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.

User behaviour modelling in a multi-dimensional environment for personalization and recommendation

Handling information overload online, from the user's point of view is a big challenge, especially when the number of websites is growing rapidly due to growth in e-commerce and other related activities. Personalization based on user needs is the key to solving the problem of information overload. Personalization methods help in identifying relevant information, which may be liked by a user. User profile and object profile are the important elements of a personalization system. When creating user and object profiles, most of the existing methods adopt two-dimensional similarity methods based on vector or matrix models in order to find inter-user and inter-object similarity. Moreover, for recommending similar objects to users, personalization systems use the users-users, items-items and users-items similarity measures. In most cases similarity measures such as Euclidian, Manhattan, cosine and many others based on vector or matrix methods are used to find the similarities. Web logs are high-dimensional datasets, consisting of multiple users, multiple searches with many attributes to each. Two-dimensional data analysis methods may often overlook latent relationships that may exist between users and items. In contrast to other studies, this thesis utilises tensors, the high-dimensional data models, to build user and object profiles and to find the inter-relationships between users-users and users-items. To create an improved personalized Web system, this thesis proposes to build three types of profiles: individual user, group users and object profiles utilising decomposition factors of tensor data models. A hybrid recommendation approach utilising group profiles (forming the basis of a collaborative filtering method) and object profiles (forming the basis of a content-based method) in conjunction with individual user profiles (forming the basis of a model based approach) is proposed for making effective recommendations. A tensor-based clustering method is proposed that utilises the outcomes of popular tensor decomposition techniques such as PARAFAC, Tucker and HOSVD to group similar instances. An individual user profile, showing the user's highest interest, is represented by the top dimension values, extracted from the component matrix obtained after tensor decomposition. A group profile, showing similar users and their highest interest, is built by clustering similar users based on tensor decomposed values. A group profile is represented by the top association rules (containing various unique object combinations) that are derived from the searches made by the users of the cluster. An object profile is created to represent similar objects clustered on the basis of their similarity of features. Depending on the category of a user (known, anonymous or frequent visitor to the website), any of the profiles or their combinations is used for making personalized recommendations. A ranking algorithm is also proposed that utilizes the personalized information to order and rank the recommendations. The proposed methodology is evaluated on data collected from a real life car website. Empirical analysis confirms the effectiveness of recommendations made by the proposed approach over other collaborative filtering and content-based recommendation approaches based on two-dimensional data analysis methods.

Leveraging the network information for evaluating answer quality in a collaborative question answering portal

Collaborative question answering (cQA) portals such as Yahoo! Answers allow users as askers or answer authors to communicate, and exchange information through the asking and answering of questions in the network. In their current set-up, answers to a question are arranged in chronological order. For effective information retrieval, it will be advantageous to have the users’ answers ranked according to their quality. This paper proposes a novel approach of evaluating and ranking the users’answers and recommending the top-n quality answers to information seekers. The proposed approach is based on a user-reputation method which assigns a score to an answer reflecting its answer author’s reputation level in the network. The proposed approach is evaluated on a dataset collected from a live cQA, namely, Yahoo! Answers. To compare the results obtained by the non-content-based user-reputation method, experiments were also conducted with several content-based methods that assign a score to an answer reflecting its content quality. Various combinations of non-content and content-based scores were also used in comparing results. Empirical analysis shows that the proposed method is able to rank the users’ answers and recommend the top-n answers with good accuracy. Results of the proposed method outperform the content-based methods, various combinations, and the results obtained by the popular link analysis method, HITS.

Really really

In “Really Really”, a series of words float through animated star constellations. While apparently drifting aimlessly, the words can be rearranged in numerous ways to recount roughly the same sappy avowal of long-distance love. The many combinations of words, and their predictable associations, call attention to the thresholds of verbal expression. When some sincere words are needed most it seems as if they’ve all been said before. And so, this declaration of love is both enabled and haunted by the tension between sentiment and formula. “Really Really” continues my interests in how meaning is constructed and circulated, and in particular, how common models of communication continue to frame and yet fall short of our personal experiences.

ArtConnexions series

The exhibition consists of a series of 9 large-scale cotton rag prints, printed from digital files, and a sound and picture animation on DVD composed of drawings, sound, analogue and digital photographs, and Super 8 footage. The exhibition represents the artist’s experience of Singapore during her residency. Source imagery was gathered from photographs taken at the Bukit Brown abandoned Chinese Cemetery in Singapore, and Australian native gardens in Parkville Melbourne. Historical sources include re-photographed Singapore 19th and early 20th century postcard images. The works use analogue, hand-drawn and digital imaging, still and animated, to explore the digital interface’s ability to combine mixed media. This practice stems from the digital imaging practice of layering, using various media editing software. The work is innovative in that it stretches the idea of the layer composition in a single image by setting each layer into motion using animation techniques. This creates a multitude of permutations and combinations as the two layers move in different rhythmic patterns. The work also represents an innovative collaboration between the photographic practitioner and a sound composer, Duncan King-Smith, who designed sound for the animation based on concepts of trance, repetition and abstraction. As part of the Art ConneXions program, the work travelled to numerous international venues including: Space 217 Singapore, RMIT Gallery Melbourne, National Museum Jakarta, Vietnam Fine Arts Museum Hanoi, and ifa (Institut fur Auslandsbeziehungen) Gallery in both Stuttgart and Berlin.

