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.

Atomistic investigations of single-crystal silicon with pre-existing defects

Molecular dynamics (MD) simulations have been employed to investigate the single-crystal Si properties with different pre-existing cavities under nanoindentation. Cavities with different radii and positions have been considered. It is found that pre-existing cavities in the Si substrate would obviously influence the mechanical properties of Si under nanoindentation. Furthermore, pre-existing cavities would absorb part of the strain energy during loading and then release during unloading. It would decrease plastic deformation to the substrate. Particularly, the larger of the cavity or the nearer of the cavity to the substrate’s top surface, the larger decrease of Young’s modulus and hardness is usually observed. Just as expected, the larger offset of the cavity in the lateral direction, the less influence is usually seen.

Challenging the authority of the autopsy in coronial investigations

The central purpose of this chapter is to address the tension between legal and medical discourses within the coronial/medico-legal system. In the context of a death investigation, medical expertise, manifest through the knowledge gained in an internal autopsy, is positioned as contributing the more valuable facts of the case, especially when contrasted with the evidence gathered at the scene of the death. We challenge this taken for granted understanding of medical knowledge in three ways: first, we examine the aspects of the history, philosophy and consequences of the processes by which the medical model gained its current dominance; second, we challenge the assumption that internal autopsy adds value to the death investigation, by utilising data from our own research in Australia; and finally, we engage with the debate about the purpose of a coronial/medico-legal investigation and role of an internal autopsy within that system.

Performance evaluation of onsite sewage treatment : results of soil investigations

Background The onsite treatment of sewage and effluent disposal is widely prevalent in rural and urban fringe areas due to the general unavailability of reticulated wastewater collection systems. Despite the low technology of the systems, failure is common and in many cases leading to adverse public health and environmental consequences. It is important therefore that careful consideration is given to the design and location of onsite sewage treatment systems. This requires an understanding of the factors that influence treatment performance. The use of subsurface absorption systems is the most common form of effluent disposal for onsite sewage treatment, particularly for septic tanks. Also, in the case of septic tanks, a subsurface disposal system is generally an integral component of the sewage treatment process. Site specific factors play a key role in the onsite treatment of sewage. The project The primary aims of the research project were: • to relate treatment performance of onsite sewage treatment systems to soil conditions at site; • to evaluate current research relating to onsite sewage treatment; and, • to identify key issues where currently there is a lack of relevant research. These tasks were undertaken with the objective of facilitating the development of performance based planning and management strategies for onsite sewage treatment. The primary focus of this research project has been on septic tanks. By implication, the investigation has been confined to subsurface soil absorption systems. The design and treatment processes taking place within the septic tank chamber itself did not form a part of the investigation. Five broad categories of soil types prevalent in the Brisbane region have been considered in this project. The number of systems investigated was based on the proportionate area of urban development within the Brisbane region located on each of the different soil types. In the initial phase of the investigation, the majority of the systems evaluated were septic tanks. However, a small number of aerobic wastewater treatment systems (AWTS) were also included. The primary aim was to compare the effluent quality of systems employing different generic treatment processes. It is important to note that the number of each different type of system investigated was relatively small. Consequently, this does not permit a statistical analysis to be undertaken of the results obtained for comparing different systems. This is an important issue considering the large number of soil physico-chemical parameters and landscape factors that can influence treatment performance and their wide variability. The report This report is the last in a series of three reports focussing on the performance evaluation of onsite treatment of sewage. The research project was initiated at the request of the Brisbane City Council. The project component discussed in the current report outlines the detailed soil investigations undertaken at a selected number of sites. In the initial field sampling, a number of soil chemical properties were assessed as indicators to investigate the extent of effluent flow and to help understand what soil factors renovate the applied effluent. The soil profile attributes, especially texture, structure and moisture regime were examined more in an engineering sense to determine the effect of movement of water into and through the soil. It is important to note that it is not only the physical characteristics, but also the chemical characteristics of the soil as well as landscape factors play a key role in the effluent renovation process. In order to understand the complex processes taking place in a subsurface effluent disposal area, influential parameters were identified using soil chemical concepts. Accordingly, the primary focus of this final phase of the research project was to identify linkages between various soil chemical parameters and landscape patterns and their contribution to the effluent renovation process. The research outcomes will contribute to the development of robust criteria for evaluating the performance of subsurface effluent disposal systems. The outcomes The key findings from the soil investigations undertaken are: • Effluent renovation is primarily undertaken by a combination of various soil physico-chemical parameters and landscape factors, thereby making the effluent renovation processes strongly site dependent. • Decisions regarding site suitability for effluent disposal should not be based purely in terms of the soil type. A number of other factors such as the site location in the catena, the drainage characteristics and other physical and chemical characteristics, also exert a strong influence on site suitability. • Sites, which are difficult to characterise in terms of suitability for effluent disposal, will require a detailed soil physical and chemical analysis to be undertaken to a minimum depth of at least 1.2 m. • The Ca:Mg ratio and Exchangeable Sodium Percentage are important parameters in soil suitability assessment. A Ca:Mg ratio of less than 0.5 would generally indicate a high ESP. This in turn would mean that Na and possibly Mg are the dominant exchangeable cations, leading to probable clay dispersion. • A Ca:Mg ratio greater than 0.5 would generally indicate a low ESP in the profile, which in turn indicates increased soil stability. • In higher clay percentage soils, low ESP can have a significant effect. • The presence of high exchangeable Na can be counteracted by the presence of swelling clays, and an exchange complex co-dominated by exchangeable Ca and exchangeable Mg. This aids absorption of cations at depth, thereby reducing the likelihood of dispersion. • Salt is continually added to the soil by the effluent and problems may arise if the added salts accumulate to a concentration that is harmful to the soil structure. Under such conditions, good drainage is essential in order to allow continuous movement of water and salt through the profile. Therefore, for a site to be sustainable, it would have a maximum application rate of effluent. This would be dependent on subsurface characteristics and the surface area available for effluent disposal. • The dosing regime for effluent disposal can play a significant role in the prevention of salt accumulation in the case of poorly draining sites. Though intermittent dosing was not considered satisfactory for the removal of the clogging mat layer, it has positive attributes in the context of removal of accumulated salts in the soil.

