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.

M-ary trees for combinatorial asset management decision problems

A novel m-ary tree based approach is presented to solve asset management decisions which are combinatorial in nature. The approach introduces a new dynamic constraint based control mechanism which is capable of excluding infeasible solutions from the solution space. The approach also provides a solution to the challenges with ordering of assets decisions.

Verifiability in Markets for Credence Goods

Theory predicts that efficiency prevails on credence goods markets if customers are able to verify which quality they receive from an expert seller. In a series of experiments with endogenous prices we observe that variability fails to result in efficient provision behavior and leads to very similar results as a setting without variability. Some sellers always provide appropriate treatment even if own money maximization calls for over- or undertreatment. Overall our endogenous price-results suggests that both inequality aversion and a taste for efficiency play an important role for experts provision behavior. We contrast the implications of those two motivations theoretically and discriminate between them empirically using a �xed-price design. We then classify experimental experts according to their provision behavior.

Delegation framework for federated systems

It is not uncommon for enterprises today to be faced with the demand to integrate and incor- porate many different and possibly heterogeneous systems which are generally independently designed and developed, to allow seamless access. In effect, the integration of these systems results in one large whole system that must be able, at the same time, to maintain the local autonomy and to continue working as an independent entity. This problem has introduced a new distributed architecture called federated systems. The most challenging issue in federated systems is to find answers for the question of how to efficiently cooperate while preserving their autonomous characteristic, especially the security autonomy. This thesis intends to address this issue. The thesis reviews the evolution of the concept of federated systems and discusses the organisational characteristics as well as remaining security issues with the existing approaches. The thesis examines how delegation can be used as means to achieve better security, especially authorisation while maintaining autonomy for the participating member of the federation. A delegation taxonomy is proposed as one of the main contributions. The major contribution of this thesis is to study and design a mechanism to support dele- gation within and between multiple security domains with constraint management capability. A novel delegation framework is proposed including two modules: Delegation Constraint Man- agement module and Policy Management module. The first module is designed to effectively create, track and manage delegation constraints, especially for delegation processes which require re-delegation (indirect delegation). The first module employs two algorithms to trace the root authority of a delegation constraint chain and to prevent the potential conflict when creating a delegation constraint chain if necessary. The first module is designed for conflict prevention not conflict resolution. The second module is designed to support the first module via the policy comparison capability. The major function of this module is to provide the delegation framework the capability to compare policies and constraints (written under the format of a policy). The module is an extension of Lin et al.'s work on policy filtering and policy analysis. Throughout the thesis, some case studies are used as examples to illustrate the discussed concepts. These two modules are designed to capture one of the most important aspects of the delegation process: the relationships between the delegation transactions and the involved constraints, which are not very well addressed by the existing approaches. This contribution is significant because the relationships provide information to keep track and en- force the involved delegation constraints and, therefore, play a vital role in maintaining and enforcing security for transactions across multiple security domains.

Behavioural evidence for social preferences

A central topic in economics is the existence of social preferences. Behavioural economics in general has approached the issue from several angles. Controlled experimental settings, surveys, and field experiments are able to show that in a number of economic environments, people usually care about immaterial things such as fairness or equity of allocations. Findings from experimental economics specifically have lead to large increase in theories addressing social preferences. Most (pro)social phenomena are well understood in the experimental settings but very difficult to observe 'in the wild'. One criticism in this regard is that many findings are bound by the artificial environment of the computer lab or survey method used. A further criticism is that the traditional methods also fail to directly attribute the observed behaviour to the mental constructs that are expected to stand behind them. This thesis will first examine the usefulness of sports data to test social preference models in a field environment, thus overcoming limitations of the lab with regards to applicability to other - non-artificial - environments. The second major contribution of this research establishes a new neuroscientific tool - the measurement of the heart rate variability - to observe participants' emotional reactions in a traditional experimental setup.

Three carrier ambiguity resolution : distance-independent performance demonstrated using semi-generated triple frequency GPS signals

In spite of significant research in the development of efficient algorithms for three carrier ambiguity resolution, full performance potential of the additional frequency signals cannot be demonstrated effectively without actual triple frequency data. In addition, all the proposed algorithms showed their difficulties in reliable resolution of the medium-lane and narrow-lane ambiguities in different long-range scenarios. In this contribution, we will investigate the effects of various distance-dependent biases, identifying the tropospheric delay to be the key limitation for long-range three carrier ambiguity resolution. In order to achieve reliable ambiguity resolution in regional networks with the inter-station distances of hundreds of kilometers, a new geometry-free and ionosphere-free model is proposed to fix the integer ambiguities of the medium-lane or narrow-lane observables over just several minutes without distance constraint. Finally, the semi-simulation method is introduced to generate the third frequency signals from dual-frequency GPS data and experimentally demonstrate the research findings of this paper.