Everything

Everything (2008) is a looped 3 channel digital video (extracted from a 3D computer animation) that appropriates a range of media including photography, drawing, painting, and pre-shot video. The work departs from traditional time-based video which is generally based on a recording of an external event. Instead, “Everything” constructs an event and space more like a painting or drawing might. The works combines constructed events (including space, combinations of objects, and aesthetic relationship of forms) with pre-recorded video footage and pre-made paintings and drawings. The result is a montage of objects, images – both still and moving – and abstracted ‘painterly’ gestures. This technique creates a complex temporal displacement. 'Past' refers to pre-recorded media such as painting and photography, and 'future' refers to a possible virtual space not in the present, that these objects may occupy together. Through this simultaneity between the real and the virtual, the work comments on a disembodied sense of space and time, while also puncturing the virtual with a sense of materiality through the tactility of drawing and painting forms and processes. In so doing, te work challenges the perspectival Cartesian space synonymous with the virtual. In this work the disembodied wandering virtual eye is met with an uncanny combination of scenes, where scale and the relationships between objects are disrupted and changed. Everything is one of the first international examples of 3D animation technology being utilised in contemporary art. The work won the inaugural $75,000 Premier of Queensland National New Media Art Award and was subsequently acquired by the Queensland Art Gallery. The work has been exhibited and reviewed nationally and internationally.

Rare chicken rescue

Sound design for documentary is an under-researched field. The specific context of representation of emotional or mental states is particularly open to clichéd treatment. Such treatment in the media often ‘perpetuates inaccurate or negative assumptions about mental health issues in the wider community’ (Francis et al 2005: 11) by employing, for example, either jarring sound/music combinations to signify ‘madness’ or overtly saccharine music to educe sympathy. This project adopted a practice-based approach to discovering a considered aesthetic treatment designed to elicit a more empathetic audience response. A more discriminating engagement with the intentions of the film was cultivated by abandoning both the ‘representational naturalism’ and the ‘distilled, evocative realism’ of documentary sound design (Davies 2007: 18) in favour of a more lyrical or musical approach. To achieve this we manipulated perspective, tonal character and perceptions of space in the final mixing stage. The project was funded by the Film Australia National Interest Program, ABC TV and the Pacific Film and Television Commission. As a crucial contributor to the aesthetic of the project I was nominated in the funding application, and ultimately received an AFI Award for Best Sound in a Documentary in 2008. The film was honoured by The Film Critics Circle of Australia, The Slamdance Film Festival in Utah and The Sydney Film Festival. It has been favourably reviewed in national and international print media (The Age, Sydney Morning Herald, among others) as well as online film/culture zines and blogs.

Development of novel vaccine strategies to prevent genital tract chlamydial infections

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

A subject-specific model of human buttocks and thighs in a seated posture

Finite element analyses of the human body in seated postures requires digital models capable of providing accurate and precise prediction of the tissue-level response of the body in the seated posture. To achieve such models, the human anatomy must be represented with high fidelity. This information can readily be defined using medical imaging techniques such as Magnetic Resonance Imaging (MRI) or Computed Tomography (CT). Current practices for constructing digital human models, based on the magnetic resonance (MR) images, in a lying down (supine) posture have reduced the error in the geometric representation of human anatomy relative to reconstructions based on data from cadaveric studies. Nonetheless, the significant differences between seated and supine postures in segment orientation, soft-tissue deformation and soft tissue strain create a need for data obtained in postures more similar to the application posture. In this study, we present a novel method for creating digital human models based on seated MR data. An adult-male volunteer was scanned in a simulated driving posture using a FONAR 0.6T upright MRI scanner with a T1 scanning protocol. To compensate for unavoidable image distortion near the edges of the study, images of the same anatomical structures were obtained in transverse and sagittal planes. Combinations of transverse and sagittal images were used to reconstruct the major anatomical features from the buttocks through the knees, including bone, muscle and fat tissue perimeters, using Solidworks® software. For each MR image, B-splines were created as contours for the anatomical structures of interest, and LOFT commands were used to interpolate between the generated Bsplines. The reconstruction of the pelvis, from MR data, was enhanced by the use of a template model generated in previous work CT images. A non-rigid registration algorithm was used to fit the pelvis template into the MR data. Additionally, MR image processing was conducted to both the left and the right sides of the model due to the intended asymmetric posture of the volunteer during the MR measurements. The presented subject-specific, three-dimensional model of the buttocks and thighs will add value to optimisation cycles in automotive seat development when used in simulating human interaction with automotive seats.