Many paths lead to discovery : analogical retrieval of cancer therapies

This paper addresses the issue of analogical inference, and its potential role as the mediator of new therapeutic discoveries, by using disjunction operators based on quantum connectives to combine many potential reasoning pathways into a single search expression. In it, we extend our previous work in which we developed an approach to analogical retrieval using the Predication-based Semantic Indexing (PSI) model, which encodes both concepts and the relationships between them in high-dimensional vector space. As in our previous work, we leverage the ability of PSI to infer predicate pathways connecting two example concepts, in this case comprising of known therapeutic relationships. For example, given that drug x TREATS disease z, we might infer the predicate pathway drug x INTERACTS WITH gene y ASSOCIATED WITH disease z, and use this pathway to search for drugs related to another disease in similar ways. As biological systems tend to be characterized by networks of relationships, we evaluate the ability of quantum-inspired operators to mediate inference and retrieval across multiple relations, by testing the ability of different approaches to recover known therapeutic relationships. In addition, we introduce a novel complex vector based implementation of PSI, based on Plate’s Circular Holographic Reduced Representations, which we utilize for all experiments in addition to the binary vector based approach we have applied in our previous research.

The late discovery of adoptive and donor insemination offspring status : ethical implications for conceptual understandings of the ‘best interests of the child’ principle

Late discovery is a term used to describe the experience of discovering the truth of one’s genetic origins as an adult. Following discovery, late discoverers face a lack of recognition and acknowledgment of their concerns from family, friends, community and institutions. They experience pain, anger, loss, grief and frustration. This presentation shares the findings of the first qualitative study of both late discovery of adoptive and donor insemination offspring (heterosexual couple use only) experiences. It is also the first study of late discovery experiences undertaken from an ethical perspective. While this study recruited new participants, it also included an ethical re-analysis of existing late discovery accounts across both practices. The findings of this study (a) draws links between past adoption and current donor insemination (heterosexual couple only) practices, (b) reveals that late discoverers are demanding acknowledgment and recognition of the particularity of their experiences, and (c) offers insights into conceptual understandings of the ‘best interests of the child’ principle. These insights derive from the lived experiences of those whose biological and social worlds have been sundered and secrecy and denial of difference used to conceal this. It suggests that acknowledging the equal moral status of the child may be useful in strengthening conceptual understandings of the ‘best interests of the child’ principle. This equal moral status involves ensuring that personal autonomy and the ability to exercise free will is protected; that the integrity of the relationships of trust expected and demanded between parent/s and children is defended and supported; and that equal access to normative socio-cultural practices, that is; non-fictionalised birth certificates and open records, is guaranteed.

