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.

XML data clustering: An overview

In the last few years we have observed a proliferation of approaches for clustering XML docu- ments and schemas based on their structure and content. The presence of such a huge amount of approaches is due to the different applications requiring the XML data to be clustered. These applications need data in the form of similar contents, tags, paths, structures and semantics. In this paper, we first outline the application contexts in which clustering is useful, then we survey approaches so far proposed relying on the abstract representation of data (instances or schema), on the identified similarity measure, and on the clustering algorithm. This presentation leads to draw a taxonomy in which the current approaches can be classified and compared. We aim at introducing an integrated view that is useful when comparing XML data clustering approaches, when developing a new clustering algorithm, and when implementing an XML clustering compo- nent. Finally, the paper moves into the description of future trends and research issues that still need to be faced.

Enriching XML documents clustering by using concise structure and content

With the growing number of XML documents on theWeb it becomes essential to effectively organise these XML documents in order to retrieve useful information from them. A possible solution is to apply clustering on the XML documents to discover knowledge that promotes effective data management, information retrieval and query processing. However, many issues arise in discovering knowledge from these types of semi-structured documents due to their heterogeneity and structural irregularity. Most of the existing research on clustering techniques focuses only on one feature of the XML documents, this being either their structure or their content due to scalability and complexity problems. The knowledge gained in the form of clusters based on the structure or the content is not suitable for reallife datasets. It therefore becomes essential to include both the structure and content of XML documents in order to improve the accuracy and meaning of the clustering solution. However, the inclusion of both these kinds of information in the clustering process results in a huge overhead for the underlying clustering algorithm because of the high dimensionality of the data. The overall objective of this thesis is to address these issues by: (1) proposing methods to utilise frequent pattern mining techniques to reduce the dimension; (2) developing models to effectively combine the structure and content of XML documents; and (3) utilising the proposed models in clustering. This research first determines the structural similarity in the form of frequent subtrees and then uses these frequent subtrees to represent the constrained content of the XML documents in order to determine the content similarity. A clustering framework with two types of models, implicit and explicit, is developed. The implicit model uses a Vector Space Model (VSM) to combine the structure and the content information. The explicit model uses a higher order model, namely a 3- order Tensor Space Model (TSM), to explicitly combine the structure and the content information. This thesis also proposes a novel incremental technique to decompose largesized tensor models to utilise the decomposed solution for clustering the XML documents. The proposed framework and its components were extensively evaluated on several real-life datasets exhibiting extreme characteristics to understand the usefulness of the proposed framework in real-life situations. Additionally, this research evaluates the outcome of the clustering process on the collection selection problem in the information retrieval on the Wikipedia dataset. The experimental results demonstrate that the proposed frequent pattern mining and clustering methods outperform the related state-of-the-art approaches. In particular, the proposed framework of utilising frequent structures for constraining the content shows an improvement in accuracy over content-only and structure-only clustering results. The scalability evaluation experiments conducted on large scaled datasets clearly show the strengths of the proposed methods over state-of-the-art methods. In particular, this thesis work contributes to effectively combining the structure and the content of XML documents for clustering, in order to improve the accuracy of the clustering solution. In addition, it also contributes by addressing the research gaps in frequent pattern mining to generate efficient and concise frequent subtrees with various node relationships that could be used in clustering.

Reduced variance in monozygous twins for multiple MR parameters : implications for disease studies and the genetic basis of brain structure

Twin studies offer the opportunity to determine the relative contribution of genes versus environment in traits of interest. Here, we investigate the extent to which variance in brain structure is reduced in monozygous twins with identical genetic make-up. We investigate whether using twins as compared to a control population reduces variability in a number of common magnetic resonance (MR) structural measures, and we investigate the location of areas under major genetic influences. This is fundamental to understanding the benefit of using twins in studies where structure is the phenotype of interest. Twenty-three pairs of healthy MZ twins were compared to matched control pairs. Volume, T2 and diffusion MR imaging were performed as well as spectroscopy (MRS). Images were compared using (i) global measures of standard deviation and effect size, (ii) voxel-based analysis of similarity and (iii) intra-pair correlation. Global measures indicated a consistent increase in structural similarity in twins. The voxel-based and correlation analyses indicated a widespread pattern of increased similarity in twin pairs, particularly in frontal and temporal regions. The areas of increased similarity were most widespread for the diffusion trace and least widespread for T2. MRS showed consistent reduction in metabolite variation that was significant in the temporal lobe N-acetylaspartate (NAA). This study has shown the distribution and magnitude of reduced variability in brain volume, diffusion, T2 and metabolites in twins. The data suggest that evaluation of twins discordant for disease is indeed a valid way to attribute genetic or environmental influences to observed abnormalities in patients since evidence is provided for the underlying assumption of decreased variability in twins.

