Fault Driven Supervised Tie Breaking for Test Case Prioritization

Regression test suites are an excellent tool to validate the existing functionality of an application during the development process. However, they can be large and time consuming to execute, thus making them inefficient in finding faults. Test Case Prioritization is an area of study that looks to improve the fault detection rates of these test suites by re-ordering execution sequence of the test cases. It attempts to execute the test cases that have the highest probability of detecting faults first. Most prioritization techniques base their decisions on the coverage information gathered from running the test cases. These coverage-based techniques however have a high probability of encountering ties in coverages between two or more test cases. Most studies employ a random selection to break these ties despite it being considered a lower bound method. This thesis designs and develops a framework to supervise the tie breaking in coverage based Test Case Prioritization using fault predictor models. Fault predictor models can assist in identifying the modules in the application that are most prone to containing faults. By selecting test cases that cover modules most prone to faults, the fault detection rate of the test cases can be improved. A fault prediction framework is also introduced in this thesis that supervises the tie breaking for coverage-based techniques. The framework employs an ensemble learner that aggregates results from multiple predictors. To date, no single predictor has been found that can perform consistently on all datasets. Numerous predictors have also required expert knowledge to make them performant. An ensemble learner is a reliable technique to mitigate the problems and bias faced by single predictors and disregard results from poorly performing predictors. In order to evaluate the supervised tie breaking, empirical studies were conducted on two large scale applications, Cassandra and Tomcat. As part of the evaluation, real faults that existed in the application during development were used instead of hand seeded faults or mutation faults as used by many other studies. The data used for fault prediction were also not groomed or marked by experts, unlike other studies. Results from the studies showed significant improvements in the fault detection rates for both case studies when using the fault driven supervision for tie breaking

Vulnerability and robustness in the essential gene complement of two bacterial species, profiled with CRISPRi

Bacterial essential genes contribute to the most fundamental processes of cellular life. The study of their functions in vivo has long been intractable to systematic genetic approaches, which are fundamental to understanding pathway level connections that govern cellular life and are a requirement for dissecting the complex cellular processes to which essential genes contribute. In Chapter 1 of this work I review recent advances in mapping gene-phenotype relationships in bacteria using the CRISPR-based technology, CRISPR interference (CRISPRi) for titratable gene knockdowns, focusing on their applications to the studies of essential genes, the exploration of chemical-genetic interactions, and the prospects for disentangling complex phenotypes in diverse bacterial species. In Chapter 2 I describe my analysis of the essential gene functions in the model Gram-negative bacterium Escherichia coli and the model Gram-positive Bacillus subtilis using datasets from paired chemical-genetic screens. In this work I identify both shared and Gram-negative specific mechanisms of collateral sensitization to antibiotic action. In Chapter 3 I investigate a fundamental property of essential genes, which is the relationship between their expression level and the cellular growth rate. Here, further developing CRISPRi tools in bacteria to predictably titrate knockdown efficacy, I interpret the knockdown-fitness relationships of each essential gene in E. coli and B. subtilis, discovering broad conservation of constraints setting and maintaining expression levels across these diverged species

CODE-CHANGE AWARE MUTATION BASED TESTING IN CONTINUOUSLY EVOLVING SYSTEMS

In modern software development practices, testing activities must be carried out frequently and preferably after each code change to bring confidence in anticipated system behaviour and, more importantly, to avoid introducing faults. When it comes to software testing, it is not only about what we are expecting; it is equally about what we are not expecting. Developers desire to test and assess the testing adequacy of the delta of behaviours between stable and modified software versions. Many test adequacy criteria have been proposed through the years, yet very few have been placed for continuous development. Among all proposed, one has been empirically verified to be the most effective in finding faults and evaluating test adequacy. Mutation Testing has been widely studied, but its current traditional form is impractical to keep up with the rapid pace of modern software development standards and code evolution due to a large number of test requirements, i.e., mutants. This dissertation proposes change-aware mutation testing, a novel approach that points to relevant change-aware test requirements, allows reasoning to what extent code modification is tested and captures behavioural relations of changed and unchanged code from which faults often arise. In particular, this dissertation builds contributions around challenges related to the code-mutants' behavioural properties, testing regular code modifications and mutants' fault detection effectiveness. First, this dissertation examines the ability of the mutants to capture the behaviour of regression faults and evaluates the relationship between the syntactic and semantic distance metrics often used to capture mutant-real fault similarity. Second, this dissertation proposes a commit-aware mutation testing approach that focuses rather on change-aware mutants that bring significant values in capturing regression faults. The approach shows 30\% higher fault detection in comparison with baselines and sheds light on the suitability of commit-aware mutation testing in the context of evolving systems. Third, this dissertation proposes the usage of high-order mutations to identify change-impacted mutants, resulting in the most extensive dataset, to date, of commit-relevant mutants, which are further thoroughly studied to provide the understanding and elicit properties of this particular novel category. The studies led to the discovery of long-standing mutants, demonstrated as suitable to maintain a high-quality test suite for a series of code releases. Fourth, this dissertation proposes the usage of learning-based mutant selection strategies when questioning how effective are the mutants of fundamentally different mutation generation approaches in finding faults. The outcomes raise awareness of the risk that the suitability of different kinds of mutants can be misinterpreted if not using intelligent approaches to remove the noise of impractical mutants. Overall, this dissertation proposes a novel change-aware testing approach and provides insights for software testing gatekeepers towards more effective mutation testing in the context of continuously evolving systems

