in health services

Purpose - This paper seeks to understand the impact of financial cost on customer value in health prevention services by comparing free government services with private fee-charging providers. This is important as there is a common belief that a free health service is of lower quality and thus lower value than a paid service. However there is no evidence to verify this notion. Design / Methodology / Approach - A large-scale online survey was administered nationwide to Australian women. The respondents were asked about the functional and emotional value derived from their service experiences. Findings - Structural equation modelling (SEM) revealed non significant relationships between fee/free services and functional and emotional value (FV/EV). The non-significant relationship with FV is contrary to the theory of price quality relationship in services. This could be attributed to consumer perceptions that the technical quality of health professionals is comparable across free and paid services. The non-significant relationship with EV could be explained by the indicators used to reflect EV. These indicators were reflective of breast screening behaviour, not breast screening services. Subsequently, it may be posited that the act of having a breast screen is sufficient for consumers to derive emotional value, regardless of the financial cost. Originality / Value - This research fills an important gap in the literature by investigating the impact of financial cost on a service that consumers use proactively(prevention), rather than reactively (treatment). Insights are provided into the impact of cost on customer value in preventive health services, which are valuable to social marketing academics, health practitioners, and governments

Pedigree-free animal models : the relatedness matrix reloaded

Animal models typically require a known genetic pedigree to estimate quantitative genetic parameters. Here we test whether animal models can alternatively be based on estimates of relatedness derived entirely from molecular marker data. Our case study is the morphology of a wild bird population, for which we report estimates of the genetic variance-covariance matrices (G) of six morphological traits using three methods: the traditional animal model; a molecular marker-based approach to estimate heritability based on Ritland's pairwise regression method; and a new approach using a molecular genealogy arranged in a relatedness matrix (R) to replace the pedigree in an animal model. Using the traditional animal model, we found significant genetic variance for all six traits and positive genetic covariance among traits. The pairwise regression method did not return reliable estimates of quantitative genetic parameters in this population, with estimates of genetic variance and covariance typically being very small or negative. In contrast, we found mixed evidence for the use of the pedigree-free animal model. Similar to the pairwise regression method, the pedigree-free approach performed poorly when the full-rank R matrix based on the molecular genealogy was employed. However, performance improved substantially when we reduced the dimensionality of the R matrix in order to maximize the signal to noise ratio. Using reduced-rank R matrices generated estimates of genetic variance that were much closer to those from the traditional model. Nevertheless, this method was less reliable at estimating covariances, which were often estimated to be negative. Taken together, these results suggest that pedigree-free animal models can recover quantitative genetic information, although the signal remains relatively weak. It remains to be determined whether this problem can be overcome by the use of a more powerful battery of molecular markers and improved methods for reconstructing genealogies.

A variable-stepsize Jacobian-free exponential integrator for simulating transport in heterogeneous porous media : application to wood drying

A Jacobian-free variable-stepsize method is developed for the numerical integration of the large, stiff systems of differential equations encountered when simulating transport in heterogeneous porous media. Our method utilises the exponential Rosenbrock-Euler method, which is explicit in nature and requires a matrix-vector product involving the exponential of the Jacobian matrix at each step of the integration process. These products can be approximated using Krylov subspace methods, which permit a large integration stepsize to be utilised without having to precondition the iterations. This means that our method is truly "Jacobian-free" - the Jacobian need never be formed or factored during the simulation. We assess the performance of the new algorithm for simulating the drying of softwood. Numerical experiments conducted for both low and high temperature drying demonstrates that the new approach outperforms (in terms of accuracy and efficiency) existing simulation codes that utilise the backward Euler method via a preconditioned Newton-Krylov strategy.