Emotional arousal of beginning physics teachers during extended experimental investigations

Teachers often have difficulty implementing inquiry-based activities, leading to the arousal of negative emotions. In this multicase study of beginning physics teachers in Australia, we were interested in the extent to which their expectations were realized and how their classroom experiences while implementing extended experimental investigations (EEIs) produced emotional states that mediated their teaching practices. Against rhetoric of fear expressed by their senior colleagues, three of the four teachers were surprised by the positive outcomes from their supervision of EEIs for the first time. Two of these teachers experienced high intensity positive emotions in response to their students’ success. When student actions / outcomes did not meet their teachers’ expectations, frustration, anger, and disappointment were experienced by the teachers, as predicted by a sociological theory of human emotions (Turner, 2007). Over the course of the EEI projects, the teachers’ practices changed along with their emotional states and their students’ achievements. We account for similarities and differences in the teachers’ emotional experiences in terms of context, prior experience, and expectations. The findings from this study provide insights into effective supervision practices that can be used to inform new and experienced teachers alike.

Genetic polymorphisms in the human tissue kallikrein (KLK) locus and their implication in various malignant and non-malignant diseases

The Kallikrein (KLK) gene locus encodes a family of serine proteases and is the largest contiguous cluster of protease-encoding genes attributed an evolutionary age of 330 million years. The KLK locus has been implicated as a high susceptibility risk loci in numerous cancer studies through the last decade. The KLK3 gene already has established clinical relevance as a biomarker in prostate cancer prognosis through its encoded protein, prostate-specific antigen. Data mined through genome-wide association studies (GWAS) and next-generation sequencing point to many important candidate single nucleotide polymorphisms (SNPs) in KLK3 and other KLK genes. SNPs in the KLK locus have been found to be associated with several diseases including cancer, hypertension, cardiovascular disease and atopic dermatitis. Moreover, introducing a model incorporating SNPs to improve the efficiency of prostate-specific antigen in detecting malignant states of prostate cancer has been recently suggested. Establishing the functional relevance of these newly-discovered SNPs, and their interactions with each other, through in silico investigations followed by experimental validation, can accelerate the discovery of diagnostic and prognostic biomarkers. In this review, we discuss the various genetic association studies on the KLK loci identified either through candidate gene association studies or at the GWAS and post-GWAS front to aid researchers in streamlining their search for the most significant, relevant and therapeutically promising candidate KLK gene and/or SNP for future investigations.

Cross-Lingual knowledge discovery : Chinese-to-English article linking in Wikipedia

In this paper we examine automated Chinese to English link discovery in Wikipedia and the effects of Chinese segmentation and Chinese to English translation on the hyperlink recommendation. Our experimental results show that the implemented link discovery framework can effectively recommend Chinese-to-English cross-lingual links. The techniques described here can assist bi-lingual users where a particular topic is not covered in Chinese, is not equally covered in both languages, or is biased in one language; as well as for language learning.

Link discovery for Chinese/English cross-language web information retrieval

Nowadays people heavily rely on the Internet for information and knowledge. Wikipedia is an online multilingual encyclopaedia that contains a very large number of detailed articles covering most written languages. It is often considered to be a treasury of human knowledge. It includes extensive hypertext links between documents of the same language for easy navigation. However, the pages in different languages are rarely cross-linked except for direct equivalent pages on the same subject in different languages. This could pose serious difficulties to users seeking information or knowledge from different lingual sources, or where there is no equivalent page in one language or another. In this thesis, a new information retrieval task—cross-lingual link discovery (CLLD) is proposed to tackle the problem of the lack of cross-lingual anchored links in a knowledge base such as Wikipedia. In contrast to traditional information retrieval tasks, cross language link discovery algorithms actively recommend a set of meaningful anchors in a source document and establish links to documents in an alternative language. In other words, cross-lingual link discovery is a way of automatically finding hypertext links between documents in different languages, which is particularly helpful for knowledge discovery in different language domains. This study is specifically focused on Chinese / English link discovery (C/ELD). Chinese / English link discovery is a special case of cross-lingual link discovery task. It involves tasks including natural language processing (NLP), cross-lingual information retrieval (CLIR) and cross-lingual link discovery. To justify the effectiveness of CLLD, a standard evaluation framework is also proposed. The evaluation framework includes topics, document collections, a gold standard dataset, evaluation metrics, and toolkits for run pooling, link assessment and system evaluation. With the evaluation framework, performance of CLLD approaches and systems can be quantified. This thesis contributes to the research on natural language processing and cross-lingual information retrieval in CLLD: 1) a new simple, but effective Chinese segmentation method, n-gram mutual information, is presented for determining the boundaries of Chinese text; 2) a voting mechanism of name entity translation is demonstrated for achieving a high precision of English / Chinese machine translation; 3) a link mining approach that mines the existing link structure for anchor probabilities achieves encouraging results in suggesting cross-lingual Chinese / English links in Wikipedia. This approach was examined in the experiments for better, automatic generation of cross-lingual links that were carried out as part of the study. The overall major contribution of this thesis is the provision of a standard evaluation framework for cross-lingual link discovery research. It is important in CLLD evaluation to have this framework which helps in benchmarking the performance of various CLLD systems and in identifying good CLLD realisation approaches. The evaluation methods and the evaluation framework described in this thesis have been utilised to quantify the system performance in the NTCIR-9 Crosslink task which is the first information retrieval track of this kind.