Organization of Retinal Ganglion Cell Axons in the Developing Mouse Retinogeniculate Pathway

Appropriately organized synaptic connections are essential for proper neural circuit function. Prior to forming and refining synaptic connections, axons of projection neurons must first navigate long distances to their targets. Research in the axon guidance field has generated a great deal of knowledge about how axons successfully navigate through intermediate choice points and form initial connections with their synaptic targets. One aspect of neural circuit development that has been less well studied is whether axons are organized within their tracts. Axons could be highly ordered, or arranged haphazardly, to be sorted out within their destination target zone. Findings from several systems indicate that axon tracts are organized and, furthermore, that pre-target organization is important for accurate targeting. Chapter 1 will survey these findings as an introduction to my thesis. The remaining chapters present my research in the mouse retinogeniculate pathway, in which I examine three aspects of pre-target axon organization: the organization of cohorts of retinal ganglion cell (RGC) axons in the optic nerve and tract; the role of axon self association in tract organization; and the relationship between tract order and targeting. RGC axons project either ipsi- or contralaterally at the optic chiasm. In the first thalamic target, the dorsal lateral geniculate nucleus (dLGN), RGC axon terminals are organized based on retinotopy and laterality (i.e., into ipsi- and contralateral zones). Chapter 2 presents my findings on the organization of ipsilateral (ipsi) and contralateral (contra) RGC axons in the optic nerve and tract. Ipsilateral RGC axons cluster together in the optic nerve, are less tightly bundled in the optic chiasm, and once in the optic tract, again bundle together and are segregated from contralateral axons. Topographic and ipsi/contra axon order in the optic tract are largely in register, although ipsi- and contralateral axons from the same topographic region maintain distinct ipsi/contra segregation in the tract. Chapter 3 explores one potential mechanism involved in creating the organization between ipsi and contra RGC axons in the tract: differential fasciculation behavior between RGC axon cohorts. I used in vitro retinal explant culture systems to test the hypothesis that ipsilateral RGC axons have a greater preference to self-fasciculate than contralateral axons. Ipsilateral neurites display greater self-association/fasciculation than contralateral neurites, indicating an axon-intrinsic mechanism of ipsilateral-specific self-association. Chapter 4 examines tract organization and fasciculation in the EphB1 mutant retinogeniculate pathway. EphB1 is expressed exclusively by ipsilateral RGCs, and loss of EphB1 leads to a reduced ipsilateral projection and increased contralateral projection. However, aberrantly crossing axons project to the ipsilateral zone in the dLGN. Given its combination of an aberrant decussation phenotype with a grossly normal targeting phenotype, I used this mutant to explore the relationship between midline choice, tract organization, and targeting. First, remaining ipsilateral axons in the EphB1-/- optic tract largely retain their position in the lateral optic tract, but appear splayed apart, suggestive of aberrant fasciculation. In vitro, EphB1-/- ipsilateral neurites still bundle more than EphB1-/- contralateral neurites, although the magnitude of this difference is less striking than in wild-type retinal explants. Thus, EphB1 may be involved in preferential ipsilateral RGC axon fasciculation. In vivo, the aberrantly crossing axons in the EphB1 mutant grossly maintain their position in the ipsilateral zone of the optic tract (i.e., the lateral aspect), indicating a preservation of ipsilateral segregation in the tract. This is in line with a model in which bundling partners in the tract may help guide axons to the correct zone in the target. The data presented in this thesis detail two organizational modes of RGC axons in the developing optic nerve and tract, eye-specific (typographic) and topographic, and suggest that axon-intrinsic factors mediate ipsilateral-specific self-association. Axon-intrinsic factors likely act alongside extrinsic cellular and molecular cues in the developing retinogeniculate pathway to facilitate pre-target axon organization, which may in turn facilitate accurate formation of synaptic connections in the dLGN