Internet/computer addiction among juveniles in Turkey

Internet and computer addiction has been a popular research area since the 90s. Studies on Internet and computer addiction have usually been conducted in the US, and the investigation of computer and Internet addiction at different countries is an interesting area of research. This study investigates computer and Internet addiction among teenagers and Internet cafe visitors in Turkey. We applied a survey to 983 visitors in the Internet cafes. The results show that the Internet cafe visitors are usually teenagers, mostly middle and high-school students and usually are busy with computer and Internet applications like chat, e-mail, browsing and games. The teenagers come to the Internet cafe to spend time with friends and the computers. In addition, about 30% of cafe visitors admit to having an Internet addiction, and about 20% specifically mention the problems that they are having with the Internet. It is rather alarming to consider the types of activities that the teenagers are performing in an Internet cafe, their reasons for being there, the percentage of self-awareness about Internet addiction, and the lack of control of applications in the cafe.

Using incomplete online metric maps for topological exploration with the Gap Navigation Tree

This paper presents a general, global approach to the problem of robot exploration, utilizing a topological data structure to guide an underlying Simultaneous Localization and Mapping (SLAM) process. A Gap Navigation Tree (GNT) is used to motivate global target selection and occluded regions of the environment (called “gaps”) are tracked probabilistically. The process of map construction and the motion of the vehicle alters both the shape and location of these regions. The use of online mapping is shown to reduce the difficulties in implementing the GNT.

A phenomenological model for the structure-composition relationship of the high Tc cuprates based on simple chemical principles

A simple phenomenological model for the relationship between structure and composition of the high Tc cuprates is presented. The model is based on two simple crystal chemistry principles: unit cell doping and charge balance within unit cells. These principles are inspired by key experimental observations of how the materials accommodate large deviations from stoichiometry. Consistent explanations for significant HTSC properties can be explained without any additional assumptions while retaining valuable insight for geometric interpretation. Combining these two chemical principles with a review of Crystal Field Theory (CFT) or Ligand Field Theory (LFT), it becomes clear that the two oxidation states in the conduction planes (typically d8 and d9) belong to the most strongly divergent d-levels as a function of deformation from regular octahedral coordination. This observation offers a link to a range of coupling effects relating vibrations and spin waves through application of Hund’s rules. An indication of this model’s capacity to predict physical properties for HTSC is provided and will be elaborated in subsequent publications. Simple criteria for the relationship between structure and composition in HTSC systems may guide chemical syntheses within new material systems.

Molecular structure of the phosphate mineral brazilianite NaAl3(PO4)2(OH)4 : a semi precious jewel

The phosphate mineral brazilianite NaAl3(PO4)2(OH)4 is a semi precious jewel. There are almost no minerals apart from brazilianite which are used in jewellery. Vibrational spectroscopy was used to characterize the mol. structure of brazilianite. Brazilianite is composed of chains of edge-sharing Al-O octahedra linked by P-O tetrahedra, with Na located in cavities of the framework. An intense sharp Raman band at 1019 cm-1 is attributed to the PO43- sym. stretching mode. Raman bands at 973 and 988 cm-1 are assigned to the stretching vibrations of the HOPO33- units. The IR spectra compliment the Raman spectra but show greater complexity. Multiple Raman bands are obsd. in the PO43- and HOPO33- bending region. This observation implies that both phosphate and hydrogen phosphate units are involved in the structure. Raman OH stretching vibrations are found at 3249, 3417 and 3472 cm-1. These peaks show that the OH units are not equiv. in the brazilianite structure. Vibrational spectroscopy is useful for increasing the knowledge of the mol. structure of brazilianite.

