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.

Control, performance management systems and identification processes

Is it possible to control identities using performance management systems (PMSs)? This paper explores the theoretical fusion of management accounting and identity studies, providing a synthesised view of control, PMSs and identification processes. It argues that the effective use of PMSs generates a range of obtrusive mechanistic and unobtrusive organic controls that mediate identification processes to achieve a high level of identity congruency between individuals and collectives—groups and organisations. This paper contends that mechanistic control of PMSs provides sensebreaking effects and also creates structural conditions for sensegiving in top-down identification processes. These processes encourage individuals to continue the bottom-up processes of sensemaking, enacting identity and constructing identity narratives. Over time, PMS activities and conversations periodically mediate several episode(s) of identification to connect past, current and future identities. To explore this relationship, the dual locus of control—collectives and individuals—is emphasised to explicate their interplay. This multidisciplinary approach contributes to explaining the multidirectional effects of PMSs in obtrusive as well as unobtrusive ways, in order to control the nature of collectives and individuals in organisations.

Control, performance management system and identification processes

Is it possible to control identities using performance management systems (PMSs)? This paper explores the theoretical fusion of management accounting and identity studies, providing a synthesised view of control, PMSs and identification processes. It argues that the effective use of PMSs generates a range of obtrusive mechanistic and unobtrusive organic controls that mediate identification processes to achieve a high level of identity congruency between individuals and collectives—groups and organisations. This paper contends that mechanistic control of PMSs provides sensebreaking effects and also creates structural conditions for sensegiving in top-down identification processes. These processes encourage individuals to continue the bottom-up processes of sensemaking, enacting identity and constructing identity narratives. Over time, PMS activities and conversations periodically mediate several episode(s) of identification to connect past, current and future identities. To explore this relationship, the dual locus of control—collectives and individuals—is emphasised to explicate their interplay. This multidisciplinary approach contributes to explaining the multidirectional effects of PMSs in obtrusive as well as unobtrusive ways, in order to control the nature of collectives and individuals in organisations.

Problematising the processes of participation in networks : working through the rhetoric

Participation in networks, both as a concept and process, is widely supported in environmental education as a democratic and equitable pathway to individual and social change for sustainability. However, the processes of participation in networks are rarely problematized. Rather, it is assumed that we inherently know how to participate in networks. This assumption means that participation is seldom questioned. Underlying support for participation in networks is a belief that it allows individuals to connect in new and meaningful ways, that individuals can engage in making decisions and in bringing about change in arenas that affect them, and that they will be engaging in new, non-hierarchical and equitable relationships. In this paper we problematize participation in networks. As an example we use research into a decentralized network – described as such in its own literature - the Queensland Environmentally Sustainable Schools Initiative Alliance in Australia – to argue that while network participants were engaged and committed to participation in this network, 'old' forms of top-down engagement and relationships needed to be unlearnt. This paper thus proposes that for participation in decentralized networks to be meaningful, new learning about how to participate needs to occur.

Reflecting on the ‘Dream Circle’ : urban Indigenous education processes designed for student and community empowerment

The ‘Dream Circle’ is a space designed by and operated through Indigenous educator footprints as a safe space for the school’s deadly jarjums (Indigenous children). The ‘Dream Circle’ uses a kinnected methodology drawing on the rich vein of Murri cultural knowledges and Torres Strait Islander supports within the local community to provide a safe and supportive circle. The ‘Dream Circle’ operates on a school site in the Logan area as an after school homework and cultural studies class. The ‘Dream Circle’ embodies practices and ritualises processes which ensure cultural safety and integrity. In this way the ‘Dream Circle’ balances the measures that Sarra (2005) purports are the stronger, smarter realities needed for positive change in Indigenous education.