Mining the Pseudomonas virulence factor pathway for novel small molecules

Bioactive small molecules often play an important role in bacterial virulence. Identification and exploration of these molecules will develop an understanding of new mechanisms for infection and potentially identify novel targets to inhibit virulence. Analysis of bacterial genomes identifies a large number of gene clusters that encode for small molecule-synthesizing enzymes (biosynthetic gene clusters), many of which are unexplored. Through genetic manipulation of these clusters, we can discover their small molecule products and determine their biological roles. We have identified a biosynthetic gene cluster conserved in over 300 strains of Pseudomonads. Many of these strains engage in pathogenic or symbiotic relationships with a range of human, animal, and plant hosts. We have shown that deletions within this gene cluster, named the Pseudomonas virulence factor (pvf), reduce fly infection by the pathogen Pseudomonas entomophila. In particular, the small molecule products of the pvf pathway are suggested to play a role in bacterial signaling and activation of virulence. Through overexpression of the pvf cluster in its native P. entomophila strain, we have discovered and characterized a number of small molecules that the pvf gene cluster is responsible for. These molecules include a class of pyrazine compounds, some of which are new to biology. We use fly infection and promoter-reporter assays to determine the biological roles of these molecules in virulence and cell-to-cell signaling of P. entomophila. Through proteomic analysis of the secretome, we have identified over 500 proteins that are differentially expressed between wildtype and pvf deletion strains, many which are known to be important to virulence and competition. To identify the active molecule responsible for pvf signaling activity we developed an optimized bioactivity-guided extraction and purification method. We can compare active purified fraction from spent media extracts of wildtype and pvf deletion strains using nuclear magnetic resonance (NMR) and liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) to identify the active pvf molecule. By characterizing the structures and functions of bioactive small molecules from pathogens, our work has the potential to develop an understanding for the production of these compounds and reveal novel and useful antimicrobial targets.Doctor of Philosoph

Surface Patterns On Single Cells: A Consequence Of A Phase Transition To Modulated Phases

Patterns are ubiquitous in the world around us, and we have only begun to scratch the surface of understanding their complexity and formation. In this thesis, we draw inspiration from rigid, extracellular surface patterns found on single living cells in many taxa and try to understand if there is a common thread in their pattern formation mechanisms that can be described by a single physical formalism. Pollen grains, butterfly wing scales, and deep-sea protists called phaeodarians all have beautifully ornate and varied hard surface structures that are likely patterned by the deposition of some soft organic matrix originating inside of the cell. We focus on pollen grain surfaces because of their remarkable geometric variety that is well documented. We find, through our own electron microscopy and careful histological techniques, that the patterns arise due to a phase separation of a transient polysaccharide material mechanically coupled to the underlying elastic cell membrane. We then show that the entire evolved diversity of patterns can be recapitulated by exploring both the equilibrium states and the dynamics of a modified Landau-Ginzburg model of phase transitions to modulated phases. We observe the surprising fact that only ~10% of extant species exhibit patterns that reach equilibrium. Furthermore, we find that although these patterns have evolved many times in seed plants, they are not, on average, selected for. The remaining 90% of pollen grain surfaces resemble arrested intermediate states of the phase transition process. We then document the pattern formation process in butterfly wing scales and show that a transient, spatially periodic surface material sits between the global surface features of the scales (the ridges). We postulate that the phase transition of this material may also contribute to the regular patterns on wing scale cells. We finally image the full three-dimensional features of geodesic phaeodarians tests using x-ray-computed tomography

