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.

Rolling in the aisles : a comparative study of male and female grocery shopper typologies

The purpose of this paper is to segment male and female grocery shoppers based on store and product attribute evaluations. A rich profile for each segment is developed. Gender comparisons are operationalised and these developed contemporary shopper typologies are further contrasted against earlier works. Data of 560 grocery shoppers was attained by a survey questionnaire. Factor analysis, cluster analysis and ANOVA were employed to develop specific segments of shoppers. Four distinct cohorts of male shoppers and three cohorts of female shoppers emerge from the data of eight constructs, measured by 46 items. One new shopper type, not found in earlier typology literature, emerged from this research. This shopper presented as a young, well educated, at the commencement of their career and family lifecycle, attracted by a strong value offer and willingness to share the family food shopping responsibilities. This research makes a contribution to segmentation literature and grocery retail practice in several ways. It presents the first retail typology of male supermarket shoppers, employing a cluster analysis technique. Comparisons between male and female grocery shopping typologies are accordingly facilitated. The research provides insights into the modern family food shopping behaviour of men; a channel in which men are now recognised as equal contributors. Research outcomes encourage supermarket retailers to implement targeted marketing and rationalized operational strategies that deliver on attributes of importance. Finally, this research provides the basis for further cross-cultural, cross-contextual comparative studies.

Comparative effects of posterior eye cup tissues from Myopic and Hyperopic chick eyes on cultured scleral fibroblasts

The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and -smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post hatch day 5 to 8) (n=6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9±2.6 µg and 8.4±1.1 µg, p=0.12; GAG 11.2±0.6 µg and 10.1±1.0 µg, p=0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2±1.7 µg versus 23.2±1.5 µg, p=0.21; GAG 28.1±1.7 µg versus. 28.7±1.2 µg, p=0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA, LIM 46.9±6.4 versus LIH 51.5±4.7 µg, p=0.31), whereas GAG content was higher for cells in co-culture with choroid from LIH eyes (GAG 32.5±0.7 µg versus 18.9±1.2 µg, F1,6=9.210, p=0.0002). In contrast, fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4±6.3 µg versus Empty basket: 46.03±1.0 µg; F1,6=69.99, p=0.0005 and LIH: 27.9±2.3 µg versus empty basket: 46.03±1.0 µg; p=0.0004). GAG content was higher with RPE from LIH eyes (LIH: 33.7±1.9 µg versus empty basket: 29.5±0.8 µg, F1,6=13.99, p=0.010) and lower with RPE from LIM eyes (LIM: 27.7±0.9 µg versus empty basket: 29.5±0.8 µg, p=0.021). GAG content of cells in co-culture with choroid from LIH eyes was higher compared to co-culture with choroid from LIM eyes (32.5±0.7 µg versus 18.9±1.2 µg respectively, F1,6=9.210, p=0.0002). In conclusion, these experiments provide evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.

The Growing Pains of Data Breach Notification Law

Mandatory data breach notification laws are a novel statutory solution in relation to organizational protections of personal information. They require organizations which have suffered a breach of security involving personal information to notif'y those persons whose information may have been affected. These laws originated in the state based legislatures of the United States during the last decade and have subsequently garnered worldwide legislative interest. Despite their perceived utility, mandatory data breach notification laws have several conceptual and practical concems that limit the scope of their applicability, particularly in relation to existing information privacy law regimes. We outline these concerns, and in doing so, we contend that while mandatory data breach notification laws have many useful facets, their utility as an 'add-on' to enhance the failings of current information privacy law frameworks should not necessarily be taken for granted.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

Comparative eye tracking of experts and novices in web single sign-on

Security indicators in web browsers alert users to the presence of a secure connection between their computer and a web server; many studies have shown that such indicators are largely ignored by users in general. In other areas of computer security, research has shown that technical expertise can decrease user susceptibility to attacks. In this work, we examine whether computer or security expertise affects use of web browser security indicators. Our study takes place in the context of web-based single sign-on, in which a user can use credentials from a single identity provider to login to many relying websites; single sign-on is a more complex, and hence more difficult, security task for users. In our study, we used eye trackers and surveyed participants to examine the cues individuals use and those they report using, respectively. Our results show that users with security expertise are more likely to self-report looking at security indicators, and eye-tracking data shows they have longer gaze duration at security indicators than those without security expertise. However, computer expertise alone is not correlated with recorded use of security indicators. In survey questions, neither experts nor novices demonstrate a good understanding of the security consequences of web-based single sign-on.

