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.

Outlearning

Making the University Matter investigates how academics situate themselves simultaneously in the university and the world and how doing so affects the viability of the university setting. The university stands at the intersection of two sets of interests, needing to be at one with the world while aspiring to stand apart from it. In an era that promises intensified political instability, growing administrative pressures, dwindling economic returns and questions about economic viability, lower enrolments and shrinking programs, can the university continue to matter into the future? And if so, in which way? What will help it survive as an honest broker? What are the mechanisms for ensuring its independent voice? Barbie Zelizer brings together some of the leading names in the field of media and communication studies from around the globe to consider a multiplicity of answers from across the curriculum on making the university matter, including critical scholarship, interdisciplinarity, curricular blends of the humanities and social sciences, practical training and policy work. The collection is introduced with an essay by the editor and each section has a brief introduction to contextualise the essays and highlight the issues they raise.

Fear of Crime in Expatriate Australians in Ho Chi Minh City Vietnam

This is an analytical report of a qualitative study of fear of crime in six Australian expatriates living in Ho Chi Minh City (HCMC) Vietnam. Addressing the primary question of what changes, or impacts upon, fear of crime in Australian expatriates in HCMC Vietnam, the research paid particular attention to studying the differences in fear of crime when respondents became expatriates, and the impact of incivilities and access to media. Each of the respondents indicated that they felt safer in Vietnam than in Australia. An analysis of the respondents’ responses indicates that this feeling of safety did not occur on arrival but after a short period of adjustment. The findings of this research support the existing theories on fear of crime and highlight the importance of context in predicting the impact of such factors as media and incivilities. The study has practical applications for both private and public sector organisations seeking to deploy staff to HCMC and adds to the current significant body of fear of crime research by specifically examining the issue of fear of crime amongst expatriates.

Proteasome inhibitors as anti-cancer agents

The ubiquitin (Ub)-proteasome pathway is the major nonlysosomal pathway of proteolysis in human cells and accounts for the degradation of most short-lived, misfolded or damaged proteins. This pathway is important in the regulation of a number of key biological regulatory mechanisms. Proteins are usually targeted for proteasome-mediated degradation by polyubiquitinylation, the covalent addition of multiple units of the 76 amino acid protein Ub, which are ligated to 1-amino groups of lysine residues in the substrate. Polyubiquitinylated proteins are degraded by the 26S proteasome, a large, ATP-dependent multicatalytic protease complex, which also regenerates monomeric Ub. The targets of this pathway include key regulators of cell proliferation and cell death. An alternative form of the proteasome, termed the immunoproteasome, also has important functions in the generation of peptides for presentation by MHC class I molecules. In recent years there has been a great deal of interest in the possibility that proteasome inhibitors, through elevation of the levels of proteasome targets, might prove useful as a novel class of anti-cancer drugs. Here we review the progress made to date in this area and highlight the potential advantages and weaknesses of this approach.

Evolutionary placement of continuously operating reference stations of network Real-Time Kinematic

Network RTK (Real-Time Kinematic) is a technology that is based on GPS (Global Positioning System) or more generally on GNSS (Global Navigation Satellite System) observations to achieve centimeter-level accuracy positioning in real time. It is enabled by a network of Continuously Operating Reference Stations (CORS). CORS placement is an important problem in the design of network RTK as it directly affects not only the installation and running costs of the network RTK, but also the Quality of Service (QoS) provided by the network RTK. In our preliminary research on the CORS placement, we proposed a polynomial heuristic algorithm for a so-called location-based CORS placement problem. From a computational point of view, the location-based CORS placement is a largescale combinatorial optimization problem. Thus, although the heuristic algorithm is efficient in computation time it may not be able to find an optimal or near optimal solution. Aiming at improving the quality of solutions, this paper proposes a repairing genetic algorithm (RGA) for the location-based CORS placement problem. The RGA has been implemented and compared to the heuristic algorithm by experiments. Experimental results have shown that the RGA produces better quality of solutions than the heuristic algorithm.