Design and implementation of a virtual world training simulation of ICU first hour handover processes

Nursing training for an Intensive Care Unit (ICU) is a resource intensive process. High demands are made on staff, students and physical resources. Interactive, 3D computer simulations, known as virtual worlds, are increasingly being used to supplement training regimes in the health sciences; especially in areas such as complex hospital ward processes. Such worlds have been found to be very useful in maximising the utilisation of training resources. Our aim is to design and develop a novel virtual world application for teaching and training Intensive Care nurses in the approach and method for shift handover, to provide an independent, but rigorous approach to teaching these important skills. In this paper we present a virtual world simulator for students to practice key steps in handing over the 24/7 care requirements of intensive care patients during the commencing first hour of a shift. We describe the modelling process to provide a convincing interactive simulation of the handover steps involved. The virtual world provides a practice tool for students to test their analytical skills with scenarios previously provided by simple physical simulations, and live on the job training. Additional educational benefits include facilitation of remote learning, high flexibility in study hours and the automatic recording of a reviewable log from the session. To the best of our knowledge, we believe this is a novel and original application of virtual worlds to an ICU handover process. The major outcome of the work was a virtual world environment for training nurses in the shift handover process, designed and developed for use by postgraduate nurses in training.

Stability of hydrotalcites formed from Bayer refinery environmental control processes

Bauxite refinery residues (red mud) are derived from the Bayer process by the digestion of crushed bauxite in concentrated sodium hydroxide at elevated temperatures and pressures. This slurry residue, if untreated, is unsuitable for discharge directly into the environment and is usually stored in tailing dams. The liquid portion has the potential for discharge, but requires pre-treatment before this can occur. The seawater neutralisation treatment facilitates a significant reduction in pH and dissolved metal concentrations, through the precipitation of hydrotalcite-like compounds and some other Mg, Ca, and Al hydroxide and carbonate minerals. The hydrotalcite-like compounds, precipitated during seawater neutralisation, also remove a range of transition metals, oxy-anions and other anionic species through a combination of intercalation and adsorption reactions: smaller anions are intercalated into the hydrotalcite matrix, while larger molecules are adsorbed on the particle surfaces. A phenomenon known as ‘reversion’ can occur if the seawater neutralisation process is not properly controlled. Reversion causes an increase in the pH and dissolved impurity levels of the neutralised effluent, rendering it unsuitable for discharge. It is believed that slow dissolution of components of the red mud residue and compounds formed during the neutralisation process are responsible for reversion. This investigation looked at characterising natural hydrotalcite (Mg6Al2(OH)16(CO3)∙4H2O) and ‘Bayer’ hydrotalcite (synthesised using the seawater neutralisation process) using a variety of techniques including X-ray diffraction, infrared and Raman spectroscopy, and thermogravimetric analysis. This investigation showed that Bayer hydrotalcite is comprised of a mixture of 3:1 and 4:1 hydrotalcite structures and exhibited similar chemical characteristic to the 4:1 synthetic hydrotalcite. Hydrotalcite formed from the seawater neutralisation of Bauxite refinery residues has been found not to cause reversion. Other components in red mud were investigated to determine the cause of reversion and this investigation found three components that contributed to reversion: 1) tricalcium aluminate, 2) hydrocalumite and 3) calcium hydroxide. Increasing the amount of magnesium in the neutralisation process has been found to be successful in reducing reversion.

From supply to demand-driven opportunities : unravelling the role of the customer in opportunity identification processes

Successful identification and exploitation of opportunities has been an area of interest to many entrepreneurship researchers. Since Shane and Venkataraman’s seminal work (e.g. Shane and Venkataraman, 2000; Shane, 2000), several scholars have theorised on how firms identify, nurture and develop opportunities. The majority of this literature has been devoted to understanding how entrepreneurs search for new applications of their technological base or discover opportunities based on prior knowledge (Zahra, 2008; Sarasvathy et al., 2003). In particular, knowledge about potential customer needs and problems that may present opportunities is vital (Webb et al., 2010). Whereas the role of prior knowledge of customer problems (Shane, 2003; Shepherd and DeTienne, 2005) and positioning oneself in a so-called knowledge corridor (Fiet, 1996) has been researched, the role of opportunity characteristics and their interaction with customer-related mechanisms that facilitate and hinder opportunity identification has received scant attention.

