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.

Developing a simulator application to test the billing platform in telecom industry

Billing Mediation Platform (BMP) in telecommunication industry is used to process real-time streams of Call Detail Records (CDRs) which can be a massive number a day. The generated records by BMP can be deployed for billing purposes, fraud detection, spam filtering, traffic analysis, and churn forecast. Several of these applications are distinguished by real-time processing requiring low-latency analysis of CDRs. Testing of such a platform carries diverse aspects like stress testing of analytics for scalability and what-if scenarios which require generating of CDRs with realistic volumetric and appropriate properties. The approach of this project is to build user friendly and flexible application which assists the development department to test their billing solution occasionally. These generators projects have been around for a while the only difference are the potions they cover and the purpose they will be used for. This paper proposes to use a simulator application to test the BMPs with simulating CDRs. The Simulated CDRs are modifiable based on the user requirements and represent real world data.

Maintenance test section survey : data collection and analysis

A Maintenance Test Section Survey (MTSS) was conducted as part of a Peer State Review of the Texas Maintenance Program conducted October 5–7, 2010. The purpose of the MTSS was to conduct a field review of 34 highway test sections and obtain participants’ opinions about pavement, roadside, and maintenance conditions. The goal was to cross reference or benchmark TxDOT’s maintenance practices based on practices used by selected peer states. Representatives from six peer states (California, Georgia, Kansas, Missouri, North Carolina, and Washington) were invited to Austin to attend a 3-day Peer State Review of TxDOT Maintenance Practices Workshop and to participate in a field survey of a number of pre-selected one-mile roadway sections. It should be emphasized that the objective of the survey was not to evaluate and grade or score TxDOT’s road network but rather to determine whether the selected roadway sections met acceptable standards of service as perceived by Directors of Maintenance or senior maintenance managers from the peer states...

Mass transfer properties (permeability and mass diffusivity) of four australian hardwood species

Characterization of mass transfer properties was achieved in the longitudinal, radial, and tangential directions for four Australian hardwood species: spotted gum, blackbutt, jarrah, and messmate. Measurement of mass transfer properties for these species was necessary to complement current vacuum drying modeling research. Water-vapour diffusivity was determined in steady state using a specific vapometer. Permeability was determined using a specialized device developed to measure over a wide range of permeability values. Permeability values of some species and material directions were extremely low and undetectable by the mass flow meter device. Hence, a custom system based on volume evolution was conceived to determine very low, previously unpublished, wood permeability values. Mass diffusivity and permeability were lowest for spotted gum and highest for messmate. Except for messmate in the radial direction, the four species measured were less permeable in all directions than the lowest published figures, demonstrating the high impermeability of Australian hardwoods and partly accounting for their relatively slow drying rates. Permeability, water-vapour diffusivity, and associated anisotropic ratio data obtained for messmate were extreme or did not follow typical trends and is consequently the most difficult of the four woods to dry in terms of collapse and checking degradation. © The State of Queensland, Department of Agriculture, Fisheries and Forestry, 2012.

Are insect frugivores always plant pests? The impact of fruit fly (Diptera: Tephritidae) larvae on host plant fitness

Herbivory is generally regarded as negatively impacting on host plant fitness. Frugivorous insects, which feed directly on plant reproductive tissues, are predicted to be particularly damaging to hosts. We tested this prediction with the fruit fly, Bactrocera tryoni, by recording the impact of larval feeding on two direct (seed number and germination) and two indirect (fruit decay rate and attraction/deterrence of vertebrate frugivores) measures of host plant fitness. Experiments were done in the laboratory, glasshouse and tropical rainforest. We found no negative impact of larval feeding on seed number or germination for three test plants: tomato, capsicum and eggplant. Further, larval feeding accelerated the initiation of decay and increased the final level of fruit decay in tomatoes, apples, pawpaw and pear, a result considered to be beneficial to the fruit. In rainforest studies, native rodents preferred infested apple and pears compared to uninfested control fruit; however, there were no differences observed between treatments for tomato and pawpaw. For our study fruits, these results demonstrate that fruit fly larval infestation has neutral or beneficial impacts on the host plant, an outcome which may be largely influenced by the physical properties of the host. These results may contribute to explaining why fruit flies have not evolved the same level of host specialization generally observed for other herbivore groups.

Learning to think spatially : what do students 'see' in numeracy test items?

Learning to think spatially in mathematics involves developing proficiency with graphics. This paper reports on 2 investigations of spatial thinking and graphics. The first investigation explored the importance of graphics as 1 of 3 communication systems (i.e. text, symbols, graphics) used to provide information in numeracy test items. The results showed that graphics were embedded in at least 50 % of test items across 3 year levels. The second investigation examined 11 – 12-year-olds’ performance on 2 mathematical tasks which required substantial interpretation of graphics and spatial thinking. The outcomes revealed that many students lacked proficiency in the basic spatial skills of visual memory and spatial perception and the more advanced skills of spatial orientation and spatial visualisation. This paper concludes with a reaffirmation of the importance of spatial thinking in mathematics and proposes ways to capitalize on graphics in learning to think spatially.

Evaluation of screening tests for predicting older driver performance and safety assessed by an on-road test

A number of tests and test batteries are available for the prediction of older driver safety, but many of these have not been validated against standardized driving outcome measures. The aim of this study was to evaluate a series of previously described screening tests in terms of their ability to predict the potential for safe and unsafe driving. Participants included 79 community-dwelling older drivers (M=72.16 years, SD=5.46; range 65-88 years; 57 males and 22 females) who completed a previously validated multi-disciplinary driving assessment, a hazard perception test, a hazard change detection test and a battery of vision and cognitive tests. Participants also completed a standardized on-road driving assessment. The multi-disciplinary test battery had the highest predictive ability with a sensitivity of 80% and a specificity of 73%, followed by the hazard perception test which demonstrated a sensitivity of 75% and a specificity of 61%. These findings suggest that a relatively simple and practical battery of tests from a range of domains has the capacity to predict safe and unsafe driving in older adults.