Minimization of railhead edge stresses through shape optimization

The railhead is severely stressed under the localized wheel contact patch close to the gaps in insulated rail joints. A modified railhead profile in the vicinity of the gapped joint, through a shape optimization model based on a coupled genetic algorithm and finite element method, effectively alters the contact zone and reduces the railhead edge stress concentration significantly. Two optimization methods, a grid search method and a genetic algorithm, were employed for this optimization problem. The optimal results from these two methods are discussed and, in particular, their suitability for the rail end stress minimization problem is studied. Through several numerical examples, the optimal profile is shown to be unaffected by either the magnitude or the contact position of the loaded wheel. The numerical results are validated through a large-scale experimental study.

Symptoms of heat illness in surface mine workers

Objective: To assess the symptoms of heat illness experienced by surface mine workers. Methods: Ninety-one surface mine workers across three mine sites in northern Australia completed a heat stress questionnaire evaluating their symptoms for heat illness. A cohort of 56 underground mine workers also participated for comparative purposes. Participants were allocated into asymptomatic, minor or moderate heat illness categories depending on the number of symptoms they reported. Participants also reported the frequency of symptom experience, as well as their hydration status (average urine colour). Results: Heat illness symptoms were experienced by 87 and 79 % of surface and underground mine workers, respectively (p = 0.189), with 81–82 % of the symptoms reported being experienced by miners on more than one occasion. The majority (56 %) of surface workers were classified as experiencing minor heat illness symptoms, with a further 31 % classed as moderate; 13 % were asymptomatic. A similar distribution of heat illness classification was observed among underground miners (p = 0.420). Only 29 % of surface miners were considered well hydrated, with 61 % minimally dehydrated and 10 % significantly dehydrated, proportions that were similar among underground miners (p = 0.186). Heat illness category was significantly related to hydration status (p = 0.039) among surface mine workers, but only a trend was observed when data from surface and underground miners was pooled (p = 0.073). Compared to asymptomatic surface mine workers, the relative risk of experiencing minor and moderate symptoms of heat illness was 1.5 and 1.6, respectively, when minimally dehydrated. Conclusions: These findings show that surface mine workers routinely experience symptoms of heat illness and highlight that control measures are required to prevent symptoms progressing to medical cases of heat exhaustion or heat stroke.

Computationally sound automated proofs of cryptographic schemes

Proving security of cryptographic schemes, which normally are short algorithms, has been known to be time-consuming and easy to get wrong. Using computers to analyse their security can help to solve the problem. This thesis focuses on methods of using computers to verify security of such schemes in cryptographic models. The contributions of this thesis to automated security proofs of cryptographic schemes can be divided into two groups: indirect and direct techniques. Regarding indirect ones, we propose a technique to verify the security of public-key-based key exchange protocols. Security of such protocols has been able to be proved automatically using an existing tool, but in a noncryptographic model. We show that under some conditions, security in that non-cryptographic model implies security in a common cryptographic one, the Bellare-Rogaway model [11]. The implication enables one to use that existing tool, which was designed to work with a different type of model, in order to achieve security proofs of public-key-based key exchange protocols in a cryptographic model. For direct techniques, we have two contributions. The first is a tool to verify Diffie-Hellmanbased key exchange protocols. In that work, we design a simple programming language for specifying Diffie-Hellman-based key exchange algorithms. The language has a semantics based on a cryptographic model, the Bellare-Rogaway model [11]. From the semantics, we build a Hoare-style logic which allows us to reason about the security of a key exchange algorithm, specified as a pair of initiator and responder programs. The other contribution to the direct technique line is on automated proofs for computational indistinguishability. Unlike the two other contributions, this one does not treat a fixed class of protocols. We construct a generic formalism which allows one to model the security problem of a variety of classes of cryptographic schemes as the indistinguishability between two pieces of information. We also design and implement an algorithm for solving indistinguishability problems. Compared to the two other works, this one covers significantly more types of schemes, but consequently, it can verify only weaker forms of security.