Electronic functionality in graphene-based nanoarchitectures : discovery and design via first-principles modeling

Graphene has promised many novel applications in nanoscale electronics and sustainable energy due to its novel electronic properties. Computational exploration of electronic functionality and how it varies with architecture and doping presently runs ahead of experimental synthesis yet provides insights into types of structures that may prove profitable for targeted experimental synthesis and characterization. We present here a summary of our understanding on the important aspects of dimension, band gap, defect, and interfacial engineering of graphene based on state-of-the-art ab initio approaches. Some most recent experimental achievements relevant for future theoretical exploration are also covered.

Slice, mine and dice : complexity-aware automated discovery of business process models

Automated process discovery techniques aim at extracting models from information system logs in order to shed light into the business processes supported by these systems. Existing techniques in this space are effective when applied to relatively small or regular logs, but otherwise generate large and spaghetti-like models. In previous work, trace clustering has been applied in an attempt to reduce the size and complexity of automatically discovered process models. The idea is to split the log into clusters and to discover one model per cluster. The result is a collection of process models -- each one representing a variant of the business process -- as opposed to an all-encompassing model. Still, models produced in this way may exhibit unacceptably high complexity. In this setting, this paper presents a two-way divide-and-conquer process discovery technique, wherein the discovered process models are split on the one hand by variants and on the other hand hierarchically by means of subprocess extraction. The proposed technique allows users to set a desired bound for the complexity of the produced models. Experiments on real-life logs show that the technique produces collections of models that are up to 64% smaller than those extracted under the same complexity bounds by applying existing trace clustering techniques.

Using Wikipedia to improve web service discovery

Building and maintaining software are not easy tasks. However, thanks to advances in web technologies, a new paradigm is emerging in software development. The Service Oriented Architecture (SOA) is a relatively new approach that helps bridge the gap between business and IT and also helps systems remain exible. However, there are still several challenges with SOA. As the number of available services grows, developers are faced with the problem of discovering the services they need. Public service repositories such as Programmable Web provide only limited search capabilities. Several mechanisms have been proposed to improve web service discovery by using semantics. However, most of these require manually tagging the services with concepts in an ontology. Adding semantic annotations is a non-trivial process that requires a certain skill-set from the annotator and also the availability of domain ontologies that include the concepts related to the topics of the service. These issues have prevented these mechanisms becoming widespread. This thesis focuses on two main problems. First, to avoid the overhead of manually adding semantics to web services, several automatic methods to include semantics in the discovery process are explored. Although experimentation with some of these strategies has been conducted in the past, the results reported in the literature are mixed. Second, Wikipedia is explored as a general-purpose ontology. The benefit of using it as an ontology is assessed by comparing these semantics-based methods to classic term-based information retrieval approaches. The contribution of this research is significant because, to the best of our knowledge, a comprehensive analysis of the impact of using Wikipedia as a source of semantics in web service discovery does not exist. The main output of this research is a web service discovery engine that implements these methods and a comprehensive analysis of the benefits and trade-offs of these semantics-based discovery approaches.

The naturally imperfect form : investigations of the application of digital sculpting methods - extracted art : incorporating and translating ‘found art’ into the medium of digital sculpture

This dissertation analyses how physical objects are translated into digital artworks using techniques which can lead to ‘imperfections’ in the resulting digital artwork that are typically removed to arrive at a ‘perfect’ final representation. The dissertation discusses the adaptation of existing techniques into an artistic workflow that acknowledges and incorporates the imperfections of translation into the final pieces. It presents an exploration of the relationship between physical and digital artefacts and the processes used to move between the two. The work explores the 'craft' of digital sculpting and the technology used in producing what the artist terms ‘a naturally imperfect form’, incorporating knowledge of traditional sculpture, an understanding of anatomy and an interest in the study of bones (Osteology). The outcomes of the research are presented as a series of digital sculptural works, exhibited as a collection of curiosities in multiple mediums, including interactive game spaces, augmented reality (AR), rapid prototype prints (RP) and video displays.