Prediction of transcriptional regulatory interactions in bacteria : a comparative genomics approach

Exponential growth of genomic data in the last two decades has made manual analyses impractical for all but trial studies. As genomic analyses have become more sophisticated, and move toward comparisons across large datasets, computational approaches have become essential. One of the most important biological questions is to understand the mechanisms underlying gene regulation. Genetic regulation is commonly investigated and modelled through the use of transcriptional regulatory network (TRN) structures. These model the regulatory interactions between two key components: transcription factors (TFs) and the target genes (TGs) they regulate. Transcriptional regulatory networks have proven to be invaluable scientific tools in Bioinformatics. When used in conjunction with comparative genomics, they have provided substantial insights into the evolution of regulatory interactions. Current approaches to regulatory network inference, however, omit two additional key entities: promoters and transcription factor binding sites (TFBSs). In this study, we attempted to explore the relationships among these regulatory components in bacteria. Our primary goal was to identify relationships that can assist in reducing the high false positive rates associated with transcription factor binding site predictions and thereupon enhance the reliability of the inferred transcription regulatory networks. In our preliminary exploration of relationships between the key regulatory components in Escherichia coli transcription, we discovered a number of potentially useful features. The combination of location score and sequence dissimilarity scores increased de novo binding site prediction accuracy by 13.6%. Another important observation made was with regards to the relationship between transcription factors grouped by their regulatory role and corresponding promoter strength. Our study of E.coli ��70 promoters, found support at the 0.1 significance level for our hypothesis | that weak promoters are preferentially associated with activator binding sites to enhance gene expression, whilst strong promoters have more repressor binding sites to repress or inhibit gene transcription. Although the observations were specific to �70, they nevertheless strongly encourage additional investigations when more experimentally confirmed data are available. In our preliminary exploration of relationships between the key regulatory components in E.coli transcription, we discovered a number of potentially useful features { some of which proved successful in reducing the number of false positives when applied to re-evaluate binding site predictions. Of chief interest was the relationship observed between promoter strength and TFs with respect to their regulatory role. Based on the common assumption, where promoter homology positively correlates with transcription rate, we hypothesised that weak promoters would have more transcription factors that enhance gene expression, whilst strong promoters would have more repressor binding sites. The t-tests assessed for E.coli �70 promoters returned a p-value of 0.072, which at 0.1 significance level suggested support for our (alternative) hypothesis; albeit this trend may only be present for promoters where corresponding TFBSs are either all repressors or all activators. Nevertheless, such suggestive results strongly encourage additional investigations when more experimentally confirmed data will become available. Much of the remainder of the thesis concerns a machine learning study of binding site prediction, using the SVM and kernel methods, principally the spectrum kernel. Spectrum kernels have been successfully applied in previous studies of protein classification [91, 92], as well as the related problem of promoter predictions [59], and we have here successfully applied the technique to refining TFBS predictions. The advantages provided by the SVM classifier were best seen in `moderately'-conserved transcription factor binding sites as represented by our E.coli CRP case study. Inclusion of additional position feature attributes further increased accuracy by 9.1% but more notable was the considerable decrease in false positive rate from 0.8 to 0.5 while retaining 0.9 sensitivity. Improved prediction of transcription factor binding sites is in turn extremely valuable in improving inference of regulatory relationships, a problem notoriously prone to false positive predictions. Here, the number of false regulatory interactions inferred using the conventional two-component model was substantially reduced when we integrated de novo transcription factor binding site predictions as an additional criterion for acceptance in a case study of inference in the Fur regulon. This initial work was extended to a comparative study of the iron regulatory system across 20 Yersinia strains. This work revealed interesting, strain-specific difierences, especially between pathogenic and non-pathogenic strains. Such difierences were made clear through interactive visualisations using the TRNDifi software developed as part of this work, and would have remained undetected using conventional methods. This approach led to the nomination of the Yfe iron-uptake system as a candidate for further wet-lab experimentation due to its potential active functionality in non-pathogens and its known participation in full virulence of the bubonic plague strain. Building on this work, we introduced novel structures we have labelled as `regulatory trees', inspired by the phylogenetic tree concept. Instead of using gene or protein sequence similarity, the regulatory trees were constructed based on the number of similar regulatory interactions. While the common phylogentic trees convey information regarding changes in gene repertoire, which we might regard being analogous to `hardware', the regulatory tree informs us of the changes in regulatory circuitry, in some respects analogous to `software'. In this context, we explored the `pan-regulatory network' for the Fur system, the entire set of regulatory interactions found for the Fur transcription factor across a group of genomes. In the pan-regulatory network, emphasis is placed on how the regulatory network for each target genome is inferred from multiple sources instead of a single source, as is the common approach. The benefit of using multiple reference networks, is a more comprehensive survey of the relationships, and increased confidence in the regulatory interactions predicted. In the present study, we distinguish between relationships found across the full set of genomes as the `core-regulatory-set', and interactions found only in a subset of genomes explored as the `sub-regulatory-set'. We found nine Fur target gene clusters present across the four genomes studied, this core set potentially identifying basic regulatory processes essential for survival. Species level difierences are seen at the sub-regulatory-set level; for example the known virulence factors, YbtA and PchR were found in Y.pestis and P.aerguinosa respectively, but were not present in both E.coli and B.subtilis. Such factors and the iron-uptake systems they regulate, are ideal candidates for wet-lab investigation to determine whether or not they are pathogenic specific. In this study, we employed a broad range of approaches to address our goals and assessed these methods using the Fur regulon as our initial case study. We identified a set of promising feature attributes; demonstrated their success in increasing transcription factor binding site prediction specificity while retaining sensitivity, and showed the importance of binding site predictions in enhancing the reliability of regulatory interaction inferences. Most importantly, these outcomes led to the introduction of a range of visualisations and techniques, which are applicable across the entire bacterial spectrum and can be utilised in studies beyond the understanding of transcriptional regulatory networks.