Calculation of volume from crank angle using reconfigurable hardware

Fast calculation of quantities such as in-cylinder volume and indicated power is important in internal combustion engine research. Multiple channels of data including crank angle and pressure were collected for this purpose using a fully instrumented diesel engine research facility. Currently, existing methods use software to post-process the data, first calculating volume from crank angle, then calculating the indicated work and indicated power from the area enclosed by the pressure-volume indicator diagram. Instead, this work investigates the feasibility of achieving real-time calculation of volume and power via hardware implementation on Field Programmable Gate Arrays (FPGAs). Alternative hardware implementations were investigated using lookup tables, Taylor series methods or the CORDIC (CoOrdinate Rotation DIgital Computer) algorithm to compute the trigonometric operations in the crank angle to volume calculation, and the CORDIC algorithm was found to use the least amount of resources. Simulation of the hardware based implementation showed that the error in the volume and indicated power is less than 0.1%.

Accountable-eHealth systems : the next step forward for privacy

EHealth systems promise enviable benefits and capabilities for healthcare. But, the technologies that make these capabilities possible brings with them undesirable drawback such as information security related threats which need to be appropriately addressed. Lurking in these threats are patient privacy concerns. Fulfilling these privacy concerns have proven to be difficult since they often conflict with information requirements of care providers. It is important to achieve a proper balance between these requirements. We believe that information accountability can achieve this balance. In this paper we introduce accountable-eHealth systems. We will discuss how our designed protocols can successfully address the aforementioned requirement. We will also compare characteristics of AeH systems with Australia’s PCEHR system and identify similarities and highlight the differences and the impact those differences would have to the eHealth domain.

The promotion of sustainability agenda for facilities management through developing knowledge capabilities

The growing awareness of sustainability issues around the world has put extensive pressure on the construction industry to improve its sustainable practice. Sustainability principles need to be applied to not just during design and construction phase but the entire life cycle of a construction project. Compared to sustainability endeavours on earlier development phases, the pace to implement sustainability agenda during the operation and maintenance phase has not been as fast during past practices of facilities management (FM). Literature study suggests that sustainable practices in FM activities can bring substantial benefits such as reducing energy consumptions and waste, while increasing productivity, financial return and standing in the community. It also suggests several barriers which inhibit the implementation of sustainability in FM practices, including the lack of knowledge, discrepancy between capability and skills, and unwillingness of the FM personnel and organizations to adapt to new routines in order to implement sustainability in their business. The capabilities of FM personnel and organizations were regarded as the key enablers in managing sustainability knowledge. In a sustainable development context, capabilities are vital to the fostering of competency in an organization to innovate in a more sustainable way and support the agenda in an organization. Additionally, research which focused on people’s capabilities and skills is still lagging behind the efforts to develop guidelines, technical manuals and knowledge portals. Therefore, it is beneficial to explore the issues of capabilities in dealing with the implementation of sustainable practices in FM. This paper introduces a research project which is aimed at establishing a knowledge capabilities framework for promoting sustainability measures in FM practices. It will explore and highlight challenges to integrate sustainability as well as the personnel and organizational capabilities that are vital in dealing with knowledge issues in implementing sustainability agenda in FM practices. The expected outcome of this research has the potential to further sustainability endeavours in FM practices, while providing a useful source of knowledge to the FM personnel and organizations.