Virtual worlds as a model-view approach to the communication of business processes models

The improvement and optimization of business processes is one of the top priorities in an organization. Although process analysis methods are mature today, business analysts and stakeholders are still hampered by communication issues. That is, analysts cannot effectively obtain accurate business requirements from stakeholders, and stakeholders are often confused about analytic results offered by analysts. We argue that using a virtual world to model a business process can benefit communication activities. We believe that virtual worlds can be used as an efficient model-view approach, increasing the cognition of business requirements and analytic results, as well as the possibility of business plan validation. A healthcare case study is provided as an approach instance, illustrating how intuitive such an approach can be. As an exploration paper, we believe that this promising research can encourage people to investigate more research topics in the interdisciplinary area of information system, visualization and multi-user virtual worlds.

How novices design business processes

Process modeling is an important design practice in organizational improvement projects. In this paper, we examine the design of business process diagrams in contexts where novice analysts only have basic design tools such as paper and pencils available, and little to no understanding of formalized modeling approaches. Based on a quasi-experimental study with 89 BPM students, we identify five distinct process design archetypes ranging from textual to hybrid and graphical representation forms. We examine the quality of the designs and identify which representation formats enable an analyst to articulate business rules, states, events, activities, temporal and geospatial information in a process model. We found that the quality of the process designs decreases with the increased use of graphics and that hybrid designs featuring appropriate text labels and abstract graphical forms appear well-suited to describe business processes. We further examine how process design preferences predict formalized process modeling ability. Our research has implications for practical process design work in industry as well as for academic curricula on process design.

A graphical notation for modelling events in business processes

Using complex event rules for capturing dependencies between business processes is an emerging trend in enterprise information systems. In previous work we have identified a set of requirements for event extensions for business process modeling languages. This paper introduces a graphical language for modeling composite events in business processes, namely BEMN, that fulfills all these requirements. These include event conjunction, disjunction and inhibition as well as cardinality of events whose graphical expression can be factored into flow-oriented process modeling and event rule modeling. Formal semantics for the language are provided.

Complex events in business processes

Flow-oriented process modeling languages have a long tradition in the area of Business Process Management and are widely used for capturing activities with their behavioral and data dependencies. Individual events were introduced for triggering process instantiation and activities. However, real-world business cases drive the need for also covering complex event patterns as they are known in the field of Complex Event Processing. Therefore, this paper puts forward a catalog of requirements for handling complex events in process models, which can be used as reference framework for assessing process definition languages and systems. An assessment of BPEL and BPMN is provided.

Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing processes

The crosstalk between fibroblasts and keratinocytes is a vital component of the wound healing process, and involves the activity of a number of growth factors and cytokines. In this work, we develop a mathematical model of this crosstalk in order to elucidate the effects of these interactions on the regeneration of collagen in a wound that heals by second intention. We consider the role of four components that strongly affect this process: transforming growth factor-beta, platelet-derived growth factor, interleukin-1 and keratinocyte growth factor. The impact of this network of interactions on the degradation of an initial fibrin clot, as well as its subsequent replacement by a matrix that is mainly comprised of collagen, is described through an eight-component system of nonlinear partial differential equations. Numerical results, obtained in a two-dimensional domain, highlight key aspects of this multifarious process such as reepithelialisation. The model is shown to reproduce many of the important features of normal wound healing. In addition, we use the model to simulate the treatment of two pathological cases: chronic hypoxia, which can lead to chronic wounds; and prolonged inflammation, which has been shown to lead to hypertrophic scarring. We find that our model predictions are qualitatively in agreement with previously reported observations, and provide an alternative pathway for gaining insight into this complex biological process.

