Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

Rational design of serine protease inhibitors

Proteases regulate a spectrum of diverse physiological processes, and dysregulation of proteolytic activity drives a plethora of pathological conditions. Understanding protease function is essential to appreciating many aspects of normal physiology and progression of disease. Consequently, development of potent and specific inhibitors of proteolytic enzymes is vital to provide tools for the dissection of protease function in biological systems and for the treatment of diseases linked to aberrant proteolytic activity. The studies in this thesis describe the rational design of potent inhibitors of three proteases that are implicated in disease development. Additionally, key features of the interaction of proteases and their cognate inhibitors or substrates are analysed and a series of rational inhibitor design principles are expounded and tested. Rational design of protease inhibitors relies on a comprehensive understanding of protease structure and biochemistry. Analysis of known protease cleavage sites in proteins and peptides is a commonly used source of such information. However, model peptide substrate and protein sequences have widely differing levels of backbone constraint and hence can adopt highly divergent structures when binding to a protease’s active site. This may result in identical sequences in peptides and proteins having different conformations and diverse spatial distribution of amino acid functionalities. Regardless of this, protein and peptide cleavage sites are often regarded as being equivalent. One of the key findings in the following studies is a definitive demonstration of the lack of equivalence between these two classes of substrate and invalidation of the common practice of using the sequences of model peptide substrates to predict cleavage of proteins in vivo. Another important feature for protease substrate recognition is subsite cooperativity. This type of cooperativity is commonly referred to as protease or substrate binding subsite cooperativity and is distinct from allosteric cooperativity, where binding of a molecule distant from the protease active site affects the binding affinity of a substrate. Subsite cooperativity may be intramolecular where neighbouring residues in substrates are interacting, affecting the scissile bond’s susceptibility to protease cleavage. Subsite cooperativity can also be intermolecular where a particular residue’s contribution to binding affinity changes depending on the identity of neighbouring amino acids. Although numerous studies have identified subsite cooperativity effects, these findings are frequently ignored in investigations probing subsite selectivity by screening against diverse combinatorial libraries of peptides (positional scanning synthetic combinatorial library; PS-SCL). This strategy for determining cleavage specificity relies on the averaged rates of hydrolysis for an uncharacterised ensemble of peptide sequences, as opposed to the defined rate of hydrolysis of a known specific substrate. Further, since PS-SCL screens probe the preference of the various protease subsites independently, this method is inherently unable to detect subsite cooperativity. However, mean hydrolysis rates from PS-SCL screens are often interpreted as being comparable to those produced by single peptide cleavages. Before this study no large systematic evaluation had been made to determine the level of correlation between protease selectivity as predicted by screening against a library of combinatorial peptides and cleavage of individual peptides. This subject is specifically explored in the studies described here. In order to establish whether PS-SCL screens could accurately determine the substrate preferences of proteases, a systematic comparison of data from PS-SCLs with libraries containing individually synthesised peptides (sparse matrix library; SML) was carried out. These SML libraries were designed to include all possible sequence combinations of the residues that were suggested to be preferred by a protease using the PS-SCL method. SML screening against the three serine proteases kallikrein 4 (KLK4), kallikrein 14 (KLK14) and plasmin revealed highly preferred peptide substrates that could not have been deduced by PS-SCL screening alone. Comparing protease subsite preference profiles from screens of the two types of peptide libraries showed that the most preferred substrates were not detected by PS SCL screening as a consequence of intermolecular cooperativity being negated by the very nature of PS SCL screening. Sequences that are highly favoured as result of intermolecular cooperativity achieve optimal protease subsite occupancy, and thereby interact with very specific determinants of the protease. Identifying these substrate sequences is important since they may be used to produce potent and selective inhibitors of protolytic enzymes. This study found that highly favoured substrate sequences that relied on intermolecular cooperativity allowed for the production of potent inhibitors of KLK4, KLK14 and plasmin. Peptide aldehydes based on preferred plasmin sequences produced high affinity transition state analogue inhibitors for this protease. The most potent of these maintained specificity over plasma kallikrein (known to have a very similar substrate preference to plasmin). Furthermore, the efficiency of this inhibitor in blocking fibrinolysis in vitro was comparable to aprotinin, which previously saw clinical use to reduce perioperative bleeding. One substrate sequence particularly favoured by KLK4 was substituted into the 14 amino acid, circular sunflower trypsin inhibitor (SFTI). This resulted in a highly potent and selective inhibitor (SFTI-FCQR) which attenuated protease activated receptor signalling by KLK4 in vitro. Moreover, SFTI-FCQR and paclitaxel synergistically reduced growth of ovarian cancer cells in vitro, making this inhibitor a lead compound for further therapeutic development. Similar incorporation of a preferred KLK14 amino acid sequence into the SFTI scaffold produced a potent inhibitor for this protease. However, the conformationally constrained SFTI backbone enforced a different intramolecular cooperativity, which masked a KLK14 specific determinant. As a consequence, the level of selectivity achievable was lower than that found for the KLK4 inhibitor. Standard mechanism inhibitors such as SFTI rely on a stable acyl-enzyme intermediate for high affinity binding. This is achieved by a conformationally constrained canonical binding loop that allows for reformation of the scissile peptide bond after cleavage. Amino acid substitutions within the inhibitor to target a particular protease may compromise structural determinants that support the rigidity of the binding loop and thereby prevent the engineered inhibitor reaching its full potential. An in silico analysis was carried out to examine the potential for further improvements to the potency and selectivity of the SFTI-based KLK4 and KLK14 inhibitors. Molecular dynamics simulations suggested that the substitutions within SFTI required to target KLK4 and KLK14 had compromised the intramolecular hydrogen bond network of the inhibitor and caused a concomitant loss of binding loop stability. Furthermore in silico amino acid substitution revealed a consistent correlation between a higher frequency of formation and the number of internal hydrogen bonds of SFTI-variants and lower inhibition constants. These predictions allowed for the production of second generation inhibitors with enhanced binding affinity toward both targets and highlight the importance of considering intramolecular cooperativity effects when engineering proteins or circular peptides to target proteases. The findings from this study show that although PS-SCLs are a useful tool for high throughput screening of approximate protease preference, later refinement by SML screening is needed to reveal optimal subsite occupancy due to cooperativity in substrate recognition. This investigation has also demonstrated the importance of maintaining structural determinants of backbone constraint and conformation when engineering standard mechanism inhibitors for new targets. Combined these results show that backbone conformation and amino acid cooperativity have more prominent roles than previously appreciated in determining substrate/inhibitor specificity and binding affinity. The three key inhibitors designed during this investigation are now being developed as lead compounds for cancer chemotherapy, control of fibrinolysis and cosmeceutical applications. These compounds form the basis of a portfolio of intellectual property which will be further developed in the coming years.