Open design of high pressure ratio radial-inflow turbine for academic validation

Computational Fluid Dynamics (CFD) simulations are widely used in mechanical engineering. Although achieving a high level of confidence in numerical modelling is of crucial importance in the field of turbomachinery, verification and validation of CFD simulations are very tricky especially for complex flows encountered in radial turbines. Comprehensive studies of radial machines are available in the literature. Unfortunately, none of them include enough detailed geometric data to be properly reproduced and so cannot be considered for academic research and validation purposes. As a consequence, design improvements of such configurations are difficult. Moreover, it seems that well-developed analyses of radial turbines are used in commercial software but are not available in the open literature especially at high pressure ratios. It is the purpose of this paper to provide a fully open set of data to reproduce the exact geometry of the high pressure ratio single stage radial-inflow turbine used in the Sundstrand Power Systems T-100 Multipurpose Small Power Unit. First, preliminary one-dimensional meanline design and analysis are performed using the commercial software RITAL from Concepts-NREC in order to establish a complete reference test case available for turbomachinery code validation. The proposed design of the existing turbine is then carefully and successfully checked against the geometrical and experimental data partially published in the literature. Then, three-dimensional Reynolds-Averaged Navier-Stokes simulations are conducted by means of the Axcent-PushButton CFDR CFD software. The effect of the tip clearance gap is investigated in detail for a wide range of operating conditions. The results confirm that the 3D geometry is correctly reproduced. It also reveals that the turbine is shocked while designed to give a high-subsonic flow and highlight the importance of the diffuser.

Teamworking for security : the collaborative approach

Collaborative methods are promising tools for solving complex security tasks. In this context, the authors present the security overlay framework CIMD (Collaborative Intrusion and Malware Detection), enabling participants to state objectives and interests for joint intrusion detection and find groups for the exchange of security-related data such as monitoring or detection results accordingly; to these groups the authors refer as detection groups. First, the authors present and discuss a tree-oriented taxonomy for the representation of nodes within the collaboration model. Second, they introduce and evaluate an algorithm for the formation of detection groups. After conducting a vulnerability analysis of the system, the authors demonstrate the validity of CIMD by examining two different scenarios inspired sociology where the collaboration is advantageous compared to the non-collaborative approach. They evaluate the benefit of CIMD by simulation in a novel packet-level simulation environment called NeSSi (Network Security Simulator) and give a probabilistic analysis for the scenarios.

Designing a new robust on-line secondary path modeling technique for feedforward active noise control systems

Several approaches have been introduced in the literature for active noise control (ANC) systems. Since the filtered-x least-mean-square (FxLMS) algorithm appears to be the best choice as a controller filter, researchers tend to improve performance of ANC systems by enhancing and modifying this algorithm. This paper proposes a new version of the FxLMS algorithm, as a first novelty. In many ANC applications, an on-line secondary path modeling method using white noise as a training signal is required to ensure convergence of the system. As a second novelty, this paper proposes a new approach for on-line secondary path modeling on the basis of a new variable-step-size (VSS) LMS algorithm in feed forward ANC systems. The proposed algorithm is designed so that the noise injection is stopped at the optimum point when the modeling accuracy is sufficient. In this approach, a sudden change in the secondary path during operation makes the algorithm reactivate injection of the white noise to re-adjust the secondary path estimate. Comparative simulation results shown in this paper indicate the effectiveness of the proposed approach in reducing both narrow-band and broad-band noise. In addition, the proposed ANC system is robust against sudden changes of the secondary path model.

Qualitative methods in information systems research

Qualitative research methods are widely accepted in Information Systems and multiple approaches have been successfully used in IS qualitative studies over the years. These approaches include narrative analysis, discourse analysis, grounded theory, case study, ethnography and phenomenological analysis. Guided by critical, interpretive and positivist epistemologies (Myers 1997), qualitative methods are continuously growing in importance in our research community. In this special issue, we adopt Van Maanen's (1979: 520) definition of qualitative research as an umbrella term to cover an “array of interpretive techniques that can describe, decode, translate, and otherwise come to terms with the meaning, not the frequency, of certain more or less naturally occurring phenomena in the social world”. In the call for papers, we stated that the aim of the special issue was to provide a forum within which we can present and debate the significant number of issues, results and questions arising from the pluralistic approach to qualitative research in Information Systems. We recognise both the potential and the challenges that qualitative approaches offers for accessing the different layers and dimensions of a complex and constructed social reality (Orlikowski, 1993). The special issue is also a response to the need to showcase the current state of the art in IS qualitative research and highlight advances and issues encountered in the process of continuous learning that includes questions about its ontology, epistemological tenets, theoretical contributions and practical applications.