Working with actors in open disclosure simulations

A key strategy in facilitating learning in Open Disclosure training is the use of hypothetical, interactive scenarios called ‘simulations’. According to Clapper (2010), the ‘advantages of using simulation are numerous and include the ability to help learners make meaning of complex tasks, while also developing critical thinking and cultural skills’. Simulation, in turn, functions largely through improvisation and role-play, in which participants ‘act out’ particular roles and characters according to a given scenario, without recourse to a script. To maximise efficacy in the Open Disclosure training context, role-play requires the specialist skills of professionally trained actors. Core capacities that professional actors bring to the training process include (among others) believability, an observable and teachable skill which underpins the western traditions of actor training; and flexibility, which pertains to the actor’s ability to vary performance strategies according to the changing dynamics of the learning situation. The Patient Safety and Quality Improvement Service of Queensland Health utilises professional actors as a key component of their Open Disclosure Training Program. In engaging actors in this work, it is essential that Facilitators of Open Disclosure training have a solid understanding of the acting process: what acting is; how actors work to a brief; how they improvise; and how they sustainably manage a wide range of emotional states. In the simulation context, the highly skilled actor can optimise learning outcomes by adopting or enacting – in collaboration with the Facilitator - a pedagogical function.

Micromarrows—Three-dimensional coculture of hematopoietic stem cells and mesenchymal stromal cells

Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates—which we term “micromarrows.” Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.

A duty of care

Mass production of PhD training compromises graduate quality. As PhD quality becomes more stratified, industry will increasingly turn to quality-branded institutions and programs when distinguishing among job candidates.

Between formal and informal : the shakeout in China’s online video industry

While a rich body of literature in television and film studies and media policy studies has tended to focus on the media activities in the formal sector, we know much less about informal media activities, its influence on state policies, as well as the dynamics between the formal and the informal sectors. This article examines these issues with reference to a particularly revealing period following a large-scale government crackdown on peer-to-peer video sharing sites in China in 2008. By analyzing the aim and consequences of the state action, I point to the counter-productive effect in terms of cultural loss and the resurgence of offline piracy; and show the positive impact on forcing the informal into the formal sector, and pressuring the formal to innovate. Meanwhile, an increasing rapprochement between professional and user-created content leads to a new relationship between formal and informal sectors. This case demonstrates the importance of considering the dynamics between the two sectors. It also offers compelling evidence of the role of the informal sector in engendering state action, which in turn impacted on the co-evolution of the formal and the informal sectors.