Managing large collections of business process models - Current techniques and challenges

Nowadays, business process management is an important approach for managing organizations from an operational perspective. As a consequence, it is common to see organizations develop collections of hundreds or even thousands of business process models. Such large collections of process models bring new challenges and provide new opportunities, as the knowledge that they encapsulate requires to be properly managed. Therefore, a variety of techniques for managing large collections of business process models is being developed. The goal of this paper is to provide an overview of the management techniques that currently exist, as well as the open research challenges that they pose.

Using turning technologies to engage tutors and facilitate effective marking and moderation in large classes

Audience Response Systems (ARS) have been successfully used by academics to facilitate student learning and engagement, particularly in large lecture settings. However, in large core subjects a key challenge is not only to engage students, but also to engage large and diverse teaching teams in order to ensure a consistent approach to grading assessments. This paper provides an insight into the ways in which ARS can be used to encourage participation by tutors in marking and moderation meetings. It concludes that ARS can improve the consistency of grading and the quality of feedback provided to students.

When the shoe doesn't quite fit: Regulating large commercial airports with expanding roles

Airports, whether publicly or privately owned or operated fill both public and private roles. They need to act as public infrastructure providers and as businesses which cover their operating costs. That leads to special governance concerns with respect to consumers and competitors which are only beginning to be addressed. These challenges are highlighted both by shifts in ownership status and by the expansion of roles performed by airports as passenger and cargo volumes continue to increase and as nearby urban areas expand outward towards airports. We survey five ways in which the regulatory shoe doesn‟t quite fit the needs. Our findings suggest that, while ad hoc measures limit political tension, new governance measures are needed.

Engaging new digital locals with interactive urban screens to collaboratively improve the city

Local governments struggle to engage time poor and seemingly apathetic citizens, as well as the city’s young digital natives, the digital locals. This project aims at providing a lightweight, technological contribution towards removing the hierarchy between those who build the city and those who use it. We aim to narrow this gap by enhancing people’s experience of physical spaces with digital, civic technologies that are directly accessible within that space. This paper presents the findings of a design trial allowing users to interact with a public screen via their mobile phones. The screen facilitated a feedback platform about a concrete urban planning project by promoting specific questions and encouraging direct, in-situ, real-time responses via SMS and twitter. This new mechanism offers additional benefits for civic participation as it gives voice to residents who otherwise would not be heard. It also promotes a positive attitude towards local governments and gathers information different from more traditional public engagement tools.