Gene Discovery in Mendelian and Complex Diseases

Through the Finding of Rare Disease Genes in Canada (FORGE Canada) initiative, individuals affected with rare Mendelian diseases were clinically ascertained with a goal of identifying the genetic origin of their disease. Herein, I describe the methods for identifying the genetic basis of four Mendelian diseases. The application of next generation sequencing led to the discovery of non-synonymous variation in the DNA of individuals affected by rare diseases. The effects of the candidate variants were assessed using a series of functional experiments to complement the human genetics data. The variants observed in patients’ cells are extremely rare, were consistently predicted to be pathogenic by multiple in silico predictive programs, segregated with disease status in the family, and affected the biological properties of their respective gene products, as measured by functional assays. Having successfully identified genetic variants underlying the Mendelian diseases, we sought to use the same approach to extract the genetic variation that may predispose individuals to complex diseases, primarily neurodegenerative disorders. We designed a neurodegeneration specific gene panel that utilizes next generation sequencing chemistry. We sequenced patients diagnosed with one of five neurodegenerative diseases: 1) Alzheimer’s disease; 2) amyotrophic lateral sclerosis (ALS); 3) frontotemporal dementia (FTD); 4) Parkinson’s disease; or 5) vascular cognitive impairment, as part of the Ontario Neurodegenerative Disease Research Initiative (ONDRI). We were successful in detecting rare variants in a large fraction of cases that may be related to the neurodegenerative phenotypes. We also independently ascertained three large, unique families affected with familial ALS and FTD across multiple generations. The three families are diagnosed with ALS and/or FTD and in which the same hexanucleotide repeat expansion in the C9orf72 gene has been observed in all three pedigrees, but their phenotypes vary significantly. We sequenced affected individuals and observed several, distinct variants in these families that may explain the additional neurodegenerative phenotypes observed. In summary, the application of next generation sequencing has successfully identified novel genetic loci in both Mendelian and complex diseases

Autism Spectrum Disorders

Estimated prevalence rates of autism spectrum disorders (ASDs) have increased at an alarming rate over the past decade; current estimates stand as high as 1 in 110 persons in the population with a higher ratio of affected males to females. In addition to their emotional impact on the affected persons and their family members (in fact, the latter are often unrecognized unaffected “patients†themselves), the economic and social impacts of ASDs on society are staggering. Persons with ASDs will need interdisciplinary approaches to complex treatment and life planning, including, but not limited to, special education, speech and language therapy, vocational skills training and rehabilitation, social skills training and cognitive remediation, in addition to pharmacotherapy. The current book highlights some of the recent research on nosology, etiology, and pathophysiology. Additionally, the book touches on the implications of new research for treatment and genetic counseling. Importantly, because the field is advancing rapidly, no book can be considered the final word or finished product; thus, the availability of open access rapid publication is a mechanism that will help to assure that readers remain current and up-to-date