Bear With Me

This project investigates musicalisation and intermediality in the writing and devising of composed theatre. Its research question asks “How does the narrative of a musical play differ when it emerges from a setlist of original songs?”, the aim being to create performance event that is neither music nor theatre. This involves composition of lyrics, music, action and spoken text, projected image: gathered in a script and presented in performance. Scholars such as Kulezic-Wilson(in Kendrick, L and Roesner, D 2011:34) outline the acoustic dimension to the ‘performative turn’ (Mungen, Ernst and Bentzweizer, 2012) as heralding “…a shift of emphasis on how meaning is created (and veiled) and how the spectrum of theatrical creation and reception is widened.” Rebstock and Roesner (2012) capture approaches similar to this, building on Lehmann’s work in the post-dramatic under the new term ‘composed theatre’. This practice led research draws influence from these new theoretical frames, pushing beyond ‘the musical’. Springing from a set of original songs in dialogue with performed narrative, Bear with Me is a 45 minute music driven work for children, involving projected image and participatory action. Bear with Me’s intermedial hybrid of theatrical, screen and concert presentations shows that a simple setlist of original songs can be the starting point for the structure of a complex intermedial performance. Bear with Me was programmed into the Queensland Performing Arts Centre’s Out of the Box Festival. It was first performed in the Tony Gould Gallery at the Queensland in June 2012. The season sold out. A masterclass on my playwriting methodology was presented at the Connecting The Dots Symposium which ran alongside the festival.

Application of Resilience concept for enhanced management of water supply systems

This paper presents an approach to developing indicators for expressing resilience of a generic water supply system. The system is contextualised as a meta-system consisting of three subsystems to represent the water catchment and reservoir, treatment plant and the distribution system supplying the end-users. The level of final service delivery to end-users is considered as a surrogate measure of systemic resilience. A set of modelled relationships are used to explore relationships between system components when placed under simulated stress. Conceptual system behaviour of specific types of simulated pressure is created for illustration of parameters for indicator development. The approach is based on the hypothesis that an in-depth knowledge of resilience would enable development of decision support system capability which in turn will contribute towards enhanced management of a water supply system. In contrast to conventional water supply system management approaches, a resilience approach facilitates improvement in system efficiency by emphasising awareness of points-of-intervention where system managers can adjust operational control measures across the meta-system (and within subsystems) rather than expansion of the system in entirety in the form of new infrastructure development.

The impact of penalty increases on speeding behaviour in Queensland and a characterisation of speeding offenders

This project examined the effects of speeding penalty changes that occurred in Queensland in 2003 on the behaviour of speeding offenders. These penalty changes included increasing the number of offence categories, and in turn narrowing the range of speeds associated with the offence categories; increasing the monetary fines for all offences, with the largest increases observed for high-range offences; and introducing automatic licence suspension and an eight demerit point penalty for the highest offence category. To explore the impact of the penalty changes, offence data collected for two cohorts of motorists in Queensland who were caught speeding prior to and subsequent to the penalty changes (N = 84,456) were compared. The first cohort consisted of individuals (operators of all vehicles including motorcycles) who committed a speeding offence in May 2001 (two years prior to the speeding penalty change); and individuals who committed a speeding offence in May 2003 (one month after the introduction of the penalty change). Four measures of recidivism were devised and used to assess the effects of the new penalties with regard to deterring the speeding behaviour of offenders. Additionally, the project investigated the relationship between speeding offences, other risky driving behaviours, crash involvement, and criminal behaviour.

WebPut : Efficient web-based data imputation

In this paper, we present WebPut, a prototype system that adopts a novel web-based approach to the data imputation problem. Towards this, Webput utilizes the available information in an incomplete database in conjunction with the data consistency principle. Moreover, WebPut extends effective Information Extraction (IE) methods for the purpose of formulating web search queries that are capable of effectively retrieving missing values with high accuracy. WebPut employs a confidence-based scheme that efficiently leverages our suite of data imputation queries to automatically select the most effective imputation query for each missing value. A greedy iterative algorithm is also proposed to schedule the imputation order of the different missing values in a database, and in turn the issuing of their corresponding imputation queries, for improving the accuracy and efficiency of WebPut. Experiments based on several real-world data collections demonstrate that WebPut outperforms existing approaches.

Curriculum, assessment teaching and learning for all

This chapter explores how the culture of classrooms and schools can acknowledge diversity and meet all learning needs. Classroom and school culture can and should enhance the belonging and learning of all students. Understanding of learning, curriculum, pedagogy and assessment influences the ways teachers consider their expectations of student achievement and participation in school. We revisit the theory of social constructionism to emphasise the development of shared and valued curriculum, which meets all learner needs. Decisions about what to teach, how to teach and assess, and what supports student needs are important considerations discussed in this chapter. Key messages drawn from the Australian and New Zealand curriculum reinforce the need to ensure education responds to the diversity of students in classrooms. A range of models of pedagogy that have influenced education in Australia and New Zealand are presented, with a particular focus on meeting the needs of students who have disabilities. In addition, the issues related to student and teacher identity, the importance of respectful partnerships that acknowledge family knowledge, and respectful collaboration are discussed. Belonging to a community of learners is made possible through teachers forming authentic relationships with students and their families. In turn, these relationships support teachers to understand how the students in their classrooms learn, and to know their students’ strengths and interests.