Screening to identify large truck potential blackspots : methodological issues and concerns

1. Overview of hotspot identification (HSID)methods 2. Challenges with HSID 3. Bringing crash severity into the ‘mix’ 4. Case Study: Truck Involved Crashes in Arizona 5. Conclusions • Heavy duty trucks have different performance envelopes than passenger cars and have more difficulty weaving, accelerating, and braking • Passenger vehicles have extremely limited sight distance around trucks • Lane and shoulder widths affect truck crash risk more than passenger cars • Using PDOEs to model truck crashes results in a different set of locations to examine for possible engineering and behavioral problems • PDOE models point to higher societal cost locations, whereas frequency models point to higher crash frequency locations • PDOE models are less sensitive to unreported crashes • PDOE models are a great complement to existing practice

Opportunities of public transport experience enhancements with mobile services and urban screens

Public transportation is an environment with great potential for applying innovative ubiquitous computing services to enhance user experiences. This paper provides the underpinning rationale for research that will be looking at how real-time passenger information system deployed by transit authorities can provide a core platform to improve commuters’ user experiences during all stages of their journey. The proposal builds on this platform to inform the design and development of innovative social media, mobile computing and geospatial information applications, with the hope to create fun and meaningful experiences for passengers during their everyday travel. Furthermore, we present the findings of our pilot study that aims to offer a better understanding of passengers’ activities and social interactions during their daily commute.

Using technology to facilitate grading consistency in large classes

University classes in marketing are often large, and therefore require teams of teachers to cover all of the necessary activities. A major problem with teaching teams is the inconsistency that results from myriad individuals offering subjective opinions. This innovation uses the latest moderation techniques along with Audience Response Technology (ART) to enhance the learning experience by providing more consistent and reliable grading in large classes. Assessment items are moderated before they are graded in meetings that employ ART. Results show the process is effective when the teaching team is very large, or there is a diverse range of experienced and inexperienced teachers. This “behind the scenes” innovation is not immediately apparent to students, but results in more consistent grades, more useful feedback for students, and more confident graders.

Taking a leap of faith : are investors left short changed?

This dissertation examines the compliance and performance of a large sample of faith based (religious) ethical funds - the Shari'ah-compliant equity funds (SEFs), which may be viewed as a form of ethical investing. SEFs screen their investment for compliance with Islamic law, where riba (conventional interest expense), maysir (gambling), gharar (excessive uncertainty), and non-halal (non-ethical) products are prohibited. Using a set of stringent Shari'ah screens similar to those of MSCI Islamic, we first examine the extent to which SEFs comply with the Shari'ah law. Results show that only about 27% of the equities held by SEFs are Shari'ah-compliant. While most of the fund holdings pass the business screens, only about 42% pass the total debt to total assets ratio screen. This finding suggests that, in order to overcome a significant reduction in the investment opportunity, Shari'ah principles are compromised, with SEFs adopting lax screening rules so as to achieve a financial performance. While younger funds and funds that charge higher fees and are domiciled in more Muslim countries are more Shari'ah-compliant, we find little evidence of a positive relationship between fund disclosure of the Shari'ah compliance framework and Shari'ah-compliance. Clearly, Shari'ah compliance remains a major challenge for fund managers and SEF investors should be aware of Shari'ah-compliance risk since the fund managers do not always fulfill their fiduciary obligation, as promised in their prospectus. Employing a matched firm approach for a survivorship free sample of 387 SEFs, we then examine an issue that has been heavily debated in the literature: Does ethical screening reduce investment performance? Results show that it does but only by an average of 0.04% per month if benchmarked against matched conventional funds - this is a relatively small price to pay for religious faith. Cross-sectional regressions show an inverse relationship between Shari'ah compliance and fund performance: every one percentage increase in total compliance decreases fund performance by 0.01% per month. However, compliance fails to explain differences in the performance between SEFs and matched funds. Although SEFs do not generally perform better during crisis periods, further analysis shows evidence of better performance relative to conventional funds only during the recent Global Financial Crisis; the latter is consistent with popular media claims.