Member moment capacities of mono-symmetric LiteSteel beam floor joists with web openings

Recently developed cold-formed LiteSteel beam (LSB) sections have found increasing popularity in residential, industrial and commercial buildings due to their light weight and cost-effectiveness. Another beneficial characteristic is that they allow torsionally rigid rectangular flanges to be combined with economical fabrication processes. Currently, there is significant interest in the use of LSB sections as flexural members in floor joist systems. When used as floor joists, these sections require openings in the web to provide access for inspection and other services. At present, however, there is no design method available that provides accurate predictions of the moment capacities of LSBs with web openings. This paper presents the results of an investigation of the buckling and ultimate strength behaviour of LSB flexural members with web openings. A detailed fine element analysis (FEA)-based parametric study was conducted with the aim of developing appropriate design rules and making recommendations for the safe design of LSB floor joists. The results include the required moment capacity curves for LSB sections with a range of web opening combinations and spans and the development of appropriate design rules for the prediction of the ultimate moment capacities of LSBs with web openings.

Restrung new chamber festival : an exploration of contemporary string practice

The Restrung New Chamber Festival was a practice-led research project which explored the intricacies of musical relationships. Specifically, it investigated the nature of contemporary string practice, with "New music" at its core. For the purposes of this project, "New music" will be defined in terms of representing a "global sonorous space" (Nancy 2007:12), which Hulse describes as "a spectacular comingling of styles and an unprecedented explosion of creative possibilities" (Hulse n.d.:2). Approaches to staging such an event are contextualised through a comparative analysis with relevant Australian and European festivals. The Restrung model derived inspiration from both art music and popular music festival models, in several aspects. One strategy was to engage audiences through combinations of musical, visual and spatial features. Another strategy was to encourage interaction by audiences with installations, workshops and forums. Restrung represents a new and distinctive model which presented art music within an immersive environment. This exegesis presents an evaluation framework which investigates the relationship between curatorial input and the experiential qualities of the festival. The context of an overview of trends in arts festival curation informs the discussion, as well as approaches to identifying new and receptive audiences. It is expected that the evaluation framework will provide a useful and practical guide for curators working in contemporary string practice, hybrid arts, experimental and cross-art form festival design.

Non-compositional concepts and quantum tests

Compositionality is a frequently made assumption in linguistics, and yet many human subjects reveal highly non-compositional word associations when confronted with novel concept combinations. This article will show how a non-compositional account of concept combinations can be supplied by modelling them as interacting quantum systems.

Project 2.7 Leveraging R&D Investment for the Australian built environment : Phase 2 Case Study Report Part 2 - Road Construction Safety

This report discusses findings of a case study into "Road Construction Safety" undertaken as a part of the retrospective analysis component of Sustainable Built Environment National Research Centre (SBEnrc) Project 2.7 Leveraging R&D investment for the Australian Built Environment. The Queensland Department of Transport and Main Roads (QTMR) has taken a leadership role in developing a safer working environment for road construction workers. In the past decades, a range of initiatives have been introduced to contribute to improved performance in this area. Several initiatives have been undertaken by QTMR as part of their overarching commitment to safety. Three such initiatives form the basis for this case study investigation, in order to better illustrate the nature of R&D investment and its impact on day-to-day operations and the supply chain. These are the development and implementation of: (i) the Mechanical Traffic Aid: (ii) the Thermal Imaging Camera; and (iii) the Trailer-based CCTV (camera). This case study should be read in conjunction with Part 1 of this suite of reports.

Strategic Management Practices in the Construction Industry : a Dynamic Capabilities View

This book is an empirical study of strategic management practices in the construction industry. It examines the dynamic capabilities paradigm within the context of the Indonesian construction industry. The characteristics of asset-capability combinations were found to be significant determinants of the competitive advantage of the Indonesian construction enterprises, and that such advantage sequentially contributes to organizational performance. In doing so, this study fills an important gap in the empirical literature and reinforces the dynamic capabilities framework’s recognition as a rigorous theory of strategic management. As the dynamic capabilities framework can work in the context of Indonesia, it suggests that the framework has potential applicability in other emerging and developing countries

Tests and Models of Non-compositional Concepts

The question of under what conditions conceptual representation is compositional remains debatable within cognitive science. This paper proposes a well developed mathematical apparatus for a probabilistic representation of concepts, drawing upon methods developed in quantum theory to propose a formal test that can determine whether a specific conceptual combination is compositional, or not. This test examines a joint probability distribution modeling the combination, asking whether or not it is factorizable. Empirical studies indicate that some combinations should be considered non-compositionally.