The microbiome of diabetic foot ulcers and the role of biofilms

Diabetic Foot Ulcers are a common precursor to the development of infection and amputations. A breach in the protective skin barrier represents a portal of entry for invading microorganisms, where infective episodes frequently pursue. Three key areas that may augment clinical care are one. understanding what microorganisms are present in Diabetic Foot Ulcers, two. differentiating if microorganisms are planktonic microbial cells or slow growing microbial biofilms and three. treating Diabetic Foot Ulcers complicated by microorganisms with effective topical agents. As part of this thesis, 16S rDNA next generation sequencing was utilised to profile the microbiota of infected Diabetic Foot Ulcers (DFUs). Clinical / laboratory data and treatment outcomes were collected and correlated against microbiota data. Thirty-nine patients with infected DFUs were recruited over twelve-months. Shorter duration DFUs (less than six weeks) all had one dominant bacterial species (n= five of five, 100%, p <⋅001), S. aureus in three cases and S. agalactiae in two. Longer duration DFUs (≥six weeks) were diversely polymicrobial (p = .01) with an average of 63 (range 19-125) bacterial species. Severe Diabetic Foot Infections (DFIs) had complex microbiota’s and were distinctly dissimilar to less severe infections (p = .02), characterised by the presence of low frequency microorganisms. Our results confirm that short DFUs have a simpler microbiota’s consisting of pyogenic cocci but chronic DFUs have a highly polymicrobial microbiota. The duration of a DFU may be useful as a guide to directing antimicrobial therapy. Secondly, we utilised Scanning electron microscopy (SEM) and Fluorescent in situ Hybridisation (FISH) techniques to determine if DFUs were complicated by sessile, slow growing bacteria referred to as biofilms. 65 DFU specimens were obtained from subjects with infected chronic ulcers. Of the 65 DFU specimens evaluated by microscopy, all were characterized as containing biofilm (100%, p < .001). Molecular analyses of DFU specimens revealed diverse polymicrobial communities. No clinical visual cues were identified in aiding clinicians identify wound biofilm. Microscopy visualization when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and a paradigm shift of managing these complicated wounds needs to consider anti-biofilm strategies. Lastly, the effectiveness of various topical antimicrobials commonly used in woundcare were tested in two separate studies by employing in vitro models, ex vivo porcine skin explant models and in vivo human studies. In the first study, 17 participants with chronic non-healing DFUs due to suspected biofilm involvement were recruited to receive one-week application of Cadexomer Iodine ointment. Real-time qPCR was used to determine the microbial load with 11 participants exhibiting one-two Log10 reductions in microbial load after treatment, in comparison to six patients who experienced less than one log10 reduction (p =.04). Scanning electron microscopy (SEM) and/or fluorescent in situ hybridisation (FISH) confirmed the presence or absence of biofilm in all 17 participants. 16SrDNAnextgenerationsequencing provided useful insights that these wounds support complex polymicrobial communities and demonstrated that Cadexomer Iodine had a broad level of antimicrobial activity in reducing both facultative anaerobes such as Staphylococcus spp., Serratia spp., aerobes including Pseudomonas spp., and obligate anaerobes including Clostridiales family XI. In the second study, a range of topical antimicrobial wound solutions were tested under three different conditions; (in vitro) 4 % w/v melaleuca oil, polyhexamethylene biguanide, chlorhexidine, povidone iodine and hypochlorous acid were tested at short duration exposure times for 15-minutes against three-day mature biofilms of S. aureus and P. aeruginosa. (ex vivo) Hypochlorous acid was tested in a porcine skin explant model with twelve cycles of tenminute exposure, over 24 hours, against three-day mature P. aeruginosa biofilms. (in vivo) 4 % w/v Melaleuca Oil was applied for 15-minutes exposure, daily, for seven days, in ten patients with chronic non-healing Diabetic Foot Ulcers (DFUs) complicated by biofilm. In vitro assessment demonstrated variable efficacy in reducing biofilms ranging between 0.5 log10 reductions to full eradication. Repeated instillation of hypochlorous acid in a porcine model achieved less than one log10 reduction (0.77 log10, p < 0.1). Application of 4 % w/v melaleuca oil in vivo, resulted in no change to the total microbial load of DFUs complicated by biofilm (median log10 microbial load pre-treatment = 4.9 log10 versus 4.8 log10 (p = .43). In conclusion, to the best of our knowledge, the in vivo human studies testing the performances of topical antimicrobials represents the first in vivo evidence employing a range of molecular and microscopy techniques. These demonstrate the ability of Cadexomer Iodine (sustained release over 48-72 hours) to reduce the microbial load of chronic non-healing DFUs complicated by biofilm. In contrast, short durations of exposure to topical antimicrobial wound solutions commonly utilised by clinicians are ineffective against microbial biofilms, particularly when used in vivo

Discursive intersections of newspapers and policy elites: a case study of genetically modified food in Britain, 1996-2000

This thesis explores the under-researched terrain of policy elite-newspaper engagements and in so doing makes a substantive contribution in formulating an original conceptual framework for understanding how the interactional dynamics of the political-media complex work. This framework is then applied to the GM food row in Britain by asking how contestation emerged, was sustained then subsided in the political-media complex. This reconstructs the processes by which the pro-GM government consensus was challenged by newspapers, conflict escalated to fever pitch, threatening policy elite agenda and was finally negotiated through key compromises. Drawing on a theoretical framework that combines participatory politics, the political-media complex and new risks, the thesis conceptualises interactional dynamics as ‘discursive intersections’. These are shifts in claims and counter-claims that emerge during engagement at the interface of different sets of knowledge, cultures and agenda in the political-media complex. However there is an element of unpredictability in discursive intersections that arises from the paradoxical interdependence-independence of the relationship in the political-media complex; the elective and episodic nature of engagement on particular issues; and the variable form this may take with potential for conflict, negotiation or consensus. Historical and wider argumentative contexts are crucial to how and what form engagement takes place but do not define it. Thus, the trajectory of discursive intersections needs to be explored empirically rather than predetermined theoretically. This is done using a hybrid methodology that draws attention to the dialogical, persuasive nature of discursive intersections. The substantive contribution of the research is the formulating of this alternative framework for the analysis of interactional dynamics and its application to the GM food row in Britain. It does this by exploring how – that is the process in which - engagement emerged, escalated into contestation, was negotiated and then subsided. What emerged were the following findings. (1) Parallel, sustained and conflictual systems of argumentation about risk were developed between media and political elites despite elite consensus, abstract debates and short news cycles. (2) Newspaper contestation was constructed around a deeply ambivalent suspended certainty based on claims that there was no evidence of risk or benefit, harm or safety and demands for elite responsiveness to acute public anxiety over this