Optimising container processes at multimodal seaport terminals : an integrated approach and application

A Multimodal Seaport Container Terminal (MSCT) is a complex system which requires careful planning and control in order to operate efficiently. It consists of a number of subsystems that require optimisation of the operations within them, as well as synchronisation of machines and containers between the various subsystems. Inefficiency in the terminal can delay ships from their scheduled timetables, as well as cause delays in delivering containers to their inland destinations, both of which can be very costly to their operators. The purpose of this PhD thesis is to use Operations Research methodologies to optimise and synchronise these subsystems as an integrated application. An initial model is developed for the overall MSCT; however, due to a large number of assumptions that had to be made, as well as other issues, it is found to be too inaccurate and infeasible for practical use. Instead, a method of developing models for each subsystem is proposed that then be integrated with each other. Mathematical models are developed for the Storage Area System (SAS) and Intra-terminal Transportation System (ITTS). The SAS deals with the movement and assignment of containers to stacks within the storage area, both when they arrive and when they are rehandled to retrieve containers below them. The ITTS deals with scheduling the movement of containers and machines between the storage areas and other sections of the terminal, such as the berth and road/rail terminals. Various constructive heuristics are explored and compared for these models to produce good initial solutions for large-sized problems, which are otherwise impractical to compute by exact methods. These initial solutions are further improved through the use of an innovative hyper-heuristic algorithm that integrates the SAS and ITTS solutions together and optimises them through meta-heuristic techniques. The method by which the two models can interact with each other as an integrated system will be discussed, as well as how this method can be extended to the other subsystems of the MSCT.

Information sharing in Onesocialweb using a multiple profile manager to better informed medical decision making processes

The study shows an alternative solution to existing efforts at solving the problem of how to centrally manage and synchronise users’ Multiple Profiles (MP) across multiple discrete social networks. Most social network users hold more than one social network account and utilise them in different ways depending on the digital context (Iannella, 2009a). They may, for example, enjoy friendly chat on Facebook1, professional discussion on LinkedIn2, and health information exchange on PatientsLikeMe3 In this thesis the researcher proposes a framework for the management of a user’s multiple online social network profiles. A demonstrator, called Multiple Profile Manager (MPM), will be showcased to illustrate how effective the framework will be. The MPM will achieve the required profile management and synchronisation using a free, open, decentralized social networking platform (OSW) that was proposed by the Vodafone Group in 2010. The proposed MPM will enable a user to create and manage an integrated profile (IP) and share/synchronise this profile with all their social networks. The necessary protocols to support the prototype are also proposed by the researcher. The MPM protocol specification defines an Extensible Messaging and Presence Protocol (XMPP) extension for sharing vCard and social network accounts information between the MPM Server, MPM Client, and social network sites (SNSs). . Therefore many web users need to manage disparate profiles across many distributed online sources. Maintaining these profiles is cumbersome, time-consuming, inefficient, and may lead to lost opportunity. The writer of this thesis adopted a research approach and a number of use cases for the implementation of the project. The use cases were created to capture the functional requirements of the MPM and to describe the interactions between users and the MPM. In the research a development process was followed in establishing the prototype and related protocols. The use cases were subsequently used to illustrate the prototype via the screenshots taken of the MPM client interfaces. The use cases also played a role in evaluating the outcomes of the research such as the framework, prototype, and the related protocols. An innovative application of this project is in the area of public health informatics. The researcher utilised the prototype to examine how the framework might benefit patients and physicians. The framework can greatly enhance health information management for patients and more importantly offer a more comprehensive personal health overview of patients to physicians. This will give a more complete picture of the patient’s background than is currently available and will prove helpful in providing the right treatment. The MPM prototype and related protocols have a high application value as they can be integrated into the real OSW platform and so serve users in the modern digital world. They also provide online users with a real platform for centrally storing their complete profile data, efficiently managing their personal information, and moreover, synchronising the overall complete profile with each of their discrete profiles stored in their different social network sites.