The role of knowledge intermediaries in developing firm learning capabilities

Regional innovation systems (RISs) literature has emphasized the critical role of interactive learning and knowledge exchange amongst firms and a variety of spatially connected innovation institutions as the foundation of regional innovation. Knowledge intermediaries have been analysed in terms of the technology-transaction services they provide firms and/or knowledge producers such as universities and therefore the role they play in facilitating interaction within the RIS. However, innovation also depends on the capability of the firm to learn. Some studies have suggested that intermediaries also play a role in that regard as participation in intermediary knowledge transfer programmes can contribute to the development of firm capabilities for problem-solving and learning. Our research is based on two case study intermediary programmes involving interviews with facilitators and participants. Our data show that knowledge intermediaries affect organizational learning capabilities by impacting on firms' network relationships, internal and external communication channels and internal learning processes which in turn affect the ability to interpret and use knowledge within the firm. This suggests that the role of knowledge intermediaries might be greater than facilitating interactions in the innovation system, as knowledge intermediation may affect the ability of firms to learn and absorb knowledge from their environment.

RcppArmadillo : accelerating R with high-performance C++ linear algebra

The R statistical environment and language has demonstrated particular strengths for interactive development of statistical algorithms, as well as data modelling and visualisation. Its current implementation has an interpreter at its core which may result in a performance penalty in comparison to directly executing user algorithms in the native machine code of the host CPU. In contrast, the C++ language has no built-in visualisation capabilities, handling of linear algebra or even basic statistical algorithms; however, user programs are converted to high-performance machine code, ahead of execution. A new method avoids possible speed penalties in R by using the Rcpp extension package in conjunction with the Armadillo C++ matrix library. In addition to the inherent performance advantages of compiled code, Armadillo provides an easy-to-use template-based meta-programming framework, allowing the automatic pooling of several linear algebra operations into one, which in turn can lead to further speedups. With the aid of Rcpp and Armadillo, conversion of linear algebra centered algorithms from R to C++ becomes straightforward. The algorithms retains the overall structure as well as readability, all while maintaining a bidirectional link with the host R environment. Empirical timing comparisons of R and C++ implementations of a Kalman filtering algorithm indicate a speedup of several orders of magnitude.

Come together : forging publics in Brisbane's Gallery of Modern Art

The Gallery of Modern Art (GoMA) in Brisbane, Australia’s third largest city, recently staged ‘21st Century: Art of the First Decade’. The gallery spaces were replete with a commissioned slide by Carsten Höller, an installation of Rivane Neuenschwande’s I Wish Your Wish (2003), a table of white Legos, a room of purple balloons and other participatory or interactive artworks designed to engage multiple publics and encourage audience participation in a variety of ways. Many of the featured projects used day-to-day experiences and offered new conceptions about art practice and what they can elicit in their public – raise awareness about local issues, help audiences imagine different ways of negotiating their environs or experi-ence a museum in a new way. At times, the bottom floor galleries resembled a theme park – adults and children playing with Legos and using Höller’s slide. This article examines the benefits and limitations of such artistic interventions by relating the GoMA exhibition to Brisbane City Council’s campaign of ‘Together Brisbane’ (featuring images of Neunenschwande’s ribbons); a response to the devastation brought to the city and its surrounds in January 2011. During the Brisbane floods, GoMA’s basement was damaged, the museum closed and upon reopening, visitor numbers soared. In this context, GoMA’s use of engaged art practice – always verging on the ephemeral and ‘fun’ – has been used to project a wider notion of a collective urban public. What questions does this raise, not only regarding the cultural politics around the social and participatory ‘turn’ in art practice, but its use to address a much wider urban public in a moment of crisis.

Cryo-electron tomography of Marburg Virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

Experience sharing

This conceptual paper continues the dialogue advancing the understanding of co-creation of value aligned to the actor-to-actor worldview (Vargo & Lusch, 2011), and builds on the work of Grönroos (2011) and Ramaswamy (2011). We introduce an effort-centric lens that interprets value as embedded in experience that is derived from individual efforts expended in co-creation processes. We conceptualise `experience sharing' as a value creation effort for the direct benefit of others, `value-in-experience' as an effort-based meaning of value creation, and `value initiators' as actors who perform `experience sharing'. In turn, we put forward an integrated framework to expand our understanding of `co-creative' interpretation of service provision.