Impact fees and new housing cost: A comparative analysis of the empirical models

Developers and policy makers are consistently at odds over the debate as to whether impact fees increase house prices. This debate continues despite the extensive body of theoretical and empirical international literature that discusses the passing on to home buyers of impact fees, and the corresponding increase to housing prices. In attempting to quantify this impact, over a dozen empirical studies have been carried out in the US and Canada since the 1980’s. However the methodologies used vary greatly, as do the results. Despite similar infrastructure funding policies in numerous developed countries, no such empirical works exist outside of the US/Canada. The purpose of this research is to analyse the existing econometric models in order to identify, compare and contrast the theoretical bases, methodologies, key assumptions and findings of each. This research will assist in identifying if further model development is required and/or whether any of these models have external validity and are readily transferable outside of the US. The findings conclude that there is very little explicit rationale behind the various model selections and that significant model deficiencies appear still to exist.

Reasonable adjustments in assessment : putting law and policy into practice in Australia

The Australian Disability Standards for Education 2005 (Cth) require education providers to make reasonable adjustments in educational assessment so that students with disability can participate on the same basis as other students and be able to demonstrate what they know and can do. Reasonableness is governed by a determination of the balance of interests, benefits and detriment to the parties involved. The Standards require providers to consult with students and associates on adjustments, although guidance on how consultation should occur and how the views of students and associates are to be taken into account is vague. In this article, we identify three principles to be considered in order to put appropriate and effective reasonable adjustments in assessment into practice. While Australian law and assessment contexts are used to examine intentions, expectations and practices in educational assessment for students with disability, we argue that these three principles must be considered in any national education system to ensure equitable assessment practices and achieve equitable educational inclusion for students with disability.

The assessment of students with disabilities : the Australian law as to reasonable adjustment and academic integrity

This paper explains the legislation which underpins the right to reasonable adjustment in education for students with disabilities in Australian schools. It gives examples of the kinds of adjustment which may be made to promote equality of opportunity in the area of assessment. It also considers how the law has constructed the border between reasonable adjustment and academic integrity.

Traffic harmony in China? Investigating driver perceptions of traffic law enforcement and social responsibility for harmonious road use.

Social harmony can manifest in many ways. In rapidly motorizing countries like China, a growing area of potential disharmony is road use. The increased ability to purchase a car for the first time and a subsequent increase in new drivers has seen several Chinese cities take unprecedented measures to manage congestion. There is a corresponding need to ensure effective traffic law enforcement in promoting a safe environment for all road users. This paper reports qualitative research conducted with Beijing car drivers to investigate perceptions of unsafe road use, penalties for traffic violations, and improvements for the current system. Overall, the findings suggest awareness among drivers of many of the key risk factors. A perceived lack of clarity in how penalties are determined was identified and drivers in-dicated a desire to know how revenue from traffic fines is used. Several suggestions for improving the current system included school/community education about road risks and traffic law. The rise of private car ownership in China may contribute to a more harmonious personal life, but at the same time, may contribute to a decrease in societal harmony. A major challenge for authorities in any country is to promote the idea of a collective responsibility for road safety (traffic harmony), especially to those who perceive that traffic rules do not apply to them. This is a potentially greater challenge for China as it strives to balance harmony on the road and harmony in the broader society.

Wrong way go back! Rediscovering the path for charity law reform

Problems with charity law jurisprudence persist. The difficulties arose in the 20th century and are fundamental to the way the doctrine is presently theorised. They grew out of the approach taken in Pemsel’s Case to the categorisation of the ‘spirit and intendment’ of the Preamble to the Statute of Charitable Uses. Recent statutory reforms, such as the Charities Act 2006 (Eng&W), have compounded the underlying problems rather than resolving them. This paper aims to stimulate thinking about a new foundation for charity jurisprudence – while the approach may seem radical, the paper argues that these new foundations can be discerned underlying the current jurisprudence. The difficulties can be overcome by rediscovering the underlying jurisprudence which is disregarded in the current approach to categorisation. Giving voice, in contemporary language, to that foundational jurisprudence, this paper provides a way out of the current problems. It also provides an alternative way of conceptualising the doctrine of charitable purpose to guide reform.

International conventions and model laws : their impact on domestic commercial law

Panellist commentary on delivered conference papers on the topic of ‘International Conventions and Model Laws - Their Impact on Domestic Commercial Law’.