Transferring a successful strategy for supporting accelerated nursing students from a small to a large cohort

In 2009 and 2010, withdrawal rates from a Pharmacology unit of accelerated QUT nursing students in the first year of their degree, were higher than for continuing students. The cohort of 216 accelerated students in 2011 had university or non-university qualification or equivalent experience and included domestic and international students. A previously tested intervention was introduced in 2011 to improve retention rates and support all Pharmacology students in their first year of nursing. The intervention involved a community website, on-line tutors and an “O week” workshop comprising information about library resources, effective learning strategies and learning tips from a previous student as well as review anatomy, physiology and microbiology lectures. Withdrawal rates for accelerated students in the Pharmacology unit improved and all students found the workshop and review lectures to be informative and valuable. The intervention was therefore successfully transferred to a large, diverse cohort of accelerated nursing students.

A critical regionalist approach to housing design in Vietnam : socio-environmental organisation of living spaces in pre- and post-reform houses

Vietnam has a unique culture which is revealed in the way that people have built and designed their traditional housing. Vietnamese dwellings reflect occupants’ activities in their everyday lives, while adapting to tropical climatic conditions impacted by seasoning monsoons. It is said that these characteristics of Vietnamese dwellings have remained unchanged until the economic reform in 1986, when Vietnam experienced an accelerated development based on the market-oriented economy. New housing types, including modern shop-houses, detached houses, and apartments, have been designed in many places, especially satisfying dwellers’ new lifestyles in Vietnamese cities. The contemporary housing, which has been mostly designed by architects, has reflected rules of spatial organisation so that occupants’ social activities are carried out. However, contemporary housing spaces seem unsustainable in relation to socio-cultural values because they has been influenced by globalism that advocates the use of homogeneous spatial patterns, modern technologies, materials and construction methods. This study investigates the rules of spaces in Vietnamese houses that were built before and after the reform to define the socio-cultural implications in Vietnamese housing design. Firstly, it describes occupants’ views of their current dwellings in terms of indoor comfort conditions and social activities in spaces. Then, it examines the use of spaces in pre-reform Vietnamese housing through occupants’ activities and material applications. Finally, it discusses the organisation of spaces in both pre- and post-reform housing to understand how Vietnamese housing has been designed for occupants to live, act, work, and conduct traditional activities. Understanding spatial organisation is a way to identify characteristics of the lived spaces of the occupants created from the conceived space, which is designed by designers. The characteristics of the housing spaces will inform the designers the way to design future Vietnamese housing in response to cultural contexts. The study applied an abductive approach for the investigation of housing spaces. It used a conceptual framework in relation to Henri Lefebvre’s (1991) theory to understand space as the main factor constituting the language of design, and the principles of semiotics to examine spatial structure in housing as a language used in the everyday life. The study involved a door-knocking survey to 350 households in four regional cities of Vietnam for interpretation of occupancy conditions and levels of occupants’ comfort. A statistical analysis was applied to interpret the survey data. The study also required a process of data selection and collection of fourteen cases of housing in three main climatic regions of the country for analysing spatial organisation and housing characteristics. The study found that there has been a shift in the relationship of spaces from the pre- to post-reform Vietnamese housing. It also indentified that the space for guest welcoming and family activity has been the central space of the Vietnamese housing. Based on the relationships of the central space with the others, theoretical models were proposed for three types of contemporary Vietnamese housing. The models will be significant in adapting to Vietnamese conditions to achieve socioenvironmental characteristics for housing design because it was developed from the occupants’ requirements for their social activities. Another contribution of the study is the use of methodological concepts to understand the language of living spaces. Further work will be needed to test future Vietnamese housing designs from the applications of the models.

How to design extended finite state machine test models in Java

This chapter is a tutorial that teaches you how to design extended finite state machine (EFSM) test models for a system that you want to test. EFSM models are more powerful and expressive than simple finite state machine (FSM) models, and are one of the most commonly used styles of models for model-based testing, especially for embedded systems. There are many languages and notations in use for writing EFSM models, but in this tutorial we write our EFSM models in the familiar Java programming language. To generate tests from these EFSM models we use ModelJUnit, which is an open-source tool that supports several stochastic test generation algorithms, and we also show how to write your own model-based testing tool. We show how EFSM models can be used for unit testing and system testing of embedded systems, and for offline testing as well as online testing.

A Reliability test system for educational purposes-basic data

The IEEE Subcommittee on the Application of Probability Methods (APM) published the IEEE Reliability Test System (RTS) [1] in 1979. This system provides a consistent and generally acceptable set of data that can be used both in generation capacity and in composite system reliability evaluation [2,3]. The test system provides a basis for the comparison of results obtained by different people using different methods. Prior to its publication, there was no general agreement on either the system or the data that should be used to demonstrate or test various techniques developed to conduct reliability studies. Development of reliability assessment techniques and programs are very dependent on the intent behind the development as the experience of one power utility with their system may be quite different from that of another utility. The development and the utilization of a reliability program are, therefore, greatly influenced by the experience of a utlity and the intent of the system manager, planner and designer conducting the reliability studies. The IEEE-RTS has proved to be extremely valuable in highlighting and comparing the capabilities (or incapabilities) of programs used in reliability studies, the differences in the perception of various power utilities and the differences in the solution techniques. The IEEE-RTS contains a reasonably large power network which can be difficult to use for initial studies in an educational environment.