Neoproterozoic glacial deposits of the Kimberly Region and northwestern Northern Territory, Australia

Neoproterozoic glacigenic formations are preserved in the Kimberley region and northwestern Northern Territory of northern Australia. They are distributed in the west Kimberley adjacent to the northern margins of the King Leopold Orogen, the Mt Ramsay area at the junction of the King Leopold and Halls Creek Orogens, and the east Kimberley, adjacent to the eastern margin of the Halls Creek Orogen. Small outlier glacigenic deposits are preserved in the Litchfield Province, Northern Territory (Uniya Formation) and Georgina Basin, western Queensland (Little Burke Formation). Glacigenic strata comprise diamictite, conglomerate, sandstone and pebbly mudstone and characterize the Walsh, Landrigan and Fargoo/Moonlight Valley formations. Thin units of laminated dolomite sit conformably at the top of the Walsh, Landrigan and Moonlight Valley formations. Glacigenic units are also interbedded with the carbonate platform deposits of the Egan Formation and Boonall Dolomite. δ13C data are available for all carbonate units. There is no direct chronological constraint on these successions. Dispute over regional correlation of the Neoproterozoic succession has been largely resolved through biostratigraphic, chemostratigraphic and lithostratigraphic analysis. However, palaeomagnetic results from the Walsh Formation are inconsistent with sedimentologically based correlations. Two stratigraphically defined glaciations are preserved in northwestern Australia: the ‘Landrigan Glaciation’, characterized by southwest-directed continental ice-sheet movement and correlated with late Cryogenian glaciation elsewhere in Australia and the world; and, the ‘Egan Glaciation’, a more localized glaciation of the Ediacaran Period. Future research focus should include chronology, palaeomagnetic constraint and tectonostratigraphic controls on deposition.

Improved facial expression recognition via uni-hyperplane classification

Large margin learning approaches, such as support vector machines (SVM), have been successfully applied to numerous classification tasks, especially for automatic facial expression recognition. The risk of such approaches however, is their sensitivity to large margin losses due to the influence from noisy training examples and outliers which is a common problem in the area of affective computing (i.e., manual coding at the frame level is tedious so coarse labels are normally assigned). In this paper, we leverage the relaxation of the parallel-hyperplanes constraint and propose the use of modified correlation filters (MCF). The MCF is similar in spirit to SVMs and correlation filters, but with the key difference of optimizing only a single hyperplane. We demonstrate the superiority of MCF over current techniques on a battery of experiments.

Design of fixed order robust power oscillation damper for TCSC

This paper illustrates robust fixed order power oscillation damper design for mitigating power systems oscillations. From implementation and tuning point of view, such low and fixed structure is common practice for most practical applications, including power systems. However, conventional techniques of optimal and robust control theory cannot handle the constraint of fixed-order as it is, in general, impossible to ensure a target closed-loop transfer function by a controller of any given order. This paper deals with the problem of synthesizing or designing a feedback controller of dynamic order for a linear time-invariant plant for a fixed plant, as well as for an uncertain family of plants containing parameter uncertainty, so that stability, robust stability and robust performance are attained. The desired closed-loop specifications considered here are given in terms of a target performance vector representing a desired closed-loop design. The performance of the designed controller is validated through non-linear simulations for a range of contingencies.

Accuracy of rate estimation using relaxed-clock models with a critical focus on the early metazoan radiation

In recent years, a number of phylogenetic methods have been developed for estimating molecular rates and divergence dates under models that relax the molecular clock constraint by allowing rate change throughout the tree. These methods are being used with increasing frequency, but there have been few studies into their accuracy. We tested the accuracy of several relaxed-clock methods (penalized likelihood and Bayesian inference using various models of rate change) using nucleotide sequences simulated on a nine-taxon tree. When the sequences evolved with a constant rate, the methods were able to infer rates accurately, but estimates were more precise when a molecular clock was assumed. When the sequences evolved under a model of autocorrelated rate change, rates were accurately estimated using penalized likelihood and by Bayesian inference using lognormal and exponential models of rate change, while other models did not perform as well. When the sequences evolved under a model of uncorrelated rate change, only Bayesian inference using an exponential rate model performed well. Collectively, the results provide a strong recommendation for using the exponential model of rate change if a conservative approach to divergence time estimation is required. A case study is presented in which we use a simulation-based approach to examine the hypothesis of elevated rates in the Cambrian period, and it is found that these high rate estimates might be an artifact of the rate estimation method. If this bias is present, then the ages of metazoan divergences would be systematically underestimated. The results of this study have implications for studies of molecular rates and divergence dates.

Tinamous and moa flock together : mitochondrial genome sequence analysis reveals independent losses of flight among ratites

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou–moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou–moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous–Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon. [Base composition; flightless; Gondwana; mitochondrial genome; Palaeognathae; phylogeny; ratites.]

A new approach to automatically producing schedules for cane railways

The scheduling of locomotive movements on cane railways has proven to be a very complex task. Various optimisation methods have been used over the years to try and produce an optimised schedule that eliminates or minimises bin supply delays to harvesters and the factory, while minimising the number of locomotives, locomotive shifts and cane bins, and also the cane age. This paper reports on a new attempt to develop an automatic scheduler using a mathematical model solved using mixed integer programming and constraint programming approaches and blocking parallel job shop scheduling fundamentals. The model solution has been explored using conventional constraint programming search techniques and found to produce a reasonable schedule for small-scale problems with up to nine harvesters. While more effort is required to complete the development of the full model with metaheuristic search techniques, the work completed to date gives confidence that the metaheuristic techniques will provide near optimal solutions in reasonable time.

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.