Morphological design control for large-scale city development : a new proposal

Whereas many good examples can be found of the study of urban morphology informing the design of new residential areas in Europe, it is much more difficult to find examples relating to other land uses and outside of Europe. This paper addresses a particular issue, the control and coordination of large and complex development schemes within cities, and, in doing so, considers commercial and mixed-use schemes outside of Europe. It is argued that urban morphology has much to offer for both the design of such development and its implementation over time. Firstly, lessons are drawn from the work of Krier and Rossi in Berlin, the form-based guidance developed in Chelmsford, UK, and the redesign and coordination of the Melrose Arch project in Johannesburg, SA. A recent development at Boggo Road in Brisbane, Australia, is then subjected to a more detailed examination. It is argued that the scheme has been unsatisfactory in terms of both design and implementation. An alternative framework based on historical morphological studies is proposed that would overcome these deficiencies. It is proposed that this points the way to a general approach that could be incorporated within the planning process internationally.

People, content, location : sweet spotting urban screens for situated engagement

A growing body of research is looking at ways to bring the processes and benefits of online deliberation to the places they are about and in turn allow a larger, targeted proportion of the urban public to have a voice, be heard, and engage in questions of city planning and design. Seeking to take advantage of the civic opportunities of situated engagement through public screens and mobile devices, our research informed a public urban screen content application DIS that we deployed and evaluated in a wide range of real world public and urban environments. For example, it is currently running on the renowned urban screen at Federation Square in Melbourne. We analysed the data from these user studies within a conceptual framework that positions situated engagement across three key parameters: people, content, and location. We propose a way to identify the sweet spot within the nexus of these parameters to help deploy and run interactive systems to maximise the quality of the situated engagement for civic and related deliberation purposes.

e. Menura Supurba: Post human dreams of ersatz animals.

This paper discusses human and post-human relationships with nature and animals, using the work e. Menura Superba1 as a focal point. This interactive artwork takes the form of a Lyre bird in a cage, that mimics it’s audience in evocative ways. It is inspired by the historical practice of displaying taxidermy specimens and live species as trophies of travels to distant lands, and as symbols of wealth and status. In both form and intent the work hybridises elements from Enlightenment culture, with materials that conjure associations with dystopic post human futures (wire, post consumer electronic & other waste, as well working parts such as mobile phone screens, LED’s, camera, and cabling etc). Speculative science fiction, such as Phillip K Dick in Do Androids Dream of Electric Sheep? (Blade Runner), provides prescient stories about future (post) human worlds. This novel remains thought provoking as it describes a world that is all to rapidly approaching: where human activity has caused the destruction of most large animal species. In this fictional world, care for animals is not only a civic duty, it is one of the ways humans distinguish themselves from androids. As in Enlightenment times, ownership of animals (real, taxidermies, ersatz) is a form of commodity fetishism indicative of social status. Though whilst well heeled Victorians may have owned an elephant or have been proud of a trophy specimen, the wealthy in Dick’s future must be content with once common, even ersatz, animals such as sheep and owls, and would be repulsed to the core by the notion of killing an animal, even an ersatz animal, for sport. In becoming post human, humans have sought to separate themselves from the natural world, destroying much of it in the process. No technical prothesis will bring back to life the species we have rendered extinct. This (evolving) relationship between humanity and other species, therefore forms a central question in this work, providing a way of approaching the post human, and problematising anthropocentric perspectives. The world promised by post-human technology is indeed rich with possibility, but without corresponding steps to ensure the sustainability of technology (human society), this paper asks whether the richness of that experience will continue to be mirrored by the richness of the environments within which we exist?

Discussions in space : interactive urban screens for enhancing citizen engagement

Local governments struggle to engage time poor and seemingly apathetic citizens, as well as the city's young digital natives, the digital locals. Capturing the attention of this digitally literate community who are technology and socially savvy adds a new quality to the challenge of community engagement for urban planning. This project developed and tested a lightweight design intervention towards removing the hierarchy between those who plan the city and those who use it. The aim is to narrow this gap by enhancing people's experience of physical spaces with digital, civic technologies that are directly accessible within that space. The study's research informed the development of a public screen system called Discussions In Space (DIS). It facilitates a feedback platform about specific topics, e.g., a concrete urban planning project, and encourages direct, in-situ, real-time user responses via SMS and Twitter. The thesis presents the findings of deploying and integrating DIS in a wide range of public and urban environments, including the iconic urban screen at Federation Square in Melbourne, to explore the Human-Computer Interaction (HCI) related challenges and implications. It was also deployed in conjunction with a major urban planning project in Brisbane to explore the system's opportunities and challenges of better engaging with Australia's new digital locals. Finally, the merits of the short-texted and ephemeral data generated by the system were evaluated in three focus groups with professional urban planners. DIS offers additional benefits for civic participation as it gives voice to residents who otherwise would not be easily heard. It also promotes a positive attitude towards local governments and gathers complementary information that is different than that captured by more traditional public engagement tools.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 μg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.