Risk control in transmission system expansion planning with wind generators

With the advent of large-scale wind farms and their integration into electrical grids, more uncertainties, constraints and objectives must be considered in power system development. It is therefore necessary to introduce risk-control strategies into the planning of transmission systems connected with wind power generators. This paper presents a probability-based multi-objective model equipped with three risk-control strategies. The model is developed to evaluate and enhance the ability of the transmission system to protect against overload risks when wind power is integrated into the power system. The model involves: (i) defining the uncertainties associated with wind power generators with probability measures and calculating the probabilistic power flow with the combined use of cumulants and Gram-Charlier series; (ii) developing three risk-control strategies by specifying the smallest acceptable non-overload probability for each branch and the whole system, and specifying the non-overload margin for all branches in the whole system; (iii) formulating an overload risk index based on the non-overload probability and the non-overload margin defined; and (iv) developing a multi-objective transmission system expansion planning (TSEP) model with the objective functions composed of transmission investment and the overload risk index. The presented work represents a superior risk-control model for TSEP in terms of security, reliability and economy. The transmission expansion planning model with the three risk-control strategies demonstrates its feasibility in the case study using two typical power systems

Comparing measures of fat-free mass in overweight older adults using three different bioelectrical impedance devices and three prediction equations

Objectives: To compare measures of fat-free mass (FFM) by three different bioelectrical impedance analysis (BIA) devices and to assess the agreement between three different equations validated in older adult and/or overweight populations. Design: Cross-sectional study. Setting: Orthopaedics ward of Brisbane public hospital, Australia. Participants: Twenty-two overweight, older Australians (72 yr ± 6.4, BMI 34 kg/m2 ± 5.5) with knee osteoarthritis. Measurements: Body composition was measured using three BIA devices: Tanita 300-GS (foot-to-foot), Impedimed DF50 (hand-to-foot) and Impedimed SFB7 (bioelectrical impedance spectroscopy (BIS)). Three equations for predicting FFM were selected based on their ability to be applied to an older adult and/ or overweight population. Impedance values were extracted from the hand-to-foot BIA device and included in the equations to estimate FFM. Results: The mean FFM measured by BIS (57.6 kg ± 9.1) differed significantly from those measured by foot-to-foot (54.6 kg ± 8.7) and hand-to-foot BIA (53.2 kg ± 10.5) (P < 0.001). The mean ± SD FFM predicted by three equations using raw data from hand-to-foot BIA were 54.7 kg ± 8.9, 54.7 kg ± 7.9 and 52.9 kg ± 11.05 respectively. These results did not differ from the FFM predicted by the hand-to-foot device (F = 2.66, P = 0.118). Conclusions: Our results suggest that foot-to-foot and hand-to-foot BIA may be used interchangeably in overweight older adults at the group level but due to the large limits of agreement may lead to unacceptable error in individuals. There was no difference between the three prediction equations however these results should be confirmed within a larger sample and against a reference standard.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.

Natural improvements of geochemical conditions of acid sulfate soils caused by free tidal inundation and its effects on the mangrove seedlings

Acid sulfate soils (ASS) are one of the stressor factors that cause many mangrove restoration projects to fail. Achieving successful rehabilitation in an ASS affected area requires an understanding of the geochemical conditions that influence the establishment and growth of mangrove seedlings. This study evaluated the effect of tidal inundation on geochemical conditions on sub layer to better understand their impacts on the density, establishment, and growth of mangrove seedlings. This study also examined the geochemical conditions under which mangrove seedlings can establish naturally, and/or be replanted in abandoned aquaculture ponds. The study area was in an area of abandoned aquaculture ponds situated in the Mare District, adjacent to Bone Bay, South Sulawesi, Indonesia.The pH, pHfox, redox potential, organic content, water soluble sulfate, SKCl, SPOS, and grain size of the soil from the sediment core at + 10 - 15 cm depth near roots were measured using. Pyrite analysis were conducted for the top and sub sediments. The density, establishment, and the relative root growth of Rhizophoraceae were also determined. Free tidal inundation at abandoned pond sites improved the sediment quality. The high density, establishment, and growth of mangrove seedlings were characterized by freely drained areas with a higher pH (field and oxidisable), lower organic content, and high proportion of silt/clay. Higher density and growth also correlated to reduced environments. Sulfur species did not influence the density, establishment, and growth of the seedlings directly. The supply of propagules from the mangrove stands, or access from good waterways were also important for seedlings to establish naturally.