Foundations for giving : why and how Australians structure their philanthropy

Philanthropic foundations in Australia have traditionally been labelled ‘icebergs’. Much of what they do and who they are is not apparent on the surface. Many are unknown and apart from an occasional biography, almost all are sparsely documented in terms of the very personal decisions behind establishing them. Practically and academically, scant data exist on the decision journeys people make into formalised philanthropy. This study seeks to fill that gap. It is believed to be the largest such study of foundation decision-making ever undertaken in this country. It is the latest in a series of ACPNS research into types of considered (versus spontaneous) giving in Australia. This research has been supported by the Perpetual Foundation, the EF and SL Gluyas Trust and the Edward Corbould Charitable Trust under the management of Perpetual Trustee Company Ltd.

A superdirective 3-element array for adaptive beamforming

A small array composed of three monopole elements with very small element spacing on the order of λ/6 to λ/20 is considered for application in adaptive beamforming. The properties of this 3-port array are governed by strong mutual coupling. It is shown that for signal-to-noise maximization, it is not sufficient to adjust the weights to compensate for the effects of mutual coupling. The necessity for a RF-decoupling network (RF-DN) and its simple realization are shown. The array with closely spaced elements together with the RF-DN represents a superdirective antenna with a directivity of more than 10 dBi. It is shown that the required fractional frequency bandwidth and the available unloaded Q of the antenna and RF-DN structure determine the lower limit for the element spacing.

Performance enhancement of smart antennas with reduced element spacing

This paper addresses the problem of degradations in adaptive digital beam-forming (DBF) systems caused by mutual coupling between array elements. The focus is on compact arrays with reduced element spacing and, hence, strongly coupled elements. Deviations in the radiation patterns of coupled and (theoretically) uncoupled elements can be compensated for by weight-adjustments in DBF, but SNR degradation due to impedance mismatches cannot be compensated for via signal processing techniques. It is shown that this problem can be overcome via the implementation of a RF-decoupling-network. SNR enhancement is achieved at the cost of a reduced frequency bandwidth and an increased sensitivity to dissipative losses in the antenna and matching network structure.

Automated cross-lingual link discovery in Wikipedia

At NTCIR-9, we participated in the cross-lingual link discovery (Crosslink) task. In this paper we describe our approaches to discovering Chinese, Japanese, and Korean (CJK) cross-lingual links for English documents in Wikipedia. Our experimental results show that a link mining approach that mines the existing link structure for anchor probabilities and relies on the “translation” using cross-lingual document name triangulation performs very well. The evaluation shows encouraging results for our system.

The market structure and characteristics of electronic games

A multi-billion dollar industry, electronic games have been experiencing strong and rapid growth in recent times. The world of games is not only exciting due to the magnificent growth of the industry however, but due to a host of other factors. This chapter explores electronic games, providing an analysis of the industry, key motivators for game play, the game medium and academic research concerning the effects of play. It also reviews the emerging relationship games share with sport, recognizing that they can replicate sports, facilitate sports participation and be played as a sport. These are complex relationships that have not yet been comprehensively studied. The current chapter serves to draw academic attention to the area and presents ideas for future research.

Fabrication of macro-mesoporous zirconia-alumina materials with a 1D hierarchical structure

A series of one dimensional (1D) zirconia/alumina nanocomposites were prepared by the deposition of zirconium species onto the 3D framework of boehmite nanofibres formed by dispersing boehmite nanofibres into butanol solution. The materials were calcined at 773K and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscope (TEM), N2 adsorption/desorption, infrared emission spectroscopy (IES). The results demonstrated that when the molar percentage X=100*Zr/(Al+Zr) was > 30 %, extremely long ZrO2/Al2O3 composite nanorods with evenly distributed ZrO2 nanocrystals on the surface were formed. The stacking of such nanorods gave rise to a new kind of macroporous material without the use of any organic space filler\template or other specific technologies. The mechanism for the formation of long ZrO2/Al2O3 composite nanorods was proposed in this work.