Markers of cutaneous human papillomavirus infection in individuals with tumor-free skin, actinic keratoses, and squamous cell carcinoma

Separately, actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) have been associated with cutaneous human papillomavirus (HPV) infections. To further explore the association between HPV infection and SCC development, we determined markers of cutaneous HPV infection within a single population in persons with precursor lesions (AK), cancerous lesions (SCC), and without. Serum and plucked eyebrow hairs were collected from 57 tumor-free controls, 126 AK, and 64 SCC cases. Presence of HPV L1 and E6 seroreactivity and viral DNA were determined for HPV types 5, 8, 15, 16, 20, 24, and 38. Significant positive associations with increasing severity of the lesions (controls, AK, and SCC, respectively) were observed for overall HPV L1 seropositivity (13%, 26%, and 37%) and for HPV8 (4%, 17%, and 30%). In parallel, the proportion of L1 seropositive individuals against multiple HPV types increased from 14% to 39% and 45%. The overall E6 seroreactivity, however, tended to decline with AK and SCC, especially for HPV8 (21%, 11%, and 2%). HPV DNA positivity was most prevalent in the AK cases (54%) compared with the SCC cases (44%) and the tumor-free controls (40%). Among all participants, there was a positive trend between overall HPV DNA positivity and L1 seropositivity, but not E6 seropositivity. Taken together, our data suggest that cutaneous HPV infections accompanied by detectable HPV DNA in eyebrow hairs and HPV L1 seropositivity, but not E6 seropositivity, are associated with an increased risk of AK and SCC.

First-principles prediction of metal-free magnetism and intrinsic half-metallicity in graphitic carbon nitride

Transition metal-free magnetism and half-metallicity recently has been the subject of intense research activity due to its potential in spintronics application. Here we, for the first time, demonstrate via density functional theory that the most recently experimentally realized graphitic carbon nitride (g-C4N3) displays a ferromagnetic ground state. Furthermore, this novel material is predicted to possess an intrinsic half-metallicity never reported to date. Our results highlight a new promising material toward realistic metal-free spintronics application.

Transmission system expansion planning considering combined operation of wind farms and energy storage systems

An energy storage system (ESS) can provide ancillary services such as frequency regulation and reserves, as well as smooth the fluctuations of wind power outputs, and hence improve the security and economics of the power system concerned. The combined operation of a wind farm and an ESS has become a widely accepted operating mode. Hence, it appears necessary to consider this operating mode in transmission system expansion planning, and this is an issue to be systematically addressed in this work. Firstly, the relationship between the cost of the NaS based ESS and its discharging cycle life is analyzed. A strategy for the combined operation of a wind farm and an ESS is next presented, so as to have a good compromise between the operating cost of the ESS and the smoothing effect of the fluctuation of wind power outputs. Then, a transmission system expansion planning model is developed with the sum of the transmission investment costs, the investment and operating costs of ESSs and the punishment cost of lost wind energy as the objective function to be minimized. An improved particle swarm optimization algorithm is employed to solve the developed planning model. Finally, the essential features of the developed model and adopted algorithm are demonstrated by 18-bus and 46-bus test systems.

Representation of assertions in clinical free-­text using SNOMED CT

Information retrieval (IR) by clinicians in the healthcare setting is critical for informing clinical decision-making. However, a large part of this information is in the form of free-text and inhibits clinical decision support and effective healthcare services. This makes meaningful use of clinical free-­text in electronic health records (EHRs) for patient care a difficult task. Within the context of IR, given a repository of free-­text clinical reports, one might want to retrieve and analyse data for patients who have a known clinical finding.